Agricultural and horticultural insecticidal composition and method for controlling agricultural and horticultural pests

Abstract

The present invention provides: an agricultural / horticultural insecticide composition which contains an insecticidal protein derived from Bacillus thuringiensis, and prumycin or a salt thereof or kanosamine or a salt thereof; or a method for controlling agricultural / horticultural insect pests which uses said agricultural / horticultural insecticide composition.

Description

[Technical field]

[0001] The present invention relates to an agricultural and horticultural insecticidal composition and a method for controlling agricultural and horticultural pests. [Background technology]

[0002] Preparations containing a culture of Bacillus thuringiensis (hereinafter referred to as "BT agents") have been widely used as microbial pesticides to control agricultural pests and sanitary pests. In general, when using chemical synthetic pesticides, a period of time must be allowed before harvesting the crop to avoid residues on the crop. On the other hand, BT agents are easily decomposed in the soil and can be used on crops just before harvesting. In addition, compared to chemical synthetic pesticides, BT agents are effective specifically against pest species, so they can control only the target pest species without harming beneficial insects. For these reasons, BT agents are used to promote reduced pesticide use and environmental conservation-oriented agriculture, without the risk of environmental pollution or substances harmful to the human body remaining on crops (Non-Patent Document 1).

[0003] Bacillus thuringiensis is a Gram-positive bacterium that is widely distributed in soil, and produces granules containing crystalline insecticidal proteins called crystals (hereinafter simply referred to as "insecticidal proteins") in the sporulation stage. It is known that this insecticidal protein is activated by proteases in the insect's intestine and exhibits insecticidal activity against insects. In addition, Bacillus thuringiensis produces different insecticidal proteins depending on its subspecies, and these insecticidal proteins have different insecticidal activities. For example, it is known that the subspecies kurstaki and aizawai are active against lepidopteran and dipteran insects, israelensis is active against dipteran insects, and tenebrionis and japonensis buibui are active against coleopteran insects (Non-Patent Document 2).

[0004] Currently, more than 400 types of insecticidal proteins have been identified, classified into Cry1 to Cry73. One strain of Bacillus thuringiensis does not necessarily produce one type of insecticidal protein; for example, the Bacillus thuringiensis HD-1 strain produces three types of insecticidal proteins, Cry1A(a), Cry1A(b), and Cry1A(c).

[0005] Insecticidal proteins are roughly classified into two types based on molecular weight: 130 kDa type and 70 kDa type. The 130 kDa type includes Cry1A, which is active against Lepidoptera, Cry4A and Cry4B, which are active against Diptera, Cry8C, which is active against Coleoptera, and Cry5, which is active against Nematoda. On the other hand, the 70 kDa type includes Cry2, which is active against both Lepidoptera and Diptera, Cry11A and Cry11B, which are active against Diptera, and Cry3B, which is active against Coleoptera (Non-Patent Document 3).

[0006] To date, many BT agents that utilize the properties of the above-mentioned insecticidal proteins have been developed and sold. However, in cultivation sites, there is a demand for a control method using BT agents that is economical, stable in effect, and fast-acting, just like chemically synthesized pesticides. Various approaches have been taken to enhance the insecticidal activity of BT agents, one of which is to mix a synergist (synergist). Methods for mixing synergists include mixing different insecticidal proteins, such as mixing Cry1A(a) and Cry1A(c) (Non-Patent Document 4), mixing Cry1A(a) and Cry1C (Non-Patent Document 5), and mixing Cry1A or Cry2 with Vip3Aa, an insecticidal protein secreted during vegetative cells (Non-Patent Documents 6 and 7). Methods for mixing synergists include mixing an insecticidal protein with spores of the genus Bacillus (Non-Patent Documents 8 and 9), mixing an insecticidal protein with Bacillus cereus ( B . candle ) and zwittermicin A produced by such strains (Patent Document 1).

[0007] The above-mentioned zwittermicin A is a low-molecular-weight, water-soluble antibiotic produced by Bacillus cereus and Bacillus thuringiensis (Non-Patent Document 10), and is known to inhibit the growth of plant pathogenic filamentous fungi and some bacteria (Non-Patent Document 11). Manker et al. (Patent Document 2) have reported that zwittermicin A does not exhibit insecticidal activity alone, but enhances the activity of Bacillus thuringiensis when mixed with Bacillus thuringiensis.

[0008] Also, Fernandez et al. have reported that Bacillus thuringiensis that produces zwittermicin A in addition to an insecticidal protein has higher insecticidal activity than conventional Bacillus thuringiensis (Patent Document 3).

[0009] Furthermore, in another document (Patent Document 4), a substance different from zwittermicin A that enhances the insecticidal activity of Bacillus thuringiensis is reported, but its structure has not yet been identified.

[0010] The pest species targeted by BT agents are mainly lepidopterous pests, among which the common cutworm ( Spodoptera cleaning ) and tobacco moth ( Helicoverpa armor-bearer ) is an extremely polyphagous insect that attacks many vegetables and field crops, causing severe damage and is considered a serious pest. The common cutworm is widely distributed throughout Asia, and even in Japan, it was previously thought that it could only survive winter outdoors in warm regions. However, due to the increase in heating facilities and global warming in recent years, the area in which the common cutworm occurs has been moving northward, and the area in which it occurs is also expanding. The genus Spodoptera, to which the common cutworm belongs, contains many species that are important agricultural pests, such as the beet armyworm ( S . small ), Striped Cutworm ( S . depraved ), Siberian armyworm ( S . fruitless ) etc.

[0011] Purmycin (4-ND-alanyl-2,4-diamino-2,4-dideoxyarabinose) is produced by bacteria of the genus Streptomyces and Bacillus, and is known as an effective drug against gray mold, sclerotinia rot, powdery mildew, etc. Purmycin was discovered by Hata et al. in 1971. Streptomyces It was isolated from the culture filtrate of sp. F-1028 strain (Non-Patent Document 12), and then in 1973, Omura et al. reported that it was effective against gray mold and sclerotinia rot of beans, and powdery mildew of cucumber (Non-Patent Document 13). The structure of prumycin was determined by Omura et al. in 1974 (Non-Patent Document 14). After that, total synthesis was achieved by three independent groups (Non-Patent Documents 15-24), and the structure of prumycin shown in the following formula was confirmed.

[0012] [ka]

[0013] Regarding naturally occurring prumycin, Streptomyces In addition to Bacillus sp., it was reported that a specific strain of Bacillus cereus produces prumycin (Patent Document 5). Furthermore, in 1989, Bacillus subtilis ( B . subtle It has been reported that a specific strain of Pseudomonas aeruginosa produces prumycin (Patent Document 6).

[0014] Kanosamine (3-amino-3-deoxy-D-glucopyranose) is a lactic acid bacteria produced by Bacillus cereus and Bacillus aminoglucosidicus ( B . aminoglycoside Kanosamine was isolated in 1958 by Cron et al. as an acid hydrolysate of kanamycin, and the structure shown in the following formula was confirmed (Non-Patent Document 25). In 1996, Milner et al. reported that kanosamine is Botrytis cinerea , Rhizoctonia solani , Alternaria alternata , Fusarium oxysporumIt has been reported that it is effective against (Non-Patent Document 26).

[0015] [ka] [Prior art documents] [Patent documents]

[0016] [Patent Document 1] Special Publication No. 8-501798 [Patent Document 2] Special Publication No. 2001-516201 [Patent Document 3] WO2020 / 102592 publication [Patent Document 4] US Patent 6,635,245 [Patent Document 5] Japanese Unexamined Patent Publication No. 54-157896 [Patent Document 6] Japanese Patent Application Publication No. Hei 2-209803 [Patent Document 7] WO2018 / 029775 publication [Non-patent literature]

[0017] [Non-Patent Document 1] Plant Biotechnology Journal., 9, 283-300 (2011) [Non-Patent Document 2] Microbiological Reviews., June, 242-255 (1989) [Non-Patent Document 3] Toxins., 6, 3296-3325 (2014) [Non-Patent Document 4] Appl. Environ. Microbiol., Feb, 583-586 (1996) [Non-Patent Document 5] Letters in Applied Microbiology., 40, 460-465 (2005)

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[0018] The above-mentioned BT agents are less stable and effective than chemical pesticides due to the effects of ultraviolet rays and uneven adhesion to plant leaves, so there is a demand for the development of BT agents with better control effects.

[0019] Under these circumstances, the inventors have conducted intensive research to create a biological preparation with high insecticidal effect, and have discovered that mixing prumycin or a salt thereof, or kanosamine or a salt thereof, with an insecticidal protein has the effect of significantly enhancing the insecticidal activity of the insecticidal protein.

[0020] There have been no reports so far on the insecticidal activity of prumycin and kanosamine, and their function as an insecticidal activity enhancer of insecticidal proteins is completely unknown.

[0021] As an agricultural and horticultural composition containing prumycin, an agricultural and horticultural fungicidal composition containing prumycin and surfactins as active ingredients has been reported (Patent Document 7). Although Patent Document 7 confirms that the composition has a fungicidal effect, it makes no mention of its insecticidal effect.

[0022] An object of the present invention is to provide a novel agricultural and horticultural insecticidal composition which has a high insecticidal effect and is environmentally friendly, and a method for controlling agricultural and horticultural pests. [Means for solving the problem]

[0023] The present inventors have studied the effects of conventional fungicidal compounds in detail and found that prumycin or its salt, or kanosamine or its salt, significantly enhances the insecticidal activity of insecticidal proteins and exhibits extremely useful insecticidal activity. As a result, the present invention has been completed, which relates to an agricultural and horticultural insecticidal composition that can more effectively control pests, particularly lepidopteran pests, that cause severe damage to many vegetables and field crops than conventional BT agents. The present invention includes the inventions exemplified below as embodiments thereof.

[0024] [1] Bacillus thuringiensis ( Bacillus Thuringian ) and prumycin or a salt thereof, or kanosamine or a salt thereof An agricultural and horticultural insecticidal composition comprising: [2] The agricultural and horticultural insecticidal composition described in [1], wherein the insecticidal protein comprises Cry1A or Cry1C. [3] Bacillus thuringiensis ( Bacillus Thuringian The agricultural and horticultural insecticidal composition according to [1] or [2], which contains a culture dried product. [4] The agricultural and horticultural insecticidal composition according to any one of [1] to [3], which contains a cultured and dried product of a fungus that produces prumycin or a salt thereof, or kanosamine or a salt thereof. [5] The agricultural and horticultural insecticidal composition according to any one of [1] to [4], further comprising 0.1 to 10 mass % calcium chloride. [6] The agricultural and horticultural insecticidal composition according to any one of [1] to [5], which is to be applied to plants using a liquid having a content of prumycin or a salt thereof, or kanosamine or a salt thereof of 0.1 ppm to 100 ppm. [7] The agricultural and horticultural insecticidal composition according to any one of [1] to [6], which is applied to a plant using a liquid having an insecticidal protein content of 0.1 ppm to 500 ppm. [8] The agricultural and horticultural insecticidal composition according to any one of [1] to [7], which contains prumycin hydrochloride. [9] The agricultural and horticultural insecticidal composition according to any one of [1] to [8], which contains kanosamine hydrochloride.

[10] Bacillus thuringiensis ( Bacillus Thuringian (a) applying to a plant an insecticidal protein derived from (b) applying prumycin or a salt thereof or kanosamine or a salt thereof to the plant simultaneously, successively or separately with said insecticidal protein; A method for controlling agricultural and horticultural pests comprising the steps of:

[11] Bacillus thuringiensis ( Bacillus Thuringian A method for controlling agricultural and horticultural pests, comprising the step of applying prumycin or a salt thereof, or kanosamine or a salt thereof to a recombinant plant producing an insecticidal protein derived from the fungus.

