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    • 专利摘要:
    "CEPA THAT BELONGS TO THE BACILLUS GENDER, MICROBIOLOGICAL AGENT AND PLANT GROWTH METHOD". The present invention relates to strains of Bacillus sp. AT-332 (NITE BP-1095) and AT-79 (NITE BP-1094) isolated from nature; and a plant growth promoter, a nematode control agent and a plant disease control agent containing the strains as active bacteria. Strains of Bacillus sp., Strains AT-332 and AT-79 can promote the growth of useful plants, and are effective in controlling both a wide range of various plant diseases and nematode damage due to a culture containing a secondary metabolite of the strains, or live bacteria cultivated and isolated from the strains being introduced to a plant body or to the soil for their growth.
    公开号:BR112013030228B1
    申请号:R112013030228-3
    申请日:2012-05-21
    公开日:2021-03-16
    发明作者:Yusuke Amaki；Keijitsu Tanaka；Motoki Tanaka；Akitomo Takahashi
    申请人:Sds Biotech K. K；
    IPC主号:
    专利说明:

    TECHNICAL FIELD
    [001] The present invention relates to a new microorganism useful for controlling plant diseases and nematode damage and promoting plant growth. Specifically, the present invention relates to strains AT-332 and AT-79 of Bacillus sp., Which are a new microorganism that exhibits many superior effects in controlling plant diseases and nematode damage and promoting plant growth compared to the microorganisms that belong to a closely related Bacillus amiloliquefaciens described in the literature; and a plant disease control agent, nematode control agent and a plant growth promoter containing the body of the fungus and the culture of the microorganisms. BACKGROUND OF THE TECHNIQUE
    [002] A primary method for controlling plant diseases and nematodes is a method using chemical pesticides, and chemical pesticides have allowed stable crop production to this day. However, recently, it has become difficult to completely control the impact on the environment due to the continued use of chemical pesticides and the appearance of drug-resistant bacteria by conventional chemical pesticides; and diseases such as a bacterial disease that is difficult to control are developing into a major problem. Consequently, biological control technology using a microorganism isolated from nature draws increasing attention and some microorganism pesticides have been commercially produced. However, conventional microbiological pesticides have a defect in which the effect is not stable and applicable diseases are less compared to chemical pesticides. In these circumstances, there was an increasing demand for a new microbiological pesticide that has new applicable diseases and exhibits a stable control effect.
    [003] As a plant disease control agent using a microorganism, a Talaromyces flavus agent, a Pseudomonas fluorescens agent, an Erwinia carotovora swelling agent, a Trichoderma atroiviride agent, a Bacillus simplex agent, a Bacillus subtilis agent and the like are registered as a microbiological pesticide and have been used.
    [004] As a nematode control agent using a microorganism, a Pasteuria penetrans agent and a Monacrosporium phymatophagums agent are registered as a microbiological pesticide and have been used.
    [005] Japanese Patent specification No. 2955655 (Patent Document 1) describes a plant disease control agent using bacteria belonging to Bacillus amiloliquefaciens. The active ingredient in the plant disease control agent is the product of the micro-organism and the bacterium itself is used as a pesticide. In addition, the target for control is a disease caused by filamentous bacteria and the document does not describe the control of bacterial disease. Publication JP-A-2009-247302 (Patent Document 2) describes a microorganism pesticide that can control filamentous bacteria disease and bacterial disease at the same time that viable bacteria cells are effective, but the document has no description on nematode control.
    [006] Japanese Patent specification No. 3471815 (Patent Document 3; WO 98/050422) describes a plant disease control agent using Bacillus bacteria that can be used for a wide range of plant diseases and effective in corn rootworms, but the document has no description on nematode control. Japanese Patent specification No. 4071036 (Patent Document 4; US 2004/265292) describes the strain of Bacillus sp. D747 that can be used to control harmful plant diseases and insects, however the document has no description on nematode control.
    [007] Japanese Patent specification No. 3471811 (Patent Document 5; WO 96/032840) describes a nematode control agent using bacteria of the genus Bacillus. The active ingredient of the nematode control agent is the bacteria or spore of the Bacillus firmus strain having an antinematode activity, however, the document has no description in the control of plant disease. Japanese Patent specification No. 4359653 (Patent Document 6; WO 1997/012980) describes a method for controlling nematodes using a toxin produced by a new strain of Bacillus thuringiensis, however, the document has no description in controlling plant.
    [008] In agriculture, chemical fertilizers are an important agricultural material that influences crop yields. However, 30 to 50% of the chemical fertilizer components are not used in crops, however, widespread in the environment, which causes river eutrophication and groundwater contamination. A large amount of fossil fuels are used in the production of chemical fertilizers and the cost of producing chemical fertilizers is increasing along with skyrocketing prices for fossil fuels. In addition, nitrogen oxide (NOx) as a decomposition product of a nitrogen fertilizer is said to be about 300 times more efficient in greenhouse emissions than carbon dioxide, and there is growing concern about the greenhouse effect. Future food shortages are expected to be due to the growth of the global population and therefore the use of material to increase crop productivity is inevitable and there is an increasing need for more environmentally friendly material to replace chemical fertilizers. conventional.
    [009] In light of such circumstances, studies have been done mainly on a wide range of Rhizobium bacteria, Pseudomonas bacteria and Bacillus bacteria. However, very little is in practical use because they are less effective.
    [0010] As discussed above, no Bacillus bacteria that are generally effective in plant diseases, available in the control of nematodes and are effective in promoting plant growth have been known to date. BACKGROUND DOCUMENTS Patent Documents
    [0011] Patent Document 1: Japanese Patent No. 2955655
    [0012] Patent Document 2: JP-A-2009-247302
    [0013] Patent Document 3: Japanese Patent No. 3471815
    [0014] Patent Document 4: Japanese Patent No. 4071036
    [0015] Patent Document 5: Japanese Patent No. 3471811
    [0016] Patent Document 6: Japanese Patent No. 4359653 DESCRIPTION OF THE INVENTION Problem to be solved by the invention
    [0017] An objective of the present invention is to isolate a new microorganism from nature to supply, which microorganism has the effects of controlling multiple plant diseases, controlling nematodes and / or promoting plant growth.