[12] The control method according to

[10] , wherein the application in step (a) or (b) includes soil incorporation, irrigation, seed smearing, seed soaking, or foliage spraying.

[13] The insecticidal protein, and The control method according to

[10] or

[12] , which uses an agricultural and horticultural insecticidal composition containing prumycin or a salt thereof, or kanosamine or a salt thereof.

[0025] [1'] Bacillus thuringiensis ( Bacillus Thuringian 2. An agricultural and horticultural insecticidal composition comprising an insecticidal protein derived from said plant, said composition being for use in combination with prumycin or a salt thereof, or kanosamine or a salt thereof. [2'] An agricultural and horticultural insecticidal composition containing prumycin or a salt thereof, or kanosamine or a salt thereof, which is characterized in that it is an insecticidal composition for agricultural and horticultural use containing prumycin or a salt thereof, Bacillus Thuringian ) for use in combination with an insecticidal protein derived from said composition. [3'] Bacillus thuringiensis ( Bacillus Thuringian ), and Purmycin or its salt or Kanosamine or its salt The agricultural and horticultural insecticidal composition according to [1'] or [2'], [4'] The agricultural and horticultural insecticidal composition according to any one of [1'] to [3'], wherein the insecticidal protein comprises Cry1A or Cry1C. [5'] Bacillus thuringiensis ( Bacillus Thuringian The agricultural and horticultural insecticidal composition according to any one of [1'] to [4'], which contains a culture dried product of [6'] The agricultural and horticultural insecticidal composition according to any one of [1'] to [5'], which contains a cultured and dried product of a fungus which produces prumycin or a salt thereof, or kanosamine or a salt thereof. [7'] The agricultural and horticultural insecticidal composition according to any one of [1'] to [6'], further comprising 0.1 to 10 mass % of calcium chloride. [8'] The agricultural and horticultural insecticidal composition according to any one of [1'] to [7'], which is applied to plants using a liquid having a content of prumycin or a salt thereof, or kanosamine or a salt thereof of 0.1 ppm to 100 ppm. [9'] The agricultural and horticultural insecticidal composition according to any one of [1'] to [8'], which is applied to a plant using a liquid having an insecticidal protein content of 0.1 ppm to 500 ppm. [10'] The agricultural and horticultural insecticidal composition according to any one of [1'] to [9'], which contains prumycin hydrochloride. [11'] The agricultural and horticultural insecticidal composition according to any one of [1'] to [10'], which contains kanosamine hydrochloride.

[0026] [1''] A method for controlling agricultural and horticultural pests using Bacillus thuringiensis ( Bacillus Thuringian ) and the use of a composition comprising an insecticidal protein derived therefrom. [2''] Bacillus thuringiensis ( Bacillus ThuringianUse of a composition comprising prumycin or a salt thereof, or kanosamine or a salt thereof, in combination with an insecticidal protein derived from prunus persicae, for controlling agricultural and horticultural pests. [3 ''] The composition is characterized in that it is produced by Bacillus thuringiensis ( Bacillus Thuringian The use described in [1''] or [2''], comprising an insecticidal protein derived from Pseudomonas aeruginosa, and prumycin or a salt thereof, or kanosamine or a salt thereof. [4''] The use described in any one of [1''] to [3''], wherein the insecticidal protein comprises Cry1A or Cry1C. [5 ''] The composition comprises Bacillus thuringiensis ( Bacillus Thuringian ) The use described in any one of [1''] to [4''], including a culture dried product. [6''] The use described in any one of [1''] to [5''], wherein the composition contains a dried culture of a bacterium that produces prumycin or a salt thereof, or kanosamine or a salt thereof. [7''] The use described in any one of [1''] to [6''], wherein the composition further contains 0.1 to 10 mass% calcium chloride. [8''] The use according to any one of [1''] to [7''], for applying to a plant a liquid having a content of prumycin or a salt thereof, or kanosamine or a salt thereof of 0.1 ppm to 100 ppm. [9''] The use according to any one of [1''] to [8''], for applying to a plant a liquid having an insecticidal protein content of 0.1 ppm to 500 ppm. [10''] The use described in any one of [1''] to [9''], wherein the composition contains prumycin hydrochloride. [11''] The use described in any one of [1''] to [10''], wherein the composition contains kanosamine hydrochloride. Effect of the Invention

[0027] In one aspect of the present invention, there are provided an agricultural and horticultural insecticidal composition having a high insecticidal effect and a method for controlling agricultural and horticultural pests, which has a high insecticidal effect and a low environmental impact. The agricultural and horticultural insecticidal composition has excellent insecticidal activity against pests that damage vegetables and field crops, particularly against Spodoptera litura and Helicoverpa armigera, and enables more effective control of agricultural and horticultural pests with a small application rate compared to the application of a conventional BT agent alone. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] (Agricultural and horticultural insecticidal composition) In one aspect of the present invention, Bacillus thuringiensis ( Bacillus Thuringian The insecticidal protein derived from Bacillus thuringiensis is used in combination with prumycin or a salt thereof, or kanosamine or a salt thereof. The insecticidal protein derived from Bacillus thuringiensis and prumycin or a salt thereof, or kanosamine or a salt thereof may be prepared separately and mixed when applied to a plant to prepare a single composition, or may be applied to a plant separately. Also, a composition containing the insecticidal protein derived from Bacillus thuringiensis and prumycin or a salt thereof, or kanosamine or a salt thereof may be prepared and stored in advance, and the composition may be applied to a plant.

[0029] In one embodiment of the present invention, Bacillus thuringiensis for producing the insecticidal protein used in the composition may be isolated from nature or purchased from a strain collection organization or the like. As Bacillus thuringiensis, for example, subspecies kurstaki, aizawai, israelensis, tenebrionis, or japonensis buibui can be preferably used. In addition, Bacillus thuringiensis may include live cells, spores, cells killed by heat or chemical means, or crushed products of these cells. As Bacillus thuringiensis, a dried culture of Bacillus thuringiensis can be used.

[0030] Furthermore, a commercially available BT preparation may be used as the composition containing an insecticidal protein derived from Bacillus thuringiensis. Examples of commercially available BT formulations include Tune Up® WG, Basilex® WG, Delfin® WG, Zentari® WG, Flowback® DF, Esmark® DF, Torrow® CT WG, Sabrina® Flowable, Agree® WG, Deliver®, Javelin® WG, Thuricide®, Biobit® DF, Crymax® WDG, Dipel® DF, Florbac®, Foray® 48B, XenTari® DF, CoStar® WG, Cordalene®, Cutlass®, BactoSpeine® DF, BMP1232X® WP, Biolep®, Lepinox Plus®, and Lipel®. SP, Turex (registered trademark) 50WG, etc.

[0031] In one embodiment of the present invention, the insecticidal protein is not particularly limited as long as it is an insecticidal protein derived from Bacillus thuringiensis, and specific examples thereof include Cry1A(a), Cry1A(b), Cry1A(c), Cry1C, Cry1D, Cry2A, Vip3A, etc. In one embodiment of the present invention, the composition may contain only one type or two or more types of these insecticidal proteins. In one embodiment, the insecticidal protein may be produced by Bacillus thuringiensis. In another embodiment, the insecticidal protein may be produced by chemical synthesis.

[0032] The insecticidal protein can also be produced by biosynthesis using bacteria such as Bacillus thuringiensis. Furthermore, the insecticidal protein may be produced by bacteria belonging to Bacillus thuringiensis that exist in nature, or may be produced by a transformant (e.g., Escherichia coli, Bacillus subtilis, plant, etc.) prepared using a conventional genetic engineering technique. The gene having a base sequence encoding the amino acid sequence of the insecticidal protein (hereinafter sometimes referred to as "the gene") used in preparing such a transformant may be (1) a gene cloned from the naturally occurring gene, (2) a gene in which deletion, substitution or addition of some bases has been artificially introduced into the base sequence of the gene cloned from the naturally occurring gene (i.e., a naturally occurring gene subjected to a mutation treatment (partial mutagenesis, mutation treatment, etc.)), or (3) a gene artificially synthesized.

[0033] In one embodiment of the present invention, prumycin or a salt thereof, or kanosamine or a salt thereof, which is used as a component for enhancing the insecticidal activity of an insecticidal protein, can be produced by the chemical synthesis shown below, or can be obtained from the culture filtrate of a microorganism capable of producing these compounds.

[0034] Purmycin or a salt thereof can be produced by chemical synthesis according to the methods described in Non-Patent Documents 13 to 24. Purmycin or a salt thereof can be produced by the following methods: Streptomyces It can be produced by biosynthesis as a fermentation product of a microorganism capable of producing prumycin or a salt thereof, such as Bacillus sp., Bacillus cereus, or Bacillus subtilis, or by using cultured cells of a microorganism capable of producing prumycin or a salt thereof.

[0035] Kanosamine or its salts can be produced by chemical synthesis according to the methods described in Non-Patent Documents 27 and 28. Kanosamine or its salts can also be produced by biosynthesis as a fermentation product of a microorganism capable of producing kanosamine or its salts, such as Bacillus cereus or Bacillus aminoglucosidicus, and cultured cells of a microorganism capable of producing kanosamine or its salts can also be used. Kanosamine or its salts can also be purchased as a reagent from Sigma-Aldrich Japan LLC.

[0036] In one embodiment of the present invention, a salt of prumycin or a salt of kanosamine may be used. The salt of prumycin or the salt of kanosamine is not particularly limited, and examples thereof include hydrochloride, phosphate, borate, formate, acetate, benzoate, etc., with hydrochloride and acetate being preferred.

[0037] The method for culturing each microorganism capable of producing prumycin or a salt thereof, or kanosamine or a salt thereof is not particularly limited, and known means such as stationary culture on a solid medium, liquid culture, etc. can be used. The type of medium and culture conditions that can be used are not particularly limited as long as the above-mentioned microorganisms survive and grow, and examples thereof include general media such as meat extract media, as well as media containing glucose, peptone, and yeast extract. In addition to liquid media, solid media such as agar-containing slants and plates may also be used.

[0038] The carbon source for the medium may be any that can be assimilated by the above-mentioned strain, including sugars such as glucose, galactose, lactose, sucrose, maltose, malt extract, blackstrap molasses, starch syrup, and starch hydrolysates, as well as various synthetic or natural carbon sources that can be used by microorganisms capable of producing prumycin or a salt thereof, or kanosamine or a salt thereof.

[0039] Similarly, the nitrogen source for the above-mentioned medium may be any of various synthetic or natural substances that can be utilized by the strain, including organic nitrogen-containing substances such as peptone, meat extract, yeast extract, soybean flour, and corn steep liquor.

[0040] In addition, according to standard methods for microbial cultivation, inorganic salts such as salt and phosphates, metal salts such as calcium, magnesium and iron, and trace nutrient sources such as vitamins and amino acids can be added to the above-mentioned medium as necessary.

[0041] The above-mentioned culture can be carried out under aerobic conditions such as shaking culture, aeration culture, etc. The culture temperature is preferably 20 to 40° C., more preferably 25 to 35° C., the pH is preferably 5 to 8, more preferably 6 to 7, and the culture period is preferably 1 to 4 days, more preferably 2 to 3 days.

[0042] When using prumycin or a salt thereof or kanosamine or a salt thereof contained in the culture solution of a bacteria producing prumycin or a salt thereof, or kanosamine or a salt thereof, the culture solution itself may be used, or the culture filtrate may be used, or the substance may be purified from the culture filtrate. The method for purifying prumycin or a salt thereof and kanosamine or a salt thereof from the culture solution is not particularly limited, and may be carried out by known methods such as acid precipitation, salting out, ultrafiltration, various types of chromatography, and electrophoresis.