    [0018] Another objective of the present invention is to provide a plant disease control agent, nematode control agent and a plant growth promoter, which contain the aforementioned microorganism as active bacteria and can be used as a biological pesticide ( microbiological agents). Means to Solve the Problem
    [0019] As a result of intensive studies to solve the problem, the present inventors have succeeded in isolating a new strain that belongs to the genus Bacillus of nature, whose strain has the effects of controlling multiple plant diseases, controlling nematodes and promoting the growth of plant, and the present invention is completed.
    [0020] The present invention relates to the strain described in 1 to 4 below, the microbiological agent in 5 to 8 below and the method for growing plants in 9 below.1. A strain comprising 16S rDNA represented by the base sequence No. 2 or 3.2. The strain as described in paragraph 1 above, in which the strain per se and / or the culture of the strain shows (m) the effects of controlling plant diseases, controlling nematodes and / or promoting plant growth.3. The strain of Bacillus sp. AT-332 as described in 1 or 2 above, containing 16S rDNA represented by base sequence No. 2.4. The strain of Bacillus sp. AT-79 as described in 1 or 2 above, containing 16S rDNA represented by the base sequence No. 3.5. A microbiological agent containing the strain and / or the culture of the strain described in any one of 1 to 4 above as an active ingredient.6. The microbiological agent as described in 5 above, which is a plant disease control agent.7. The microbiological agent as described in 5 above, which is a nematode control agent.8. The microbiological agent as described in 5 above, which is a plant growth promoter. 9. A method for growing the plants, treating the plants with the microbiological agent described in any one of 5 to 8 above. EFFECTS OF THE INVENTION
    [0021] The strains AT-332 and AT-79 of Bacillus sp. of the present invention can control a wide range of various plant diseases and nematodes and can also promote the growth of useful plants due to culture (including viable bacterial cells) or live bacteria grown and isolated from the strains that are presented to a plant body such as roots, stems, leaves, seeds and fruits or to the cultivated soil. BRIEF DESCRIPTION OF THE DRAWINGS
    [0022] Figure 1 shows the molecular phylogenetic tree using the base sequence of 16S rDNA of strains AT-332 and AT-79 from Bacillus sp .. In the figure, the numbers near the branches are the input load values and a bar of scale is shown on the bottom left.
    [0023] Figure 2 photographs (a) to (d) show the effect of promoting plant growth of strain AT-332 in the basic test (Example 12 and Comparative Examples 12-13).
    [0024] Figure 3 shows the effect of promoting the growth of Chinese pulp of the strains AT-332 and AT-79 in a pot test (Example 13). MODE FOR CARRYING OUT THE INVENTION
    [0025] The present inventors have evaluated that microorganisms from various plants, soils and the like for the purpose of recently developing a safe and superior microbial pesticide and / or microbial fertilizer that has a broad antibacterial spectrum against various plant diseases, show antinematode activity and have the effect of promoting plant growth. As a result, the present inventors made a useful discovery that the strain isolated from the soil collected in Ibaraki Prefecture shows a broad antibacterial spectrum against various plant diseases, shows high insecticidal activity against nematodes and has the effect of promoting plant growth.
    [0026] Both strains thus recently isolated (cepaAT-332 and strain AT-79) are gram-positive mobile bacilli as is clear from the bacteriological characteristics to be described later, and they grow and form spores under an aerobic condition. Both strains became positive in both the catalase reaction and the oxidase reaction. In addition, as a result of identification based on the sequence of about 1500 bp-base of the 5 'terminal side of 16S rDNA, the strains have been confirmed to be a new strain belonging to the Bacillus genus related to Bacillus amiloliquefaciens. Due to the superior characteristics of having effects on a wide range of plant diseases, the effect of high control on nematodes and the effect of promoting plant growth, strains AT-332 and AT-79 have been identified as a new strain and designated such as the AT-332 and AT-79 strains of Bacillus sp. related to Bacillus amiloliquefaciens.
    [0027] The AT-332 strain and the AT-79 strain of Bacillus sp. of the present invention were deposited as Bacillus sp. AT-332 and Bacillus sp. AT-79 with the depositary institution, Biological Resource Center, National Institute of Technology and Evaluation (2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818 JAPAN) (original filing date (accepted date): May 2, 2011; Accession number: NITE BP-1095 and NITE BP-1094).
    [0028] The bacteriological characteristics of Bacillus sp. AT-332 (NITE BP-1095) are described below. The bacteriological characteristics were determined with reference to the following documents.
    [0029] PRIEST (F.G.), GOODFELLOW (M.), SHUTE (L.A.) andBERKELEY (R.C.W.): Bacillus amiloliquefaciens sp. nov., nom. rev. Int. J. Syst. Bacteriol., 1987, 37, 69-71 and Bergey's Manual of SystematicBacteriology, Second Edition volume 3. (1) Morphological property
    [0030] Form: rod-shaped bacteria
    [0031] Size: width from 0.8 to 0.9 μm and length from 1.5 to 2.0 μm
    [0032] Mobility: +
    [0033] Epiphytic state of the scourge: peritrichous
    [0034] Presence or absence of spores: + (quasi-terminal) (2) Cultural characteristics
    [0035] Culture medium: nutrient agar medium (30 ° C)
    [0036] Shape: circular
    [0037] Prominence: flat
    [0038] Periphery: entire margin
    [0039] State of surface: smooth
    [0040] Viscosity: viscous
    [0041] Transparency: opaque
    [0042] Color shade: cream color
    [0043] Brightness: matte
    [0044] Pigment production: non-productive (3) Physiological characteristics
    [0045] Gram staining: +
    [0046] Nitrate reduction: -
    [0047] Nitrogen desorption reaction: -
    [0048] MR test: -
    [0049] VP test: +
    [0050] Generation of indole: -
    [0051] Generation of hydrogen sulfide: -
    [0052] Hydrolysis of starch: +
    [0053] Use of citric acid: - (Koser)
    [0054] + (Christensen)
    [0055] Use of inorganic nitrogen source: - (nitrate)
    [0056] + (ammonium salt)
    [0057] Urease: -
    [0058] Oxidase: +
    [0059] Catalase: +
    [0060] Range for growth pH 5: +
    [0061] pH 8: +
    [0062] pH 9: + Growth temperature 37 ° C:
    [0063] 45 ° C: +
    [0064] 50 ° C: +
    [0065] 55 ° C: -
    [0066] Growth in anaerobic condition: -
    [0067] OF test (oxidation / fermentation): - / - Acid production / gas production from sugars:
    [0068] L-arabinose: + / -
    [0069] D-glucose: + / -
    [0070] D-fructose: + / -
    [0071] Maltose: + / -
    [0072] Lactose: - / -
    [0073] D-sorbitosis: + / -
    [0074] Inositol: + / -
    [0075] D-xylose: + / -
    [0076] D-mannose: + / -
    [0077] D-galactose: - / -
    [0078] Sucrose: + / -
    [0079] Trealose: + / -
    [0080] D-mannitol: + / -
    [0081] Glycerin: + / -
    [0082] β-galactosidase activity: -
    [0083] Arginine dihydrolase activity: -
    [0084] Lysine decarboxylase activity: -
    [0085] Tryptophan deaminase activity: -
    [0086] Gelatinase activity: +
    [0087] The bacteriological characteristics of Bacillus sp. AT-79 (NITE BP-1094) are described below. (1) morphological property
    [0088] Form: rod-shaped bacteria
    [0089] Size: width from 0.8 to 0.9 μm and length from 1.5 to 2.0 μm
    [0090] Mobility: +
    [0091] Epiphytic state of the scourge: peritrichous
    [0092] Presence or absence of spores: + (quasi-terminal) (2) Cultural characteristics
    [0093] Culture medium: nutrient agar medium (30 ° C)
    [0094] Form: circular
    [0095] Prominence: flat
    [0096] Periphery: entire margin
    [0097] State of surface: smooth
    [0098] Viscosity: viscous
    [0099] Transparency: opaque
    [00100] Color shade: cream color
    [00101] Brightness: matte
    [00102] Pigment production: non-productive (3) Physiological characteristics
    [00103] Gram staining: +
    [00104] Nitrate reduction: -
    [00105] Nitrogen desorption reaction: -
    [00106] MR test: -
    [00107] VP test: +
    [00108] Generation of indole: -
    [00109] Generation of hydrogen sulfide: -
    [00110] Hydrolysis of starch: +
    [00111] Use of citric acid: - (Koher)
    [00112] + (Christensen)
    [00113] Use of inorganic nitrogen source: - (nitrate)
    [00114] + (ammonium salt)
    [00115] Urease: -
    [00116] Oxidase: +
    [00117] Catalase: + PH 5 growth range: +
    [00118] pH 8: +
    [00119] pH 9: + Growth temperature 37 ° C:
    +
    [00120] 45 ° C: +
    [00121] 50 ° C: +
    [00122] 55 ° C: -
    [00123] Growth in anaerobic condition: -
    [00124] OF test (oxidation / fermentation): - / - Acid production / gas production from sugars:
    [00125] L-arabinose: + / -
    [00126] D-glucose: + / -
    [00127] D-fructose: + / -
    [00128] Maltose: + / -
    [00129] Lactose: - / -
    [00130] D-sorbitosis: + / -
    [00131] Inositol: + / -
    [00132] D-xylose: + / -
    [00133] D-mannose: + / -
    [00134] D-galactose: - / -
    [00135] Sucrose: + / -
    [00136] Trealose: + / -
    [00137] D-mannitol: + / -
    [00138] Glycerin: + / -
    [00139] β-galactosidase activity: -
    [00140] Arginine dihydrolase activity: -
    [00141] Lysine decarboxylase activity: -
    [00142] Tryptophan deaminase activity: -
    [00143] Gelatinase activity: +
    [00144] The base sequences of the 5 'terminal side of 16S rDNA of the strain of Bacillus sp. AT-332 and AT-79 of the present invention are represented by sequence No. 2 and sequence No. 3, respectively.
    [00145] Sequence No. 2 and sequence No. 3 differ from each other only by two bases in base No. 444 and base No. 1242. Base No. 444 is guanine (g) in sequence No. 2 and adenine (a) in sequence No. 3, and base No. 1242 is adenine (a) in sequence No. 2 and guanine (g) in sequence No. 3.
    [00146] Therefore, the microorganism of the present invention is characterized by having the base sequence of the No. 1 sequence including the aforementioned No. 2 and No. 3 sequences (i.e., base No. 444 and base No. 1242 are represented by "r") from the 5 'terminal side of 16S rDNA.
    [00147] In the present invention, the 16S rDNA base sequence was analyzed as below.
    [00148] InstaGene matrix (produced by BIO RAD Laboratories, Inc., California (CA), U.S.A.) was used for DNA extraction; PrimeSTAR HS DNA Polymerase (produced by Takara Bio Inc.) was used for PCR; the BigDye Terminator Cycle Sequencing Kit v3.1 (produced by Applied Biosystems, California (CA), U.S.A.) was used to determine the cycle sequence, respectively. The primers used (according to "Gene Analysis Method - method for determining the base sequence of 16S rRNA gene", Yasuyoshi Nakagawa et al., Edited by the Society for Actinomycetes Japan, Classification and identification of Actinomycetes, pp. 88-117, Business Center for Academic Societies Japan, 2001) were 9F, 339F, 785F, 1099F, 536R, 802R, 1242R and 1541R. The sequence was identified using ABI PRISM 3100 genetic Analizer System (produced by Applied Biosystems, California (CA), U.S.A.).