[0043] In one embodiment, the composition is 1) a composition comprising a chemically synthesized insecticidal protein and chemically synthesized prumycin or a salt thereof, or kanosamine or a salt thereof, 2) a composition comprising a chemically synthesized insecticidal protein and biosynthesized prumycin or a salt thereof, or kanosamine or a salt thereof, 3) a composition comprising a chemically synthesized insecticidal protein and a dried culture of a microorganism producing prumycin or a salt thereof, or kanosamine or a salt thereof, which contains prumycin or a salt thereof, or kanosamine or a salt thereof, 4) a composition comprising a biosynthesized insecticidal protein and chemically synthesized prumycin or a salt thereof, or kanosamine or a salt thereof, 5) a composition comprising a biosynthesized insecticidal protein and biosynthesized prumycin or a salt thereof, or kanosamine or a salt thereof, 6) a composition comprising a biosynthesized insecticidal protein and a dried culture of a microorganism producing prumycin or a salt thereof, or kanosamine or a salt thereof, which contains prumycin or a salt thereof, or kanosamine or a salt thereof, 7) a Bacillus containing the insecticidal protein. the composition may be, for example, a composition comprising a dried Bacillus thuringiensis culture containing an insecticidal protein and chemically synthesized prumycin or a salt thereof, or kanosamine or a salt thereof; 8) a composition comprising a dried Bacillus thuringiensis culture containing an insecticidal protein and biosynthesized prumycin or a salt thereof, or kanosamine or a salt thereof; 9) a composition comprising a dried Bacillus thuringiensis culture containing an insecticidal protein and a dried culture of a microorganism capable of producing prumycin or a salt thereof, or kanosamine or a salt thereof, which contains prumycin or a salt thereof, or kanosamine or a salt thereof; 10) a dried culture of a microorganism capable of simultaneously producing an insecticidal protein and prumycin or a salt thereof, or kanosamine or a salt thereof; and 11) a dried culture of a microorganism capable of simultaneously producing an insecticidal protein, prumycin or a salt thereof, and kanosamine or a salt thereof.

[0044] Bacillus thuringiensis subspecies kurstakii ( Bacillus Thuringian subsp. kurstaki ) culture dried material and Bacillus thuringiensis subsp. Aizawai ( Bacillus Thuringiansubsp. aizawai ) Examples of insecticidal proteins contained in the dried culture product include Cry1A(a), Cry1A(b), Cry1A(c), Cry1C, Cry1D, Cry2A, etc. In one embodiment of the present invention, the content of the insecticidal protein that can serve as an active ingredient in the composition varies depending on the form of use and the dilution ratio, and is not particularly limited, but the total content (concentration) of the insecticidal protein in the liquid to be applied to plants is preferably 0.1 ppm to 500 ppm, more preferably 1 ppm to 50 ppm.

[0045] The content (concentration) of insecticidal proteins can be measured by purifying insecticidal proteins from a dried culture of Bacillus thuringiensis by the precipitation separation method of Goodman et al. using dextran sulfate and polyethylene glycol (Non-Patent Document 29), and then isolating and quantifying the proteins using HPLC with reference to the analytical method of Masson et al. (Non-Patent Document 30). In addition, when using a commercially available BT preparation, the content (concentration) of insecticidal proteins can be measured by diluting the BT preparation and separating and quantifying the proteins using HPLC with reference to the analytical method of Masson et al. (Non-Patent Document 30) without purifying the proteins.

[0046] In one embodiment of the present invention, the content of prumycin or its salt, or kanosamine or its salt in the composition depends on the form of use and dilution ratio, and is not particularly limited, but the content (concentration) of prumycin or its salt alone, or kanosamine or its salt alone in the liquid applied to plants is preferably 0.1 ppm to 100 ppm, more preferably 0.5 ppm to 5 ppm. When prumycin or its salt and kanosamine or its salt are used in combination, the total content (concentration) of prumycin or its salt and kanosamine or its salt in the liquid applied to plants is preferably 0.1 ppm or more, more preferably 0.5 ppm or more. When prumycin or its salt, or kanosamine or its salt is obtained as a culture dry product of a microorganism producing prumycin or its salt, or kanosamine or its salt, the content (concentration) of prumycin or its salt, or kanosamine or its salt in the culture dry product can be measured using CE-TOF-MS according to the analytical method of Tanaka et al. (Non-Patent Document 31). Then, from the content (concentration) of prumycin or a salt thereof, or kanosamine or a salt thereof in the obtained dried culture product, the content (concentration) of prumycin or a salt thereof, or kanosamine or a salt thereof in the liquid to be applied to plants can be calculated, taking into account the dilution rate, etc.

[0047] The weight ratio of the insecticidal protein to prumycin or a salt thereof, or kanosamine or a salt thereof is not particularly limited, but is preferably 1000:1 to 1:50, more preferably 50:1 to 1:5. When prumycin hydrochloride or kanosamine hydrochloride is used, the above-mentioned weight ratio range can also be used.

[0048] In one embodiment of the present invention, calcium chloride can be used as a component of the composition to improve storage stability. The content of calcium chloride in the composition varies depending on the form of use and the dilution ratio, and is not particularly limited, but in one embodiment of the present invention, the content of calcium chloride is preferably 0.1 to 10% by mass, more preferably 1 to 5% by mass, based on the composition.

[0049] In one embodiment of the present invention, the composition may not contain surfactins. Surfactins refer to cyclic lipodepsipeptide-type substances having surface activity produced by Bacillus bacteria. Examples of surfactins include surfactin, [Ala 4 ]Surfactin, [Leu 4 ]Surfactin, [Val 7 ]Surfactin, [Ile 7 ] Surfactin, etc. In this application, the term "free" means that a specific component is not intentionally added, and the component is permitted to be contained as an impurity.

[0050] When no surfactins are contained, the content of surfactins in the liquid when applied to plants can be less than 1.0 ppm, less than 0.5 ppm, or less than 0.1 ppm.

[0051] In one aspect of the present invention, a composition is provided that has high insecticidal activity compared to the case where only insecticidal protein is applied.Therefore, the composition can be used as an agricultural and horticultural insecticidal composition that is useful for agricultural and horticultural pest control.By making the composition itself or its dilution exist on plant bodies such as roots, stems, leaves, seeds, or in the cultivation soil, it can suppress crop damage caused by various agricultural pests.

[0052] In one aspect of the present invention, the composition can be used to control not only Lepidoptera pests but also a wide range of agricultural and horticultural pests (e.g., insects such as Coleoptera, Hemiptera, Thripida, and Diptera, nematodes, and harmful mites). It can be particularly preferably used against Lepidoptera pests. Examples of agricultural and horticultural pests include the following.

[0053] Lepidoptera pests: Hemophilus gracilis ( Strong islander ), Smaller Tea Tortrix ( Adoxophyes honorable ), the smaller apple tortrix ( Adoxophyes pray do it), Oriental fruit moth ( Grapholite troublesome ), Chahamaki ( Humans greatness ), Bean pulp moth ( Leguminous glycine ), Silver leafminer moth ( Lyons small plum tree little apple ), Tea leaf moth ( Caloptilia food-eater ), Golden Grass Moth ( Phyllonorycter small ring ), Citrus leafminer moth ( Phyllocnistis lemon ), Negi koga ( Acrolepiopsis from Sapporo ), Diamondback moth ( Small xylostella ), Apple fruit moth ( Argyresthia young wife ), Grape moth ( Nokona royal ), Small-headed moth ( Synanthedon Hector ), Persimmon stem moth ( Stathmopoda machine-made ), Peach fruit moth ( Carp Sasaki ), Iraga ( Monument yellowing ), rice stem borer ( Kilo suppressed ), rice leafroller ( Cnaphalocrocus Medicinal ), the gray spotted moth ( Hellila undulating ), European corn borer ( Ostrinia furnace ), Shibatsutomo ( Parapedia little bird ), Striped moth ( Ephesus kuehniella ), wax moth ( You are Gallic. melon ), Swallowtail ( Swallowtail butterfly Hippocrates ), Cabbage White Butterfly ( Piers turnips cruciferous ), Common Skipper ( Parnara drops ), Artemisia moth ( Ascot moon ), Shrimp Sparrow ( Agriculturist rolled up ), Lesser White-streaked Tussock Moth ( Orgy Thyella ), Fall Webworm ( Hyphantria wedge ), Tamanayaga ( Agrotis Y ), Cabbage moth ( Agrotis crop ), Tamanaginwaba ( Autograph black mark ), nettle silver moth ( Trichoplusia if ), Helicoverpa armigera ( Helicoverpa armor-bearer ), tobacco moth ( Helicoverpa attack ), Armyworm moth ( Mother cabbages ), armyworm ( Mythimna separated ), Two-banded moth ( Orange aging ), Spodoptera exigua ( Spodoptera small ), Spodoptera litura ( Spodoptera cleaning ), cotton ball worm ( Helicoverpa zea ), tobacco budworm ( Heliothis greening ), Cotton Leafworm ( Spodoptera coastal ), Fall Army Worm ( Spodoptera fruitless ), Southern Army Worm ( Spodoptera laughter ), tobacco hornworm ( Eat sixth ), Grapeberry Moss ( Endopiza grapevine ), pink bollworm ( Pectinophora gossypiella ), Codling moth ( Cydla apple tree ), European Corn Borer ( Ostrinia cloudy ), Soybean Looper ( Pseudoplusia including ) etc.

[0054] Coleoptera: Cuprea vitellus ( Abnormal copper ), Japanese beetle ( Abnormal red-copper ), Japanese beetle ( Popillia Japanese ), wireworm beetle ( Melanotan from Tamsui ), Himemarukatsuobushimushi ( Anthrene to be a fool ), 204-spotted ladybird ( Henosepilachna twenty-eight-pointed ), Tenebrio molitor ( Darkness inventor ), red flour beetle ( Tribolium chestnut ), Spotted longhorn beetle ( Anoplophora Malaysian ), Japanese pine sawyer ( Monochamus alternate I will ), Azuki bean weevil ( Callosobruchus Chinese ), cucumber beetle ( Aulacophora femoral ), Striped flea beetle ( Phyllotreta striped ), Sweet potato weevil ( Cylas anteater ), chestnut weevil ( Weevil Sikkimese ), rice water weevil ( Lissorhoptrus rice-loving ), rice weevils ( Sitophilus more ), Japanese sedge weevil ( Sphenophrus game more clothes ), Boll weevil ( Anthonom big ), Momochokkiri ( Rhynchites hero ), cigarette beetles ( Lasioderma serricone ), Siberian bush warbler ( Purification lady ), rice leaf beetle ( Oulema rice ), Alfalfa weevil ( Hyper back door ), Colorado potato beetle ( Lepinotarsa ten-line ), Granary Weavile ( Sitophilus granary ), Southern corn rootworm ( Diabrotica eleven-spotted ), Western corn rootworm ( Diabrotica virgin-bearing ), Northern corn rootworm ( Diabrotica barbers ), Blue-leafed rove beetle ( Paederus dark-skinned ) etc.