    [00149] As a result of the search for homology based on the international base sequence database (GenBank / DDBJ / EMBL) using BLAST (ALTSCHUL, (SF) et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acid Res. 1997.25, 3389-3402), the base sequence of 16S rDNA of strain AT-332 and strain AT-79 had a high degree of homology with the 16S rDNA derived from the genus Bacillus, and both strains had the highest homology of 99.9% with 16S rDNA from the strain of Bacillus amiloliquefaciens BCRC11601. On the other hand, as a result of the search for homology based on the database of the international base sequence (GenBank / DDBJ / EMBL), no base sequence of 16S rDNA of the strains AT-332 and AT-79 exactly compared the sequence base of 16S rDNA derived from the genus Bacillus.
    [00150] In the present invention, molecular phylogenetic analysis was performed as below.
    [00151] 16S rDNA derived from the standard strain from the strain group was assumed to be closely related was obtained from the international base sequence database (GenBank / DDBJ / EMBL) to perform the molecular phylogenetic analysis using 1500 bp of the base sequence 16S rDNA obtained in the previous.
    [00152] 16S rDNA used for molecular phylogenetic analysis was derived from the following strains. - Bacillus subtilis, IAM12118T (AB042061) - Bacillus subtilis subsp. spizizenii, NBRC101239T (AB325584) - Bacillus mojavensis, IFO15718 T (AB021191) - Bacillus vallismortis, DSM11031 T (AB021198) - Bacillus amiloliquefaciens, BCRC11601 T (EF433406) - Bacillus atropha (11) ) - Bacillus sonorensis, BCRC17416 T (EF433411) - Bacillus licheniformis, DSM13 T (AE017333) - Bacillus altitudinis, 41KF2b T (AJ831842) - Bacillus cereus ATCC14579 T (NC_004722) BSL2
    [00153] "T" at the end of the strain name means the standard strain of the species. BSL means that the strain is a biosafety level (level 2 or higher is indicated). The codes in parentheses indicate the access number.
    [00154] The molecular phylogenetic tree obtained is shown in Figure 1.
    [00155] The numbers near the branches are the load values entered and a scale bar is shown on the bottom left.
    [00156] Since the AT-332 strain and the AT-79 strain have the property that does not perform nitrate reduction as mentioned above, their mycological characteristics do not exactly compare to those of Bacillus amiloliquefaciens described in the Bergey Manual. Likewise, from the result of the analysis of 16S rDNA, strains AT-332 and AT-79 are considered to be closely related to Bacillus amiloliquefaciens, however they cannot be identified as Bacillus amiloliquefaciens and strain AT-332 and strain AT-79 was determined be a new strain that belongs to the genus Bacillus.
    [00157] The AT-332 strain and the AT-79 strain of Bacillus sp. of the present invention are allowed to grow by known means such as static culture in a solid medium and liquid culture and the type of medium available, culture conditions and the like are not particularly limited as long as they allow bacteria to survive and grow. Examples of the medium include a medium containing glucose, peptone, yeast extract and the like as well as a general medium such as a meat extract. Likewise, unlike a liquid medium, a solid medium such as an agar slant medium and a plate medium other than a liquid medium can be used.
    [00158] All carbon sources that the AT-332 strain and AT-79 strain can use can be used for the medium. Specific examples include several synthetic or natural carbon sources where the AT-332 and AT-79 strains can use different sugars such as glucose, galactose, lactose, sucrose, maltose, malt extracts, residual molasses, starch syrup and hydrolyzate of starch.
    [00159] Similarly, several synthetic and natural substances that the aforementioned strains can use, such as a substance containing organic nitrogen including peptone, meat extract, yeast extract, soybean powder and macerated maize liquor can be used for the source of nitrogen from the medium.
    [00160] According to a conventional method for cultivating microorganisms, inorganic salts such as dietary salt and phosphoric salt, metal salts such as calcium, magnesium and iron and micronutrients such as vitamins and amino acids can be added when necessary.
    [00161] The culture can be performed under an aerobic condition such as agitation culture and aeration culture. The culture temperature is 20 to 40 ° C and preferably 25 to 35 ° C, pH is 5 to 8 and preferably 6 to 7, and the culture period is one to four days and preferably two to three days.
    [00162] The culture containing the bacterial body of strain AT-332 and strain AT-79 of Bacillus sp. of the present invention has the property of controlling various plant diseases, controlling nematodes and promoting the growth of useful plants.
    [00163] Various plant diseases can be prevented and the nematodes can be controlled by allowing the processed product of the culture containing the bacterial body of strain AT-332 and strain AT-79 of Bacillus sp. of the present invention, mixing the culture and other components and the like; the processed product from separate cultured bacterial cells obtained by subjecting the culture product to centrifugal separation treatment or washing the bacterial cells, the mixture of separate cultured bacterial cells and other components, and the like; a diluent of the same with a liquid or a solid and the like exist in the body of the plant such as the roots, stems, leaves, seeds and fruits or in the grove soil.
    [00164] The AT-332 strain and the AT-79 strain of Bacillus sp. of the present invention is available as a plant disease control agent, nematode control agent and a plant disease promoter in any state of nutrient cells, spores or a mixture of both as long as the bacteria are alive. Likewise, strains can be used if the components of the culture medium are mixed since they are after cultivation or if they are in a state where the different components of bacterial cells are removed by washing with distilled water and the like. .
    [00165] The AT-332 strain and the AT-79 strain of Bacillus sp. of the present invention can control plant disease caused by fungi and bacteria belonging to Oomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes depending on the type of application and can control the phytoparasitic nematode, such as Ditilenchus dipasaci, Ditilenchus destructor, Pratilenchus sp., Meloidogyne sp. ., Heterodera sp. and Globodera spp .. Strains can promote the growth of crops, vegetables, fruits, flowers and vegetables at the same time.