[0055] Hemiptera: Leafhopper ( Arboretum apical ), Japanese giant leafhopper ( Epiacanthus straw ), Tea green leafhopper ( Empoasca grandchildren ), green rice leafhopper ( Nephotettix girth ), Small brown planthopper ( Laodelphax striped ), brown planthopper ( Nilaparvata mourning ), White-backed planthopper ( Sogatella fork-bearer ), Asian citrus psyllid ( Diaphorine lemons ), cotton aphid ( Aphid gossip ), Spiraea aphid ( Aphid small hole ), potato aphid ( Aulacorthum nightshade ), radish aphids ( Brevicoryne cabbages ), apple aphid ( Eriosoma woolen ), green peach aphid ( Myzus peaches ), wheat curl aphid ( Rhopalosiphum water lilies ), Bemisia tabaci ( Bemisia tobacco ), Greenhouse whitefly ( Trialeurodes vaporizers ), Mulberry mealybug ( Pseudococci comstock ), Ceroplastes ( Wax plastics cherry-bearing ), Ruby Cerambycidae ( Wax plastics red ), Red-bearded grasshopper ( Trigonotylus celestial ), Nasi Gunbai ( Stephanitis we are hungry ), Large-spined white-spotted stink bug ( Eysarcorus Lewis ), White-spotted stink bug ( Eysarcorus ventral ), German-winged green stink bug ( Plautia cross-legged steel ), spider bug ( Leptocorisa Chinese ), narrow-striped stink bug ( Torn nailed ), Nagame ( Eurydema wrinkled ), Brown marmorated stink bug ( Haliomorpha mixed ), Southern green stink bug ( Nezara greenish ), Japanese long-horned stink bug ( Togo half-winged ), Potato Leaf Hopper ( Empoasca beans ), citrus whitefly ( Dialeurodes lemons ), Iseria scale insect ( Iceria purchased ), Yanon scale ( One-sided from Genoa ), Asian citrus spiny whitefly ( Aleurocanthus thorny ),bed bugs( Bug bedridden ) etc.

[0056] Thripidae: Thrips palmi ( Frankliniella uncut ), Western flower thrips ( Frankliniella western ), Tea tree thrips ( Scirtothrips dorsal ), Thrips palmi ( Thrips palms ), Onion thrips ( Thrips tobacco ), etc.

[0057] Diptera: Oriental fruit fly ( Dacian dorsal ), Melon fly ( Dacian squash ), Mediterranean fruit fly ( Ceratitis capitals ), pea leafminer ( Liriomyza clover ), Phytophthora gracilis ( Chromatomyia vegetable garden ), tomato leafminer ( Liriomyza sativa ), seed flies ( Hylemya flatness ), onion flies ( Delia ancient ), Apple Maggot ( Rhagoletis apple tree ), Hessian Fry ( Mayetiola destroyer ), house fly ( Fly domestic ), stable fly ( Stmoxys kicking ), Sheep ked fly ( Melophagus sheep ), Heteroptera ( Hypodermis lined ), cowflies ( Hypodermis ox ), sheep fly ( Estrus sheep ), tsetse fly ( Glossine palpable , Glossine biting ), Yellow-legged flycatcher ( Proximum from Yezo ), Horsefly ( Horsefly triangle ), Asian butterflies ( Telematoscope white-spotted ), Tokunaga nukaka ( Leptoconops Japanese ), Culex pipiens ( Mosquito pipit pale ), Asian tiger mosquito ( House white-spotted ), Aedes aegypti ( House Egyptian ), Anopheles sinensis ( Anopheles Hyracanus Chinese ) etc.

[0058] Hymenoptera: Chestnut wasp ( I wish go ), Turnip sawfly ( Athaliah roses Japanese ), Pine sawfly ( Neodiprion necklace-bearer ), chestnut gall wasp ( Dryocosm philistine ), Army ants ( I am moving. burchelli, I am moving. to schmitt ), Camponotus nigricans ( Field-known Japanese ), Asian giant hornet ( Wasp tangerine ), Bulldog Ant ( Myrmecia spp.), fire ants ( Nightshade spp.), Pharaoh Ant ( Monomorium Pharaoh's ) etc.

[0059] Cockroach: Smoky Brown Cockroach ( Periplanet sooty ), Japanese cockroach ( Periplanet Japanese ), German cockroach ( Cockroach German ) etc.

[0060] Orthoptera: Field cricket ( Teleogryllus Emma ), Kera ( Gryllotalpa African ), Migratory locust ( Locust migratory ), Oriental grasshopper ( Oxy from Yezo ), Desert grasshopper ( Schistocerca gregarious ) etc.

[0061] Termitidae: Formosan subterranean termites ( Coptotermes handsome ), Yamato Termite ( Reticulitermes hoped for ), Formosan termite ( Toothed termites handsome ) etc.

[0062] Siphonaptera: Cat flea ( Ctenocephalidae cat ), Human flea ( Flea irritating ), Cheops rat flea ( Xenopsylla Cheops ) etc.

[0063] Order: Chicken Body Louse ( Menacanthus straw ), cattle louse ( Cowgirl ox ) etc.

[0064] Phthiraptera: Cattle lice ( Blood-stained Eurysternal ), Cattle louse ( Linognathus calves ), Hairy lice ( Solenopotes hairy ) etc.

[0065] Acari: Citrus red mite ( Panonychus lemons ), European red mite ( Panonychus elm ), Kanzawa spider mite ( Spider mite kanzawai ), Two-spotted Spider Mites ( Spider mite nettles ) etc.

[0066] Tulip Rust Mites ( Acer tulips ), tomato rust mite ( Aculops tomatoes ), Citrus rust mite ( Aculops pelekassi ) and other eriophyid mites, Rhizoglyphus robins ), spinach mite ( Tyrannosaurus similar ) and other acarid mites, Polyphaotarsonemus side ) and other dust mites, Varroa destructor mites ( Varroa Jacobson ) and other mites, Boophilus microplus ), Haemaphysalis longicornis ( Haemaphysalis long-horned ) and other ticks, Psoroptes sheep ) and other mites, Sarcoptes scabies ) and other mites. Armadillo vulgar ) and other crustaceans.

[0067] Sweet potato root-knot nematode ( Meloidogyne unknown ), Northern root-knot nematode ( Meloidogyne words), potato cyst nematode ( Globodera from Rostochi ), Soybean cyst nematode ( Heterodera wisteria ), Northern root-lesion nematode ( Prathylenchus penetrating ), Walnut Lesion Nematode ( Prathylenchus wound ), Rice root nematode ( Aphelenchoides bessey ), pinewood nematode ( Bursaphelenchus xylophilus ) and other nematodes.

[0068] Others include apple snails ( Ponacea canal-shaped ), slugs ( Insular double-lined ), Usukawamai ( Acustic despised Sieboldian ), Brown slug ( Lehmannina Valentine's Day ) and other mollusks.

[0069] In one embodiment of the present invention, the plant (crop) to which the composition is applied includes, but is not limited to, cereals (e.g., rice, wheat, barley, corn, buckwheat), tubers (e.g., potato, sweet potato, taro, yam), legumes (e.g., soybean, kidney bean, adzuki bean, pea), vegetables (e.g., melons, tomatoes, eggplants, peppers, cabbage, Chinese cabbage, radish, lettuce, carrots, leeks, onions, strawberries, spinach, celery), fruit trees (e.g., apples, pears, cherries, peaches, grapes, persimmons, citrus fruits, kiwi), specialty crops (e.g., cotton, rapeseed, sunflowers, beets, sugarcane, tobacco), turf, trees, ornamental plants (e.g., roses, chrysanthemums, tulips, baby's breath, lisianthus, etc.), and the like. In one embodiment of the present invention, the composition can also be used in crops that have been transformed by techniques such as genetic recombination.

[0070] In one embodiment of the present invention, the application amount of the composition can be appropriately determined depending on the above-mentioned pests and crops.

[0071] In one embodiment of the present invention, when the composition is used, the composition can be directly applied to plants. In addition, in one embodiment of the present invention, the composition can be used in the form of a drug suitable for agricultural and horticultural pesticides, such as granules, powders, microgranules, liquids, water-soluble agents, oils, emulsions, surf agents, emulsions, microemulsions, suspoemulsion formulations, EW (emulsion oil in water) agents, microcapsules, wettable powders, suspensions, flowable agents, tablets, granule water-dispersible agents, dry flowable agents, wettable granules, aerosols, pastes, cyclodextrin formulations, jumbo agents, pack agents, water-soluble packaging agents, powders, smoking agents, fumigants, etc. In addition, in one embodiment of the present invention, the composition can be used as a drug diluted with an inert liquid or solid carrier and, if necessary, a surfactant, a dispersant, or other auxiliary agent is added. Specific formulation examples include dosage forms such as granules, powders, wettable powders, granule water-dispersible agents, suspensions, and emulsions.

[0072] In one embodiment of the present invention, the composition may contain additives that are commonly used in agricultural chemical formulations, if necessary. Examples of such additives include carriers such as solid carriers and liquid carriers, surfactants, binders, adhesive additives, thickeners, colorants, spreading agents, antifreeze agents, anticaking agents, disintegrants, antidecomposition agents, preservatives, etc., and if necessary, preservatives, plant pieces, etc. may be used as additional components. These additives may be used alone or in combination of two or more.

[0073] Examples of solid supports include: Natural minerals such as quartz, clay, kaolinite, pyrophyllite, sericite, talc, chalk, bentonite, attapulgite, montmorillonite, acid clay, zeolite, natural rock, diatomaceous earth, calcite, marble, pumice, sepiolite, dolomite, etc.; Inorganic salts such as calcium carbonate, ammonium sulfate or other ammonium salts, sodium sulfate, potassium chloride, etc.; Organic solid carriers such as starch, cellulose, vegetable powders, etc.; Plastic carriers such as polyethylene, polypropylene, polyvinylidene chloride, etc.; Other examples include synthetic silicic acid, synthetic silicate, alumina, finely powdered silica, silicate, etc. These may be used alone or in combination of two or more.

[0074] Examples of liquid carriers include: Alcohols broadly classified into monohydric alcohols such as methanol, ethanol, propanol, isopropanol, butanol, etc., and polyhydric alcohols such as ethylene glycol, diethylene glycol, propylene glycol, hexylene glycol, polyethylene glycol, polypropylene glycol, glycerin, etc.; Polyhydric alcohol derivatives such as propylene glycol ethers; Ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, diisobutyl ketone, cyclohexanone, and isophorone; Ethers such as ethyl ether, dioxane, cellosolve, dipropyl ether, and tetrahydrofuran; Aliphatic hydrocarbons such as normal paraffin, naphthene, isoparaffin, kerosene, mineral oil, etc.; Aromatic hydrocarbons such as benzene, toluene, xylene, solvent naphtha, and alkylnaphthalenes; Halogenated hydrocarbons such as dichloroethane, chloroform, and carbon tetrachloride; Esters such as ethyl acetate, diisopropyl phthalate, dibutyl phthalate, dioctyl phthalate, and dimethyl adipate; Lactones such as γ-butyrolactone; Amides such as dimethylformamide, diethylformamide, dimethylacetamide, and N-alkylpyrrolidine; Nitriles such as acetonitrile; Sulfur compounds such as dimethyl sulfoxide; vegetable oils such as soybean oil, rapeseed oil, cottonseed oil, castor oil, etc.; These may be used alone or in combination of two or more.

[0075] The surfactant is not particularly limited, but is preferably one that gels or swells in water. Examples of the surfactant include: nonionic surfactants such as sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, sucrose fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene resin acid esters, polyoxyethylene fatty acid diesters, polyoxyethylene alkyl ethers, polyoxyethylene alkylphenol ethers, polyoxyethylene dialkyl phenyl ethers, polyoxyethylene alkylphenol formalin condensates, polyoxyethylene polyoxypropylene block polymers, alkyl polyoxyethylene polypropylene block polymer ethers, polyoxyethylene alkylamines, polyoxyethylene fatty acid amides, polyoxyethylene fatty acid bisphenyl ethers, polyalkylene benzyl phenyl ethers, polyoxyalkylene styrene phenyl ethers, acetylene diols, polyoxyalkylene-added acetylene diols, polyoxyethylene ether-type silicones, ester-type silicones, fluorine-based surfactants, polyoxyethylene castor oil, and polyoxyethylene hydrogenated castor oil; anionic surfactants such as alkyl sulfates, polyoxyethylene alkyl ether sulfates, polyoxyethylene alkyl phenyl ether sulfates, alkylbenzene sulfonates, lignin sulfonates, alkyl sulfosuccinates, naphthalene sulfonates, alkyl naphthalene sulfonates, salts of formalin condensates of naphthalene sulfonic acid, salts of formalin condensates of alkyl naphthalene sulfonic acid, fatty acid salts, polycarboxylates, N-methyl-fatty acid sarcosinates, resin salts, polyoxyethylene alkyl ether phosphates, and polyoxyethylene alkyl phenyl ether phosphates; cationic surfactants, such as alkylamine salts, such as laurylamine hydrochloride, stearylamine hydrochloride, oleylamine hydrochloride, stearylamine acetate, stearylaminopropylamine hydrochloride, alkyltrimethylammonium chloride, and alkyldimethylbenzalkonium chloride; Examples of the amphoteric surfactant include amino acid type and betaine type surfactants. These surfactants may be used alone or in combination of two or more.

[0076] Examples of binders and tackifiers include carboxymethylcellulose and its salts, dextrin, water-soluble starch, xanthan gum, guar gum, sucrose, polyvinylpyrrolidone, gum arabic, polyvinyl alcohol, polyvinyl acetate, sodium polyacrylate, polyethylene glycol having an average molecular weight of 6,000 to 20,000, polyethylene oxide having an average molecular weight of 100,000 to 5,000,000, natural phospholipids (e.g., cephalic acid, lecithinic acid, etc.), etc. These binders and tackifiers may be used alone or in combination of two or more.

[0077] Examples of thickeners include: Water-soluble polymers such as xanthan gum, guar gum, carboxymethylcellulose, polyvinylpyrrolidone, carboxyvinyl polymers, acrylic polymers, starch derivatives, polysaccharides; Examples of the thickener include inorganic fine powders such as high-purity bentonite and white carbon. These thickeners may be used alone or in combination of two or more.

[0078] Examples of colorants include: Inorganic pigments such as iron oxide, titanium oxide, and Prussian blue; Examples of the coloring agent include organic dyes such as alizarin dyes, azo dyes, and metal phthalocyanine dyes. These coloring agents may be used alone or in combination of two or more.

[0079] Examples of the spreading agent include: Examples of the spreading agent include silicone surfactants, cellulose powder, dextrin, modified starch, polyaminocarboxylic acid chelate compounds, crosslinked polyvinylpyrrolidone, maleic acid and styrene acid, methacrylic acid copolymers, half esters of polyhydric alcohol polymers and dicarboxylic acid anhydrides, water-soluble salts of polystyrene sulfonic acid, etc. These spreading agents may be used alone or in combination of two or more.

[0080] Examples of the spreading agent include: Various surfactants such as sodium dialkyl sulfosuccinate, polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, and polyoxyethylene fatty acid esters; Examples of the spreading agent include paraffin, terpene, polyamide resin, polyacrylate, polyoxyethylene, wax, polyvinyl alkyl ether, alkylphenol-formaldehyde condensate, synthetic resin emulsion, etc. These spreading agents may be used alone or in combination of two or more.

[0081] Examples of the antifreezing agent include polyhydric alcohols such as ethylene glycol, diethylene glycol, propylene glycol, glycerin, etc. These antifreezing agents may be used alone or in combination of two or more.

[0082] Examples of the anti-caking agent include polysaccharides such as starch, alginic acid, mannose, and galactose, polyvinylpyrrolidone, white carbon, ester gum, petroleum resin, etc. These anti-caking agents may be used alone or in combination of two or more.

[0083] Examples of disintegrants include sodium tripolyphosphate, sodium hexane metaphosphate, metal stearates, cellulose powder, dextrin, copolymers of methacrylic acid esters, polyvinylpyrrolidone, polyaminocarboxylic acid chelate compounds, sulfonated styrene-isobutylene-maleic anhydride copolymers, starch-polyacrylonitrile graft copolymers, etc. These disintegrants may be used alone or in combination of two or more.

[0084] Decomposition inhibitors include, for example, desiccants such as zeolite, quicklime, and magnesium oxide; Antioxidants such as phenolic, amine, sulfur, and phosphoric acid antioxidants; Examples of such anti-decomposition agents include ultraviolet absorbers of salicylic acid type, benzophenone type, etc. These anti-decomposition agents may be used alone or in combination of two or more types.

[0085] Examples of the preservative include potassium sorbate, 1,2-benzthiazolin-3-one, etc. These preservatives may be used alone or in combination of two or more.

[0086] In one embodiment of the present invention, the composition can contain, as co-ingredients, ingredients known in the Pesticide Manual (2013) published by the British Crop Protection Council, Kumiai Pesticide Directory (2018) published by the National Federation of Agricultural Cooperative Associations, and SHIBUYA INDEX (17th edition) published by the National Rural Education Association, such as insecticides, acaricides, nematicides, snailicides, and antifeedants, to form a multi-component pest control agent that provides broader agricultural protection. Examples of co-ingredients are shown below, but are not limited to these.

[0087] Examples of insecticides, acaricides, nematicides, snailicides and antifeedants include 1,2-dichloropropane, 1,3-dichloropropene, abamectin, acephate, acequinocyl, acetamiprid, acetion, acetoprole, acrinasrin, aphidopyropen, alanycarb, aldicarb, allethrin, allicin, allosamidin, alixycarb, alphacypermethrin, alphaendosulfan, amidithione, amidoflumet, aminocarb, amitraz, anabasine, alamite, atidathion, azadirachtin, azamethiphos, azinphos-ethyl, azinphos-methyl, azocyclotin, azotoate, valsuline. , benclothiaz, bendiocarb, benfuracarb, benoxafos, bensultap, benzoximate, benzyl benzoate, beta-cyfluthrin, beta-cypermethrin, bifenazate, bifenthrin, bifujunzhi, binapacryl, bioallethrin, bioresmethrin, bistrifluron, borax, boric acid, brofuranilide, bromethrin, bromfenvinphos, bromocyclen, bromophos, bromophosethyl, bromopropylate, bufencarb, buprofezin, butacarb, butathiophos, Butoxycarboxim, butonate, polyoxin complex, cadusafos, calcium polysulfate, carbinphos, campechlor, carbanolate, carbaryl, carbofuran, carbon disulfide, carbon tetrachloride, carbonyl sulfide, carbophenothione, carbosulfan, cartap, carvacrol, chinomethionate, chloramine phosphorus, chlorantraniliprole, chlordane, chlorbencide, chlorbenzuron, chlorbicyclen, chlordecone, chlorethoxyphos, chlorfenapyr, chlorphenetole, chlorphen Son, chlorfensulfide, chlorfenvinphos, chlorfluazuron, chlormephos, chlormebuform, chloromethiuron, chloropicrin, chlorpropylate, chlorphoxim, chlorprazophos, chlorpyrifos, chlorpyrifos-methyl, chlorthiophos, chromafenozide, cinerin I, cinerin II, cinerin complex, cismethrin, clempirin, cloethocarb, clofentezine, closantel, clothianidin, colophonate, copper naphthenate, copper oleate, copper sulfate, coumaphos, chumitoate,CPMC, Crotamiton, Crotoxyphos, Crufomate, Cryolite, Cyanofenphos, Cyanophos, Cyantoate, Cyantraniliprole, Cyclaniliprole, Cycloprothrin, Cyenopyrafen, Cyflumetofen, Cyfluthrin, Cyhalodiamide, Cyhalothrin, Cyhexatin, Cymiazole, Cypermethrin, Cyromazine, Cythioate, Dayoutong, Dazomet, DBCP, Chloropicrin, DCIP, Decarbofuran, Deltamethrin, Demefion, Demefion O, Demefion S, Demeton, Demeton S Methyl, Diafenthiuron, Diariphos, Diamidaphos, Diatomaceous Earth, Diazinon, Dikapton, Diclofenthion, Dichlofluanid, Dichlorvos, Dichloromezothiaz, Dicofol, Dicrotophos, Dicyclanil, Dienochlor, Diflobidazin, Diflubenzuron, Dimefluthrin, Dimefox, Dimethane, Dimethacarb, Dimethoate, Dimethrin, Dimethylvinphos, Dimethilan, Zinex, Dinobuton, Dinocap, Dinokton, Dinopenton, Dinoprop, Dinosam, Dinosulfone, Dinotefuran, Dinoterbone, Diofenolan, Diox Sabenzophos, dioxacarb, dioxathion, diphenyl sulfone, dipimethitron, disulfiram, disulfoton, dicyclophos, d-limonene, DNOC, dofenapine, doramectin, ecdysterone, emamectin, EMPC, empenthrin, endosulfan, endothion, endrin, EPN, eprinomectin, momfluorotrin, fenvalerate, esfenvalerate, etaphos, ethiofencarb, ethion, ethiprole, ethoeate methyl, ethoprophos, ethyl formate, ethylene dibromide, ethylene dichloride, ethofu Enprox, etoxazole, etrimphos, fanfur, fenamiphos, fenazaflor, fenazaquin, fenbutatin oxide, fenchlorphos, fenetacarb, fenfluthrin, fenitrothion, fenobucarb, fenothiocarb, fenoxacrim, fenoxycarb, fenpyritrin, fenpropathrin, fenpyroximate, fenthone, fensulfothion, fenthion, fentrifanil, fenvalerate, ferric phosphate, fipronil, flometoquin, flonicamid, fluacrypyrim, fluazaindolizine,Fluazuron, flubendiamide, flubenzimin, flucofuron, flucycloxuron, flucythrinate, fluenethyl, fluensulfone, flufenerim, flufenoxuron, flufenprox, flufiprole, fluhexafon, flumethrin, fluorbenside, flupyradifuron, fluralaner, flursuramide, fluvalinate, fluxamethamide, fonofos, formetanate, formetanate hydrochloride, formothion, formaparanate, fosmetiran, fospirate, fosthiazate, fostietan, furametrin, furantebufenozide, furathiocarb, furthrin, furfural, gan Masihalothrin, Halfenprox, Halofenozide, HCH, HEOD, Heptafluthrin, Heptenophos, Hexachlorophene, Hexaflumuron, Hexythiazox, HHDN, Hydramethylnon, Hydroprene, Hikincarb, Imisiaphos, Imidacloprid, Imidaclotiz, Imiprothrin, Indoxacarb, IPSP, Isamidophos, Isazophos, Isobenzan, Isocarbophos, Isodrin, Isophephos, Isophephos-methyl, Isolane, Isoprocarb, Isoprothiolane, Isothioate, Isoxathion, Iprodione, Ivermectin, Jasmolin I, Jasmolin II, Jia Huang Chong zong, iodofenphos, juvenile hormone I, juvenile hormone II, juvenile hormone III, cadethrin, bifenthrin, karanjin, kereban, kinoprene, salicylic acid, methyl salicylate, lambda-cyhalothrin, lepimectin, leptophos, lilimphos, lufenuron, ritidathion, malathion, malonoven, maltodextrin, matrine, magidox, mecarbam, mecarfone, menazone, meperfluthrin, mesulfon, mesulfon, mesulfenphos, Metaflumizone, metaldehyde, metam, methacrifos, methamidophos, methidathion, methiocarb, metoclothos, methomyl, methoprene, methothrin, methoxychlor, methoxyfenozide, methyl iodide, methyl isothiocyanate, metofluthrin, metolcarb, methoxadiazone, mevinphos, mexacarbate, milbemectin, mipafox, mirex, monocrotophos, momfluorotrin, morphothion, moxidectin, naphthalophos, naled,Naphthalene, niclosamide, nicotine, nifluridide, nitenpyram, nithiazine, nitrilacarb, novaluron, noviflumuron, omethoate, oxamyl, oxydemeton-methyl, oxydeprophos, oxydisulfoton, oxymatrine, paichondine, paradichlorobenzene, penfluron, pentachlorophenol, permethrin, fencapton, fenothrin, fenproxide, phenthoate, phorate, phosalone, phospholane, phosmet, phosphamidon, phosnichlor, phosphine, phosphocarb, phoxim, phoxim-methyl , Pyrimetaphos, Pyriminocarb, Pyrimioxyphos, Pirimiphos-ethyl, Pirimiphos-methyl, Potassium thiocyanate, Prallethrin, Precocene I, Precocene II, Precocene III, Primidophos, Proclonol, Profenofos, Profluthrin, Promacyl, Promecarb, Propaphos, Propargite, Propetamphos, Propoxur, Protidathion, Prothiofos, Protoate, Protrifenbut, Piflubumid, Pymetrozine, Pyraclofos, Pyrafluprole, Pyrazophos, Pyrazothion, Pyresmethrin, Pyrethrin, Pyridamyl Ben, pyridalyl, pyridaphenthion, pyrifluquinazon, pyrimidifen, pyrimitate, pyriprole, pyriproxyfen, pyrrolan, quinalphos, quinalphos-methyl, quinothione, quinthiophos, lafoxanide, resmethrin, rhodojaponin III, rotenone, riania, sabadilla, sanguinarine, shradan, selamectin, silafluofen, silica gel, sodium fluoride, sodium hexafluorosilicate, sodium thiocyanate, sofamid, spinetoram, spinosad, spirodiclofen, spiromethine Fen, spirotetramat, sulcofuron, sulphiram, sulfluramide, sulphotep, sulphoxaflor, sulphoxime, sulphur, sulphuryl fluoride, sulprofos, taufluvalinate, thazimcarb, TDE, tebufenozide, tebufenpyrad, tebupirimfos, teflubenzuron, tefluthrin, temephos, TEPP, teralethrin, terbufos, tetrachloroethane, tetrachlorvinphos, tetradifon, tetramethrin, tetramethylfluthrin, tetranactin, tetraniliprole, tetrasul, thiacloprid, thiamethoxam,Examples of such compounds include tiapronil, thiocarboxim, thiocyclam, thiodicarb, thiofanox, thiometon, thionazine, thioquinox, thiosultap, thioxazafen, chirpate, tolfenpyrad, tralocitrin, tralomethrin, tralopyril, transfluthrin, transpermethrin, triaratene, triazamate, triazophos, trichlorfon, trichloronate, triphenmorph, tripenophos, triflumezopyrim, triflumuron, trimethacarb, triplen, triptolide, vamidothion, vaniliprole, xiao chong liu lin, XMC, xylenols, xylylcarb, yishijing, zolaprophos, alphaecdysone, acinonapyr, flupirimine, spiropydione, benzpyrimoxane, cyclopyrazoflor, oxazosulfil, etc. These compounds may be used alone or in combination of two or more. ,