    [00166] Specifically, offensive bacteria in which strain AT-332 and strain AT-79 of Bacillus sp. of the present invention can control include Pyricularia oryzae, Cochliobolus miyabeanus, Rhizoctonia solani and Gibberella fujikuroi that infest rice; Erysiphe graminis f.sp. hordei, Erysiphe graminis f.sp. tritici, Puccinia striiformis, Puccinia graminis, Puccinia recondita f.sp. tritici, Puccinia hordei, Gibberella zeae, Pyrenophorateres, Typhula incarni nuda, Tilletia caries, Tilletia toetida, Tapesia yallundea, Phynchosporium secalis f.sp. hordei, Septoria tritici and Lentosphaeria nodorum that infest wheat; Diaporthe citri, Elsinoe fawcettii, Phytophthora citrophthora, Penicillium digitatum and Penicillium italicum from citrus plants; Monilinia mali, Waltz ceratosperma, Podosphaera leucotricha, Alternaria alternataapple pathotype, Venturia inaequalis, Gymnosporangium yamadae, Botryosphaeria berengeriana f.sp. Venturia nashicola, Alternaria alternatajapanese pear pathotype, Physalospora piricola and Gymnosporangium asiaticum of pears; Monilinia fructicola, Cladosporium carpophilum and Phomopsis sp. peaches; Pseudocercospora vitis, Marssonina viticola, Elsinoe ampelina, Glomerella cingulata, Uncinula necator, Phakopsora ampelopsidis and Phomopsis sp. of grapes; Phillactinia kakicola, Colletotrichum gloeosporioides, Cercospora kaki and Mycosphaerella nawae of persimmons; Plados Cladosporium carpophilum; Monilinia fructicola of Prunus avium; Sphaerotheca fuliginea, Didymella bryoniae, Colletotorichum legenarium of gourds; Alternaria solani, Cladosporium fulvum of tomatoes; Phomopsis vexans and Erysiphe cichoracearum from eggplants; Alternaria japonica, Alternaria bracicae, Alternaria brassicicola and Cercosporella brassicae from vegetables of the brassica genus; Pucciniaallii of green onions; Pythium ultimum and Pythium zigiberis of gingers; Sphaerotheca humuli and Strawberry Glomerella cingulata; Cercospora kikuchii, Elsinoe glycines and Diaporthe phaseolorum var. soybeans and soybeans; Cercospora canescens and Uromyces phaseoli var. azukicola from azuki beans; Colletotrichum lindemuthianum of brown beans; Cercosporidium personatum, Cercospora arachidicola and Shaceloma arachidis from peanuts; Pea Erysiphe pisi; Alternaria solani of potatoes; Exobasidium reticulatum, Elsinoe leucospila, Pestalotiopsis theae and Pestalotiopsis longiseta of teas; Alternaria longipes, Erysiphe cichoracearum and Colletotrichum gloeosporioides from tobacco; Cercospora beticola from beets; Curvularia geniculata and Ceratobasidium spp. of lawns; Diplocarpon rosae and Shaerotheca pannosa de roses; Obese Septoria and Puccinia horiana from chrysanthemums; and Botrytis cinerea and Sclerotinia sclerotiorum from several crop plants, but not limited to them.
    [00167] The plant disease control agent of the present invention includes a post-harvest disease control agent for crops stored after harvest, particularly to prevent fruits and the like from spoilage. There is no limit on the types of crops on which the post-harvest disease control agent of the present can be applied, and examples include fruits such as strawberry, grape, fig, citrus, peach, melon, watermelon, apple, pear, banana and pineapple and vegetables, such as a cucumber, tomato, Chinese cabbage, cabbage, Welsh onion, onion, carrot, Japanese radish, ginger, green pepper, eggplant, pumpkin and bean sprouts. There is no limit on the types of fungi that cause postharvest disease and examples include Botrytis cinerea, Colletotrichum gloeosporioides and Alternaria alternata.
    [00168] Examples of nematodes that the AT-332 and AT-79 strains of Bacillus sp. of the present invention can control include Meloidogyne sp., such as Meloidogyne hapla species, Meloidogyne incognita, Meloidogyne javanica and Meloidogyne; Globodera spp., Such as Globodera rostochiensis and other species of Globodera; Heterodera sp., Such as species of Heterodera avenae, Heterodera glycines, Heterodera schachtii, Heterodera trifolii and Heterodera; species of Anguiana that belong to Anguina funesta; Aphelenchoides species; Belonolaimus longicaudatus and other species that belong to Belonolaimus; Bursaphelenchus xilophilus which belongs to Bursaphelenchus xilophilus and other species of Bursaphelenchus; Criconema species, Criconemella species, Criconemoides species and Mesocriconema species that belong to Criconemoides; Ditilenchus destructor, Ditilenchus dipsaci and other species that belong to Ditilenchus; Dolichodorus species that belong to awl nematodes; Heliocotilenchus multicinctus that belongs to Helicotilenchus and other species of Helicotilenchus; species of Hemicycliophora and species of Hemicriconemoides that belong to tubular and tubular nematodes; Hirshmanniella species; Hoploaimus species that belong to Hoplolaimus; species of Nacobbus that belong to Nacobbus; Longidorus elongatus that belongs to Longidorus and other species of Longidorus; Pratilenchus neglectus, Pratilenchus penetrans, Pratilenchus curvitatus, Pratilenchus goodeyi and other species of Pratilenchus that belong to Pratylenchus sp .; Radopholus similis and other species that belong to Radopholus; Rotilenchus robustus and other species of Rotilenchus that belong to Rotilenchulus reniformis; Scutellonema species; Trichodorus primitivus and other species of Trichodorus that belong to the short and thick root nematodes; species of Paratrichodorus; Tilenchorhynchus claytoni, Tilenchorhynchus dubius and other species that belong to Tilenchorhynchus; Tilenchulus species that belong to Tilenchulus semipenetrans; and Xiphinema species that belong to Xiphinema americanum, but are not limited to these.
    [00169] The AT-332 strain and the AT-79 strain of Bacillus sp. of the present invention are especially useful for controlling Meloidogyne species, Globodera species, Heterodera species, Pratilenchus species, Radopholus species, Rotilenchus species and Tilenchulus species, and in particular can be used appropriately to exterminate Meloidogyne species, species of Pratilenchus, species of Globodera and species of Heterodera.