[0088] In one embodiment of the present invention, the composition can also optionally contain at least one biologically active ingredient, such as a plant growth regulator, e.g., a root promoter, or a fertilizer, e.g., a plant nutrient, as a co-ingredient to form a multi-component pest control agent that provides even broader agricultural protection.

[0089] In one aspect of the present invention, Bacillus thuringiensis ( Bacillus ThuringianThe present invention provides a composition containing prumycin or a salt thereof, or kanosamine or a salt thereof, for application to a plant into which a gene for expressing an insecticidal protein derived from B. cerevisiae has been introduced (hereinafter referred to as "BT plant"). Examples of BT plants include KnockOut (registered trademark), NatureGard (registered trademark), Bollgard (registered trademark), Agrisure (registered trademark) CB, YieldGard Corn Rootworm (registered trademark), Herculex I (registered trademark), Bollgard II (registered trademark), YieldGard Plus (registered trademark), VipCot (registered trademark), WideStrike (registered trademark), Herculex RW (registered trademark), Herculex Xtra (registered trademark), and the like, which control pests by expressing a single or multiple insecticidal proteins in soybean, corn, cotton, and canola.

[0090] (Methods for controlling agricultural and horticultural pests) In one aspect of the present invention, a method for controlling agricultural and horticultural pests includes: Bacillus thuringiensis ( Bacillus Thuringian (a) applying to a plant an insecticidal protein derived from Puromycin or a salt thereof, or kanosamine or a salt thereof is added to the Bacillus thuringiensis ( Bacillus Thuringian (b) applying to the plant simultaneously, sequentially or separately an insecticidal protein derived from Includes.

[0091] In one embodiment of the present invention, the control method may involve applying an insecticidal protein derived from Bacillus thuringiensis and prumycin or a salt thereof, or kanosamine or a salt thereof, separately to a plant, or may involve applying an agricultural and horticultural insecticidal composition containing an insecticidal protein derived from Bacillus thuringiensis and prumycin or a salt thereof, or kanosamine or a salt thereof, to a plant. From the viewpoints of the stability of the insecticidal effect, rapidity of action, and reduced risk of resistance development, it is preferable to use an agricultural and horticultural insecticidal composition containing an insecticidal protein derived from Bacillus thuringiensis and prumycin or a salt thereof, or kanosamine or a salt thereof.

[0092] In one embodiment of the present invention, the plant to which the control method is applied refers to a plant or a group of plants such as a wild plant, a cultivated plant, a naturally occurring plant, or a cultivated plant, and also includes plants produced by breeding methods such as introduction breeding, isolation breeding, cross breeding, hybrid vigor breeding, mutation breeding, polyploidy breeding, gene recombination (gene introduction), and marker-assisted selection. The plant to be treated may be either the whole plant or a part of the plant.

[0093] In one embodiment of the present invention, when the composition is applied to a plant in the control method, it can be applied to the plant or the area around the plant. The area around the plant refers to soil (soil in which seeds are sown), paddy fields, water for hydroponic cultivation, cultivation materials, etc.

[0094] In addition, the method of application to plants may include spraying, dispersing, dusting, spraying, diffusing, soaking, irrigating, injecting, watering (submerging), foaming, spreading, coating, dusting, coating, spraying, fumigating, smoking, misting, painting, etc., and preferably includes soil mixing, irrigation, seed smearing, seed soaking, or foliar spraying. These methods of application to plants may be used alone or in combination of two or more.

[0095] In one embodiment of the present invention, when the composition is applied to control agricultural and horticultural pests, the application can be performed throughout the growth and storage period of the plant. Here, in one embodiment of the present invention, the control method can be applied to a part or all of the plant. The part of the plant means the leaves, stems, trunks, branches, flowers, fruit bodies, fruits, seeds, roots, tubers, rhizomes, or combinations thereof.

[0096] In one embodiment of the present invention, the composition can be used in an amount adjusted to be effective but not phytotoxic, in order to control agricultural and horticultural pests. Here, the effective but not phytotoxic amount means an amount that can sufficiently control agricultural and horticultural pests and does not harm plants, and this amount can vary within a relatively wide range depending on the agricultural and horticultural pests to be controlled, the plants to which it is applied, the natural environment in which it is used, and the components of the composition.

[0097] In one embodiment of the present invention, in the control method, all of the components described in the above section (Agricultural and horticultural insecticidal composition), such as the type and content of the insecticidal protein, the type and preparation method of Bacillus thuringiensis, the type and content of prumycin or a salt thereof, and the type and content of kanosamine or a salt thereof, can be similarly applied, either alone or in combination of two or more. EXAMPLES

[0098] The present invention will be specifically described below with reference to Production Examples, Formulation Examples (Formulation Examples), and Test Examples, but the present invention is not limited to these examples.

[0099] (Production Example 1: Synthesis of prumycin hydrochloride) Puromycin hydrochloride was synthesized according to the method of Iwabuchi et al. (J. Carbohydr. Chem., 7, 605-616 (1988)). That is, D-arabinose (25 g) was treated in the following order: 1-O-benzylation, 3,4-O-isopropylidene acetal protection, 2-benzoylation, isopropylidene acetal deprotection, 4-selective azide substitution, benzoyl deprotection, epoxidation, ring-opening amination, and N-Cbz protection to give 4-azido-2-(benzyloxycarbonyl)amino-2,4-dideoxy-α-L-arabinopyranoside (9.6 g, yield after the above 9 steps: 14%). The resulting compound (984 mg) was subjected to the steps of azide selective reduction, condensation with N-carbobenzoxy-D-alanine (N-Cbz-D-alanine), and reductive deprotection in order to obtain prumycin hydrochloride (424 mg, yield after the above four steps: 59%).

[0100] (Formulation Example 1: Preparation of Btk culture dried product or Bta culture dried product) Bacillus thuringiensis subspecies kurstakii ( Bacillus Thuringian subsp. kurstaki ) culture dried product (hereinafter referred to as "Btk culture dried product") and Bacillus thuringiensis subsp. aizawai ( Bacillus Thuringian subsp. aizawai A dried Btk culture was prepared by the following procedure. The dried Btk culture and the dried Bta culture are collectively referred to as the dried Bt culture.

[0101] The strains for preparing the Btk culture dry product and the Bta culture dry product were obtained by isolating the strains from Tune Up (registered trademark) granule water dispersible agent (Bacillus thuringiensis subsp. kurstachii (10% viable spores and insecticidal protein of BT bacteria) (Agro Kanesho Co., Ltd.) and Zentari (registered trademark) granule water dispersible agent (Sumitomo Chemical Co., Ltd.). Each of the obtained strains was cultured on a slant medium and stored at 4°C. After storage, a platinum loopful of each strain was taken and inoculated into 60 mL of normal bouillon medium (Eiken Chemical Co., Ltd.), and each strain was cultured for 1 day using a rotary shaker at 180 rpm and 28°C (preculture). 60 mL of the culture solution obtained by pre-culture was inoculated into 2 L of culture solution (soybean-derived protein powder 4%, potato starch 2%, corn steep liquor 1.5%, sodium carbonate 0.0001%, pH 7.0) and cultured with shaking at 30°C for 2 days (main culture). The culture solution obtained by main culture was freeze-dried and then pulverized, and the resulting fine powders were used as the Btk and Bta culture dried products, respectively.

[0102] The total proportion of insecticidal proteins contained in the obtained Btk culture dried material or Bta culture dried material was 4% by mass when the insecticidal protein group was purified by the precipitation separation method of Goodman et al. using dextran sulfate and polyethylene glycol (Non-Patent Document 29), and then measured by HPLC according to the analytical method of Masson et al. (Non-Patent Document 30).

[0103] (Formulation Example 2: Preparation of a dried culture of a fungus producing prumycin or a salt thereof, or a dried culture of a fungus producing kanosamine or a salt thereof) With reference to the method of Stabb et al. (Non-Patent Document 32), a dried culture of a bacteria producing prumycin or a salt thereof, or a dried culture of a bacteria producing kanosamine or a salt thereof was prepared by the following procedure.

[0104] A loopful of Bacillus subtilis or Bacillus cereus strain isolated from soil was inoculated into 60 mL of normal bouillon medium (Eiken Chemical Co., Ltd.), and each strain was cultured for one day using a rotary shaker at 180 rpm and 28°C (preculture). 60 mL of the culture obtained by the preculture was inoculated into 2 L of L-Broth medium and cultured with shaking at 180 rpm and 30°C for three days (main culture). The culture obtained by the main culture was freeze-dried and then pulverized, and the resulting fine powders were used as the dried culture product of the bacteria producing prumycin or its salt and the dried culture product of the bacteria producing kanosamine or its salt, respectively.

[0105] (Formulation Example 3: Preparation of a wettable powder of Btk culture dried material and prumycin hydrochloride) In the examples, "parts" indicating the proportions of the formulations used are by weight unless otherwise specified.