    [00170] Examples of cultures that strain AT-332 and strain AT-79 of Bacillus sp. of the present invention that can promote growth include cereal crops such as rice, wheat and corn; vegetables such as carrot, cucumber, Japanese radish, pumpkin, lettuce, eggplant, tomato, cabbage, potato, Chinese cabbage, crown daisy, Japanese mustard, spinach, green pepper, Welsh onion, onion, ginger, garlic, strawberry; mushrooms such as the shiitake mushroom; fruit trees, such as persimmon, pear, orange, grape, apple and peach; ornamental flowers and plants such as chrysanthemum, tulip and rose; and vegetables such as soy, sesame and peanuts.
    [00171] The plant disease control agent, nematode control agent and plant growth promoter of the present invention contain the strain AT-332 and strain AT-79 of Bacillus sp. which can control plant diseases and nematodes and have a plant growth promoting effect as mentioned above as a suitable micro-organism.
    [00172] In the plant disease control agent, nematode control agent and plant growth promoter of the present invention, the AT-332 strain or AT-79 strain can be used alone or in combination. Likewise, the mutant of each of the strains can be used. The mutant is one that has the aforementioned bacteriological property of strain AT-332 and AT-79 and an activity to control plant diseases, control nematodes and promote plant growth. A natural mutant strain, a mutant strain caused by ultraviolet ray or chemical mutagenic agent, a cell fusion strain, and a genetically modified strain, or the like, can be used.
    [00173] When live bacteria from strain AT-332 and strain AT-79 are used in the plant disease control agent, nematode control agent and plant growth promoter of the present invention it is preferable to add the bacteria to the body of the invention. plant at a concentration of 105 to 1010 units / ml.
    [00174] When the culture product of the AT-332 strain and / or the AT-79 strain is used, the dosage can be appropriately determined in individual cases of the aforementioned viable bacteria.
    [00175] As the microbiological agent (the plant disease control agent, nematode control agent and plant growth promoter) of the present invention, the and / or bacterial cells grow product of the AT-332 strain and AT strain -79 can be used alone. Either the microbiological agent can be diluted with an inert liquid or a solid carrier can be used as a pharmacological agent with the addition of the surfactant, dispersing the agent and other adjuvant as needed. Examples of specific formulation include granular formulation, powder formulation, wettable powder, suspending agent and emulsion formulation.
    [00176] Examples of vehicle include talc, bentonite, kaolin, clay, diatomaceous earth, white carbon, vermiculite, lime hydrate, ammonium sulfate, silica sand, urea, porous solid vehicle and liquid vehicles such as water, isopropyl alcohol , methyl naphthalene, xylene, cyclohexanone and alkylene glycol. Examples of the surfactant and dispersing agent include salts of dinaftylmethanesulfonic acid, ester salts of alcohol sulfuric acid, salts of lignin sulfonic acid, salts of alkylaryl sulfonic acid, polyoxyethylene glycol ethers, polyoxyethylene sorbitan monoalkylate and polyoxyethylene alkylethyl ethers. Examples of the adjuvant include carboxymethylcellulose, polyethylene glycol, propylene glycol, gum arabic and xanthan gum; and examples of the cryoprotective agent include skim milk and pH protection agent. The amount of live bacteria and / or culture product of strain AT-332 and strain AT-79, the time of application and the amount of application can be suitably determined depending on each case of the previous viable bacteria.
    [00177] The microbiological agent (plant disease control agent, nematode control agent and plant growth promoter) of the present invention may contain active ingredients different from those of the present invention: that is, insecticides, other bactericidal agents, herbicides , plant growth regulators and fertilizers. Likewise, the plant disease control agent, nematode control agent and plant growth promoter of the present invention can contain the strain of other species in combination with the AT-332 and / or AT-79 strain.
    [00178] Examples of the bactericidal components include bitertanol, bromuconazole, cyproconazole, diphenoconazole, diniconazole, enylconazole, epoxiconazole, fluquinconazole, fenbuconazole, flusilazole, flutriafol, hexaconazole, imibenconazole, ipconazolol, proponazole, metconazole, proponazole, metconazole, triadimenol, tebuconazole, tetraconazole, triticonazole, prochloraz, pefurazoate, imazalil, triflumizole, ciazofamide, benomile, carbendazim, thiabendazole, fuberidazole, etaboxam, etridiazole, fumaric acid, oxoxoxazol, dimoxyazole, oxoxoxazole , Orizastrobina, picoxyestrobina, piracloestrobina, trifloxiestrobina, carboxina, benalaxila, boscalida, bixafeno, fenexamida, flutolanil, furametpir, mepronil, metalaxila, mefenoxam, offurace, oxadixila, oxycarfetamide, flicarboxyl, toxyamide, toxyamide, pentoxyramide , diclocimete, mandi propamide, fluazinam, pyrifenox, bupyrimide, ciprodinil, fenarimol, ferinzone, mepanipirim, nuarimol, pyrimethanil, triforin, fempiclonil, fludioxonil, aldimorph, dodemorfe, fempropimorph, tridemoron, probpropin, fampropidone, phenpropidone, procomidone, phenpropidone, prochimidone, procomidone, anilazine, diclomezine, pyroquinone, proquinazide, tricyclazole, captafol, captano, dazomete, folpete, fenoxanil, quinoxifene, amissulbrom, manzebe, manebe, metam, metindo, ferbam, propineb, tiuram, zinebe, zinc, isopropyl, propamocarb hydrochloride, methyl thiophanate, pyribencarb, Bordeaux mixture, basic copper chloride, basic copper sulfide, cupric hydroxide, copper 8-hydroxyquinoline, dodine, iminoctadine albesylate, iminoctadine acetate, guazatin, casugamycin, streptomycin, polytamycin, streptomycin oxytetracycline, validamycin A, binapacryl, dinocape, dinobutone, dithianone, isoprothiolane, edifenfos, iprobenfos, fosetil, fosetil a aluminum, pirasofos, tolclofos-methyl, chlorotalonil, diclofluanide, flussulfamide, hexiachlorobenzene, phthalide, pencicurone, quintozene, ciflufenamide, cymoxanil, dimethyrimol, ethirimol, furalaxil, metrafenone, sulfonamide, sulfoxamide, spiroxamine, potassium, spiroxamide, spiroxamine, spiroxamine, ammonium sulfate calcium bicarbonate, thiadiazine, chavephthalam, triazine, copper nonylphenol sulfonate, isoxazole hydroxy, fluoroimide, polycarbamate, metasulfocarb, EDDP, IBP, tolfempirade, fluopiram, isotianil and isopirazam, but not limited to these.