[0106] A wettable powder of the dried Btk culture and prumycin hydrochloride was obtained by mixing and grinding 45 parts of the dried Btk culture prepared in Formulation Example 1, 5 parts of prumycin hydrochloride prepared in Manufacturing Example 1, 20 parts of diatomaceous earth, 20 parts of white carbon, 8 parts of sodium ligninsulfonate, and 2 parts of sodium alkylnaphthalenesulfonate.

[0107] (Formulation Example 4: Preparation of a wettable powder of Btk culture dried material and kanosamine hydrochloride) A hydrate of the dried Btk culture and kanosamine hydrochloride was obtained by mixing and grinding 45 parts of the dried Btk culture prepared in Formulation Example 1, 5 parts of kanosamine hydrochloride (Sigma-Aldrich Japan LLC), 20 parts of diatomaceous earth, 20 parts of white carbon, 8 parts of sodium ligninsulfonate, and 2 parts of sodium alkylnaphthalenesulfonate.

[0108] [Test Example 1: Efficacy test against 3rd instar larvae of Spodoptera litura] A suspension of the dried Btk culture or a suspension of the dried Bta culture was prepared by adding distilled water to the dried Btk culture or the dried Bta culture prepared in Formulation Example 1, and prumycin hydrochloride or kanosamine hydrochloride (Sigma-Aldrich Japan LLC) prepared in Production Example 1 was added to the suspension to a predetermined concentration to prepare a solution. The obtained solution was added to an artificial feed (Insector LFS (Nihon Nosan Kogyo Co., Ltd.)) so that the content of the dried Btk culture or the dried Bta culture was 0.2 to 2 mg / g per 1 g of the artificial feed and the content of prumycin hydrochloride or kanosamine hydrochloride was 2 μg / g per 1 g of the artificial feed, as shown in Tables 1 to 6, and mixed. The artificial feed in which the dried Btk culture or the dried Bta culture was mixed with prumycin hydrochloride or kanosamine hydrochloride was placed in a petri dish (diameter 90 mm, height 15 mm), and then the mixture was used to feed Spodoptera litura ( Spodoptera cleaning Ten third-instar larvae of the species were released into a petri dish (three replicates), the dish was then covered, and the dish was kept at 25°C for five days.

[0109] Thereafter, the petri dish was observed to visually check the number of dead insects, and the number of dead insects relative to the total number of reared insects (insect death rate (%)) (actual value) was calculated.

[0110] In addition, only the suspension of the dried Btk culture product, only the suspension of the dried Bta product, only prumycin hydrochloride, only kanosamine hydrochloride, or a combination of prumycin hydrochloride and kanosamine hydrochloride was added to the artificial feed at the application concentration per 1 g of the artificial feed shown in Tables 1 to 6, and the rest was the same as in the above examples. Spodoptera cleaning Ten third-instar larvae of the species were released into petri dishes (three replicates) and incubated at 25°C for five days.

[0111] Thereafter, the inside of the petri dish was observed, and the insecticidal rate (%) (actual value) was calculated in the same manner as in the above example.

[0112] The theoretical value of the expected insecticidal rate (%) when the dried Btk culture or the dried Bta culture was mixed with prumycin hydrochloride or kanosamine hydrochloride was calculated using Colby's formula (E=X+Y-(X+Y / 100)).

[0113] In this formula, X, Y, and E have the following meanings: X: Insecticidal rate (%) when active ingredient A was applied at M ppm Y: Insecticidal rate (%) when active ingredient B was applied at N ppm E: Theoretical value (%) of the expected insecticidal rate when active ingredient A is treated at M ppm and active ingredient B is treated at N ppm.

[0114] The insecticidal rate (%) when Btk culture dried product or Bta culture dried product was used in combination with prumycin hydrochloride or kanosamine hydrochloride was compared with the theoretical value and the actual measured value to determine the presence or absence of a synergistic effect. Specifically, when the actual measured value of the insecticidal rate (%) was greater than the theoretical value, it was determined that there was a "synergistic effect", and when the actual measured value was equal to or less than the theoretical value, it was determined that there was no synergistic effect.

[0115] The test results obtained are shown in Tables 1 to 6 below.

[0116] [Table 1]

[0117] [Table 2]

[0118] [Table 3]

[0119] [Table 4]

[0120] [Table 5]

[0121] [Table 6]

[0122] As shown in the results of Tables 1 to 6, when prumycin hydrochloride or kanosamine hydrochloride was applied alone at a concentration of 2 μg / g feed, no insecticidal effect was observed against the third instar larvae of Spodoptera litura. On the other hand, by applying the combination of Btk culture dried product or Bta culture dried product with prumycin hydrochloride or kanosamine hydrochloride, the insecticidal rate of the third instar larvae of Spodoptera litura was significantly increased compared to the case of applying the Btk culture dried product alone or the Bta culture dried product alone, and an extremely high insecticidal effect was obtained. From the above results, it was found that a synergistic effect on the insecticidal effect can be obtained by applying the combination of Btk culture dried product or Bta culture dried product with prumycin hydrochloride or kanosamine hydrochloride.

[0123] [Test Example 2: Field test against Spodoptera litura] In this test example, the following test agents were used. - Bacillus thuringiensis preparation: Tune Up (registered trademark) granular water dispersible agent (Bacillus thuringiensis subspecies kurstakii (10% viable spores and insecticidal protein of Bacillus thuringiensis)) (Agro Kanesho Co., Ltd.) - Dried culture of the bacteria producing prumycin or its salt prepared in the above Preparation Example 2 Dried culture of the bacteria producing kanosamine or its salt prepared in the above Preparation Example 2

[0124] The above BT bacteria preparation and the dried culture of the bacteria producing prumycin or its salt or the dried culture of the bacteria producing kanosamine or its salt were mixed to a predetermined concentration, and then diluted with water to obtain a dilution. A spreading agent (Breakthrough (registered trademark), Sankei Chemical Co., Ltd.) was added to the obtained dilution in an amount of 1 / 5000 of the weight of the dilution to prepare a drug solution. The dilution ratio of each test agent in each obtained drug solution was 2000 times or 20000 times, as shown in Table 7.

[0125] The insecticidal proteins contained in the above-mentioned BT bacteria formulation are Cry1Aa, Cry1Ab, Cry1Ac, and Cry2A. When the concentration of the insecticidal proteins in the drug solution in which the BT bacteria formulation was diluted 2000 times was measured by HPLC according to the analytical method of Masson et al. (Non-Patent Document 30), it was 24 ppm.

[0126] The concentration of prumycin or its salt, or kanosamine or its salt in the obtained liquid, which is diluted 2000 times with the culture dried product of the bacteria producing prumycin or its salt, or the culture dried product of the bacteria producing kanosamine or its salt, was measured using CE-TOF-MS according to the analytical method of Tanaka et al. (Non-Patent Document 31), and the concentration was 5 ppm (as prumycin) and 5 ppm (as kanosamine). As a result, the concentration of prumycin in the obtained liquid, which is diluted 20000 times with the culture dried product of the bacteria producing prumycin or its salt, or the culture dried product of the bacteria producing kanosamine or its salt, is considered to be 0.5 ppm (as prumycin) and 0.5 ppm (as kanosamine).

[0127] From the above, for a liquid medicine in which the dilution ratio of the BT bacteria preparation is 2000 times, and the dilution ratio of the culture dried product of the bacteria producing prumycin or its salt, or the culture dried product of the bacteria producing kanosamine or its salt is 2000 times, the concentration ratio (mass ratio) of the insecticidal protein in the liquid medicine to prumycin or kanosamine is 4.8: 1. For a liquid medicine in which the dilution ratio of the BT bacteria preparation is 2000 times, and the dilution ratio of the culture dried product of the bacteria producing prumycin or its salt, or the culture dried product of the bacteria producing kanosamine or its salt is 20000 times, the concentration ratio (mass ratio) of the insecticidal protein in the liquid medicine to prumycin or kanosamine is 48: 1.

[0128] In addition, each of the above test agents (BT bacteria preparations, dried culture of bacteria producing prumycin or its salt, and dried culture of bacteria producing kanosamine or its salt) was used alone in the same manner as in the above examples, diluted with water, and a spreading agent was added to prepare a medicinal solution containing each test agent alone.

[0129] District 1 0.72 m 2 Four cabbage plants (variety: Saiho) with 6-7 true leaves were planted in two rows with 60 cm spacing per area (1.2 m x 0.6 m). One month after planting, each of the drug solutions prepared as described above was sprayed using a filling sprayer at an amount equivalent to 300 L / 10a. Five days after spraying, the number of parasites naturally occurring on all leaves was investigated and the corrected density index was calculated. The corrected density index is an index that represents the control effect based on the pest density in the treated area relative to the pest density in the untreated area, taking into account the density of the pests before spraying, and is calculated based on the following formula. The lower the corrected density index, the higher the control effect.

[0130]

number

[0131] [Table 7]

[0132] As shown in the results in Table 7, the corrected density index of each test agent against Spodoptera litura larvae 5 days after spraying was 19.7 in the mixed area of ​​the BT bacterial preparation (dilution ratio 2000 times) and the culture dry product of prumycin or its salt (dilution ratio 2000 times), and 29.1 in the mixed area of ​​the BT bacterial preparation (dilution ratio 2000 times) and the culture dry product of kanosamine or its salt (dilution ratio 2000 times). The corrected density index in these mixed areas was extremely low compared to the corrected density index: 67.1 in the area where only the BT bacterial preparation (dilution ratio 2000 times) was used, and an extremely excellent synergistic control effect was observed by the combined use of the BT bacterial preparation and the culture dry product of prumycin or its salt or the culture dry product of kanosamine or its salt.

[0133] On the other hand, the corrected density index of each test agent against Spodoptera litura larvae 5 days after application was 56.6 in the mixed area of ​​the BT bacterial preparation (dilution ratio 2000 times) and the dried culture product of prumycin or its salt (dilution ratio 20000 times), and 58.4 in the mixed area of ​​the BT bacterial preparation (dilution ratio 2000 times) and the dried culture product of kanosamine or its salt (dilution ratio 2000 times). The corrected density index in these mixed areas was lower than the corrected density index of 67.1 in the area where only the BT bacterial preparation (dilution ratio 2000 times) was used, indicating an improved control effect.

[0134] As described above, the corrected density index in the mixed application area where the dried culture of the producing bacteria of prumycin or its salt or the dried culture of the producing bacteria of kanosamine or its salt (corresponding to a prumycin concentration or kanosamine concentration of 5 ppm) was applied together with the BT bacteria preparation was extremely lower than the corrected density index in the area where the BT bacteria preparation was applied alone (dilution ratio 2000 times), and it was found that an excellent control effect was obtained. In addition, the corrected density index in the mixed application area where the dried culture of the producing bacteria of prumycin or its salt or the dried culture of the producing bacteria of kanosamine or its salt (corresponding to a prumycin concentration or kanosamine concentration of 0.5 ppm) was applied together with the BT bacteria preparation was also clearly lower than the corrected density index in the area where the BT bacteria preparation was applied alone (dilution ratio 2000 times), and it was found that an excellent control effect was obtained.

[0135] In addition, the corrected density index in the areas where the dried culture of the bacteria producing prumycin or its salts (dilution ratio 2000 times) was used alone and the dried culture of the bacteria producing kanosamine or its salts (dilution ratio 2000 times) was used alone was similar to the corrected density index in the untreated area, and no insecticidal effect was observed against Spodoptera litura larvae.