    [00179] Examples of the insecticidal components include acetamipride, pimetrozine, phenitrothione, acephate, carbaryl, methomyl, cartap, cyhalothrin, etofemprox, teflubenzurone, flubendiamide, fluphenoxurone, tebufenozide, methoxy, pyridabine, methazine, imidacid, buidazidine fentiona, daiazinone, oxideprofos, vamidothione, ethiofencarb, pirimicarb, permethrin, cypermethrin, bifenthrin, halfemprox, silafluofen, nitempiram, chlorfluazurone, methoxyfenzide, tebufempirade, pyrimidifen, hexane, celtane, propyl, hexane, sputino, celtano, indoxacarb, metaflumizone, chlorfenapyr, fipronil, ethoxazole, acequinocyl, pyrimiphos-methyl, acrinatrine, quinomethionate, chlorpyrifos, abamectin, emamectin benzoate, fembutatin oxide, terbufos, etoprofos, fusofos, fosfos, fosfos, fosfos, fosfos, fosfos, fosfos, fosfos, fosfos, fosfos, fosfos, fosam, fos, isoamidophos, fostietane, isazophos, thionazine, benfuracarb, spirodiclofen, etiof carbohydrate, azinfos-methyl, disulfotone, metiocarb, oxidemeton-methyl, parathione, cyfluthrin, beta-cyfluthrin, tebupyrimphos, spiromesifene, endosulfan, amitraz, tralometrine, acetoprol, etiprol, ethione, trichlorone, metamidofos, metamidofos, dichlorometho, dichlorometho, dichloro, formetanate, formothionate, mecarbam, thiometone, nalede, methyl parathion, cyanophos, diamidafos, albendazole, oxybendazole, fembendazole, oxfendazole, propafos, sulprofos, protiofos, profofenates, isofeninfos, temfosphosphosphos, fentoxide, fentoxide, fentoxide, tofo, , chlorpyrifos, pyridafentiona, fosalone, fosmete, dioxabenzofos, quinalfos, pyrethrin, aletrine, pralethrin, resmethrin, permethrin, teflutrin, fempropatrin, alpha-cypermethrin, lambda-cyhalothrin, delta-metrine, fenvalerate, fluvitrinate, fluvitrine, fluvitrine, fluvitrine, fluvitrinate, fluvitrinate , aldicarb, alanicarb, metolcarb, xylylcarb, propoxur, phenoxycarb, phenothiocarb, biphenazate, carbofuran, carbosulfan, sulfur, p irifluquinazone, furatiocarb, diafentiurone, diflubenzurone, hexaflumurone, novalurone, lufenurone, chlorfluazurone, tricyclohexylthine hydroxide, sodium oleate, potassium oleate, metoprene, hydroprene, binapacril, chlorobenzenedate, chlorobenzylate, tetraobenzylate, phenobromide, benzobenzylate, diophenolane, tolfempirade, triazamate, nicotine sulfate, thiaclopride, thiamethoxam, clothianidin, dinotefuran, fluazinam, pyriproxyphene, fluacripyrim, hydramethylnone, cyromazine, TPIC, thiocyam, phenazaquin, hydroxyphenone, hydroquinone, polynactin, hydroxyamine, hydroxyamine, hydroxyaminone aldoxicarb, metam sodium, morantel tartrate, dazomete, levamisole hydrochloride, triclamide, pyridalyl, chlorantraniliprol, cyienopyraphene and cyflumetophene, but not limited to these.
    [00180] The plant disease control agent, nematode control agent and plant growth promoter of the present invention can be directly applied since they are or applied as a solution diluted with water and the like. The method of application of the plant disease control agent, nematode control agent and plant growth promoter of the present invention is not particularly limited and examples thereof include a method of spraying the agent directly onto plants and insect pests. , a method of spraying the agent on the soil, a method of adding the agent to the water and fertilizer to be applied to plants and the soil, and a method of coating the seeds with the agent. In addition, it is desirable to adequately adjust the amount of application of the drug product since the amount of application varies depending on the disease and the insect pest to be controlled, the crops as the object of application, the method of application, tendency of occurrence diseases, degree of damage, environmental conditions and formulations to be used.
    [00181] As discussed above, strain AT-332 and strain AT-79 of the present invention have a wide disease and nematicidal spectrum and can control various types of plant diseases and nematodes, and can promote plant growth. Since the plant disease control agent, nematode control agent and plant growth promoter of the present invention comprising these strains are highly safe for the environment and have control effects on various types of diseases and nematodes, the Plant disease control can prevent a wide range of diseases and nematodes without using other means in combination and can be used as a biological pesticide and / or biological fertilizer that can promote the growth of useful plants as well. EXAMPLES
    [00182] The present invention will be described in more detail with the Production Example, Formulation Examples, Examples and Comparative Examples, however, the present invention is not limited to these examples. Culture of strain AT-332 and strain AT-79
    [00183] The AT-332 strain and the AT-79 strain were isolated from the soil containing plant roots.