[0136] [Test Example 3: Storage stability test / Effectiveness test against 3rd instar larvae of diamondback moth] As shown in Tables 8 and 9, calcium chloride was added (to a content of 1.0% by mass or 5.0% by mass relative to the total amount of the hydrate and calcium chloride) and / or heat-treated (54°C, sealed for 2 weeks) to the wettable powder containing the Btk culture dried product prepared in Formulation Examples 3 and 4, to prepare wettable powders with calcium chloride added and wettable powders that had been heat-treated. When both calcium chloride was added and heat-treated, the wettable powders after calcium chloride addition were heat-treated. In addition, wettable powders without calcium chloride added and wettable powders without heat treatment were also prepared, as shown in Tables 8 and 9. Using each wettable powder, the insecticidal activity against diamondback moth 3rd instar larvae on Chinese cabbage leaves was measured by the leaf piece drug solution immersion method as shown below.

[0137] Each wettable powder prepared as described above was diluted with distilled water to a dilution ratio of 4000 times to prepare each drug solution. The third to fourth true leaves of Chinese cabbage (variety: Nozaki No. 2) were cut into a circle with a diameter of 30 mm, and the obtained circular leaves were immersed in 20 mL of each drug solution prepared as described above for 30 seconds, and then air-dried on filter paper. After confirming that the leaf surface of the Chinese cabbage was sufficiently dry, the dried Chinese cabbage leaves were placed in a plastic petri dish (diameter 35 mm, height 10 mm) in two layers, and 3rd instar larvae of the diamondback moth were released on the leaf surface, five per petri dish, and the mixture was left at 25°C under conditions of 16 hours light / 8 hours dark (1 area, 4 repetitions). Three days after the release, the petri dish was observed to determine the number of dead insects with the naked eye, and the number of dead insects relative to the total number of insects raised (insect killing rate (%)) (actual value) was calculated.

[0138] The test results obtained are shown in Tables 8 and 9 below.

[0139] [Table 8]

[0140] [Table 9]

[0141] As shown in the results of Tables 8 and 9, the wettable powder without calcium chloride had an extremely decreased insecticidal activity due to heat treatment. On the other hand, the wettable powder containing 1.0 or 5.0% calcium chloride by mass was able to suppress the decrease in insecticidal activity due to heat treatment compared to the wettable powder without calcium chloride, and the suppression effect was particularly remarkable when the calcium chloride content was 5.0% by mass. This is thought to be because the storage stability of the wettable powder was improved by the addition of calcium chloride.

[0142] The effect of calcium chloride in improving storage stability was observed in both wettable powders containing prumycin hydrochloride and wettable powders containing kanosamine hydrochloride. Heat treatment at 54°C for two weeks is thermodynamically equivalent to a thermal history at room temperature for two years.

[0143] From the above, it has been confirmed that the composition formed by further adding calcium chloride to a wettable powder containing dried Bt culture material and prumycin hydrochloride or kanosamine hydrochloride is an agricultural and horticultural insecticidal composition having excellent insecticidal effect and excellent storage stability.

[0144] [Test Example 4: Efficacy test against third-instar larvae of Helicoverpa armigera] Using the procedure described below, the activity of various insecticidal proteins (Cry1A(a), Cry1A(b) or Cry1A(c)) was measured by an artificial diet bioassay when prumycin hydrochloride or kanosamine hydrochloride was added.

[0145] Insecticidal protein solutions were prepared by adding distilled water to each of the insecticidal proteins Cry1A(a), Cry1A(b), or Cry1A(c) expressed and purified in E. coli according to the method of Non-Patent Document 33. To each of the obtained insecticidal protein solutions, prumycin hydrochloride or kanosamine hydrochloride (Sigma-Aldrich Japan LLC) prepared in Production Example 1 was added to a predetermined concentration to prepare a solution containing the insecticidal protein and prumycin hydrochloride or kanosamine hydrochloride. The obtained solution was added to and mixed with artificial feed (Insector LFS (manufactured by Nippon Nosan Kogyo Co., Ltd.) so that the content of each insecticidal protein was 0.063 to 8 μg / g feed per 1 g of artificial feed, and the content of prumycin hydrochloride or kanosamine hydrochloride was 10 μg / g per 1 g of artificial feed, as shown in Tables 10 to 13, to prepare an artificial feed containing insecticidal protein and prumycin hydrochloride or kanosamine hydrochloride. In addition, the above-mentioned insecticidal protein solution, prumycin hydrochloride, or kanosamine hydrochloride was added to the artificial feed so that the content of each insecticidal protein was 0.063 to 8 μg / g feed per 1 g of artificial feed, and the content of prumycin hydrochloride or kanosamine hydrochloride was 10 μg / g per 1 g of artificial feed, as shown in Tables 10 to 13. The artificial diets containing the insecticidal protein, prumycin hydrochloride, or kanosamine hydrochloride alone were prepared by adding each of the substances individually and mixing them so that the total amount was μg / g of the diet. The artificial diets containing each of the substances thus obtained, or the artificial diets without these substances, were placed in a petri dish (diameter 35 mm, height 10 mm) and then used to feed the bollworms (Helicoverpa armigera ( Helicoverpa armor-bearer ) third instar larvae were released into each petri dish (10 replicates), the dishes were then capped and kept at 25°C for four days.

[0146] After that, the number of dead insects in the petri dish was visually examined, and the LC was calculated using the probit method. 50 Then, the ratio between the test area to which prumycin hydrochloride or kanosamine hydrochloride was not added and the test area to which prumycin hydrochloride and kanosamine hydrochloride were added was calculated based on the following formula, and the increase in insecticidal activity due to the addition of prumycin hydrochloride and kanosamine hydrochloride was calculated.

[0147]

number

[0148] [Table 10]

[0149] [Table 11]

[0150] [Table 12]

[0151] [Table 13]

[0152] As shown in the results of Tables 10 to 13, when prumycin hydrochloride or kanosamine hydrochloride was applied alone, no insecticidal effect was observed against third-instar larvae of Helicoverpa armigera. On the other hand, by applying the insecticidal protein: Cry1A(a), Cry1A(b), or Cry1A(c) in combination with prumycin hydrochloride or kanosamine hydrochloride, respectively, the LC 50 The value was significantly lower than when only the insecticidal protein was applied, and an extremely high insecticidal effect was obtained. From the above results, it was found that a synergistic effect on the insecticidal effect can be obtained by applying the insecticidal protein in combination with prumycin hydrochloride or kanosamine hydrochloride.

[0153] [Test Example 5: Efficacy test against 3rd instar larvae of Spodoptera litura] The activity of the insecticidal protein (Cry1C) when prumycin hydrochloride or kanosamine hydrochloride was added was measured by the artificial diet bioassay method according to the procedure described below.

[0154] According to the method of Non-Patent Document 33, distilled water was added to Cry1C expressed and purified in E. coli to prepare a Cry1C solution. Puromycin hydrochloride or kanosamine hydrochloride (Sigma-Aldrich Japan LLC) prepared in Production Example 1 was added to the obtained Cry1C solution to a predetermined concentration to prepare a solution containing Cry1C and puromycin hydrochloride or kanosamine hydrochloride. The obtained solution was added to an artificial feed (Insector LFS (Nihon Nosan Kogyo Co., Ltd.)) so that the Cry1C content was 1.25 to 40.0 μg / g feed per 1 g of artificial feed and the puromycin hydrochloride or kanosamine hydrochloride content was 10 μg / g per 1 g of artificial feed, as shown in Tables 14 and 15, and mixed to prepare an artificial feed containing Cry1C and puromycin or kanosamine. In addition, the above-mentioned Cry1C solution, prumycin hydrochloride, or kanosamine hydrochloride was added and mixed alone to the artificial diet so that the content of each insecticidal protein was 1.25 to 40.0 μg / g feed per 1 g of artificial diet, or the content of prumycin hydrochloride or kanosamine hydrochloride was 10 μg / g feed per 1 g of artificial diet, as shown in Tables 14 and 15, to prepare artificial diets containing Cry1C, prumycin hydrochloride, or kanosamine hydrochloride alone. The artificial diets containing each substance obtained in this manner, or the artificial diets without these substances added, were placed in a petri dish (diameter 90 mm, height 15 mm) and then used to feed Spodoptera litura ( Spodoptera cleaning Ten third-instar larvae of the species were released into a petri dish (three replicates), the dish was then covered, and the dish was kept at 25°C for five days.

[0155] After that, the number of dead insects in the petri dish was visually examined, and the LC was calculated using the probit method. 50 The value was calculated, and the increase in insecticidal activity due to the addition of prumycin or kanosamine was calculated based on the same formula as in Test Example 4 above.

[0156] The test results obtained are shown in Tables 14 and 15 below.

[0157] [Table 14]

[0158] [Table 15]

[0159] As shown in the results of Tables 14 and 15, when prumycin hydrochloride or kanosamine hydrochloride was applied alone, no insecticidal effect was observed against the third instar larvae of Spodoptera litura. On the other hand, when the insecticidal protein Cry1C was applied in combination with prumycin hydrochloride or kanosamine hydrochloride, respectively, the LC 50 The value was significantly lower than when only the insecticidal protein was applied, and an extremely high insecticidal effect was obtained. From the above results, it was found that a synergistic effect on the insecticidal effect can be obtained by applying the insecticidal protein in combination with prumycin hydrochloride or kanosamine hydrochloride. [Industrial Applicability]

[0160] Bacillus thuringiensis of the present invention ( Bacillus Thuringian A composition containing prumycin or a salt thereof, or kanosamine or a salt thereof has excellent insecticidal activity and can be used as a pest composition having high insecticidal effect and excellent stability.

Claims

1. An insecticidal protein derived from Bacillus thuringiensis, and Purmycin or its salt or Kanosamine or its salt An agricultural and horticultural insecticidal composition comprising:

2. 2. The agricultural and horticultural insecticidal composition according to claim 1, wherein the insecticidal protein comprises Cry1A or Cry1C.

3. 3. The agricultural and horticultural insecticidal composition according to claim 1, comprising a dried culture of Bacillus thuringiensis containing the insecticidal protein.

4. 4. The agricultural and horticultural insecticidal composition according to claim 1, comprising a culture dried product of a fungus that produces prumycin or a salt thereof, or kanosamine or a salt thereof.

5. The agricultural and horticultural insecticidal composition according to any one of claims 1 to 4, further comprising 0.1 to 10 mass% calcium chloride.

6. The agricultural and horticultural insecticidal composition according to any one of claims 1 to 5, which is applied to plants using a liquid having a content of prumycin or a salt thereof, or kanosamine or a salt thereof of 0.1 ppm to 100 ppm.

7. The agricultural and horticultural insecticidal composition according to any one of claims 1 to 6, which is applied to plants using a liquid having an insecticidal protein content of 0.1 ppm to 500 ppm.

8. The agricultural and horticultural insecticidal composition according to any one of claims 1 to 7, comprising prumycin hydrochloride.

9. The agricultural and horticultural pesticidal composition according to any one of claims 1 to 8, comprising kanosamine hydrochloride.

10. A method for controlling agricultural and horticultural pests, comprising: a step (a) of applying an insecticidal protein derived from Bacillus thuringiensis to a plant; and a step (b) of applying prumycin or a salt thereof, or kanosamine or a salt thereof to the plant simultaneously, successively, or separately from the insecticidal protein.

11. A method for controlling agricultural and horticultural pests, comprising the step of applying prumycin or a salt thereof, or kanosamine or a salt thereof to a recombinant plant producing an insecticidal protein derived from Bacillus thuringiensis.

12. The method according to claim 10, wherein the application in step (a) or (b) comprises soil incorporation, irrigation, seed smearing, seed soaking, or foliage spraying.

13. 13. The method for controlling pests according to claim 10 or 12, which uses an agricultural and horticultural insecticidal composition comprising the insecticidal protein and prumycin or a salt thereof, or kanosamine or a salt thereof.

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