    [00184] In detail, 1 g of a dry soil obtained by collecting the soil in Moriya City in Ibaraki Prefecture, Japan in August 2009 and undergoing treatment with heating (80 ° C, for 10 minutes) was suspended in sterile water. The suspension was diluted with the dilution rate of 102 to 104 times and the culture separated from the suspension was carried out in nutrient broth medium (Eiken Chemical Co., Ltd.) (28 ° C, for three days) and the colonies formed were isolated. The isolated colonies were grown on a potato dextrose agar medium and strains effective in relation to the pathogens of various plant diseases were found. The strains were also subjected to agitation culture in a liquid medium of potato dextrose, and the strain AT-332 and strain AT-79 of Bacillus sp. were isolated as a strain that has an activity against the second stage larva of Meloidogyne sp. sweet potato
    [00185] The method for identifying each of the strains, the various methods of analysis and their results, and bacteriological properties are those described in "Mode for Carrying Out the Invention". Production Example 1: Cultivation and preparation of the AT-332 strain
    [00186] As a preculture, a sediment of the preserved bacteria of the present invention (strain A-332) was inoculated in 60 ml per bottle of a nutrient broth medium (available from Eiken Chemical Co., Ltd.) in a bottle 500 ml conical with lid, and subjected to shaking culture using a rotary shaker at 180 rpm and 28 ° C for one day.
    [00187] 60 ml of the culture obtained by the previous pre-culture were inoculated in a 5000 ml jar fermenter containing 2,000 ml of LB medium (20 g of peptone, 10 g of yeast extract, 20 g of chloride of sodium and water for the rest) and grown as the main crop at 500 rpm, aeration rate of 1 l / hour and 35 ° C for three days.
    [00188] About 1,800 g of the culture was obtained by the previous main culture. The concentration of the bacterial cell was about 8.0 x 109 CFU / ml.
    [00189] About 140 g of the dry powder were obtained by freezing 1,800 g of the culture product obtained at -80 ° C, followed by freeze drying under reduced pressure and spraying. The bacterial cell concentration of the powder was about 1.0 x 1011 CFU / g. Production Example 2: Cultivation and preparation of the AT-79 strain
    [00190] As a pre-culture, a sediment of the preserved bacteria of the present invention (strain AT-79) was inoculated into 60 ml per flask of a nutrient broth medium (available from Eiken Chemical Co., Ltd.) in a flask 500 ml conical with buffer, and subjected to stirring culture using a rotary shaker at 180 rpm and 28 ° C for one day.
    [00191] 60 ml of the culture obtained by the previous preculture were inoculated in a 5000 ml jar fermenter containing 2,000 ml of LB medium (20 g of tryptone, 10 g of yeast extract, 20 g of chloride) sodium and water for the rest) and grown as the main crop at 500 rpm, aeration rate of 1 l / hour and 35 ° C for three days.
    [00192] About 1,700 g of culture was obtained by the previous main culture. The bacterial cell concentration of the powder was about 9.0 x 109 CFU / g.
    [00193] About 130 g of the dry powder were obtained by freezing 1,700 g of the culture product obtained at -80 ° C, followed by freeze drying under reduced pressure and spraying. The bacterial cell concentration of the powder was about 1.0 x 1011 CFU / g.
    [00194] Formulation examples are given below. Here, the word "part (s)" means a mass part (s). Formulation Example 1: Wetting Powder
    [00195] 60 parts of the dry powder obtained by the Formulation Example 1.25 parts of diatomaceous earth, 5 parts of white carbon, 8 parts of lignin sulfonate and 2 parts of alkyl naphthalene sulfonate were mixed and sprayed to obtain wetting powder. Formulation Example 2: Granular formulation
    [00196] 5 Parts of the dry powder obtained by the Formulation Example 1.25 parts of bentonite, 66 parts of talc, 2 parts of dodecylbenzene sulfonate and 2 parts of lignin sulfonate were mixed and pulverized. After adding about 20 parts of water to this and kneading the mixture by a kneading machine, the resultant was granulated by a granulator and dried, and then the size of the granules was adjusted to obtain a granular formulation. Formulation Example 3: Wetting Powder
    [00197] 60 Parts of the dry powder obtained by the Formulation Example 2.25 parts of diatomaceous earth, 5 parts of white carbon, 8 parts of lignin sulfonate and 2 parts of alkyl naphthalene sulfonate were mixed and sprayed to thereby obtain the wetting powder. Formulation Example 4: Granular formulation
    [00198] 5 Parts of the dry powder obtained by the Formulation Example 2.25 parts of bentonite, 66 parts of talc, 2 parts of dodecylbenzene sulfonate and 2 parts of lignin sulfonate were mixed and pulverized. After adding about 20 parts of water to this and kneading the mixture by a kneading machine, the resultant was granulated by a granulator and dried, and then the size of the granules was adjusted to obtain a granular formulation.
    [00199] Next, Examples and Comparative Examples to test the effects of the plant disease control agent, nematode control agent and plant growth promoter of the present invention are described below. Example 1 and Comparative Example 1: Test for the effects in relation to the rice explosion
    [00200] Sufficient doses of the wetting powders diluted in Formulation Examples 1 and 3 with the 250-fold dilution rate were sprayed with a spray gun on the rice (variety: Koshihikari, 15 plants per hill) grown in a greenhouse for the stage of unfolding the third sheet in a plastic pot 6 cm in diameter. As a comparative example, Impression wettable powder (produced by SDS Biotech KK) with a 250-fold dilution rate was similarly tested in the same way. The following day, the spore suspension of the rice blast pathogen (Pyricularia oryzae) was sprayed and inoculated. After keeping the pots in a humid environment at 22 ° C for 24 hours, the pots were allowed to remain in the greenhouse for seven days and the number of lesions on the inoculated leaves was investigated to thereby determine the preventive value. The preventive value (%) was calculated based on the number of leaf lesions in the untreated region. As can be seen from the results shown in Table 1, by treatment with the microbiological agent of the present invention, the incidence of the rice explosion was very low compared to the untreated region, and significantly high control effects were obtained. Table 1
    Example 2 and Comparative Example 2: Test for anthracnose effects of cucumber
    [00201] Sufficient doses of the wetting powders in Formulation Examples 1 and 3 with a 250-fold dilution rate were sprayed on the first and second cucumber leaves (variety: Tokiwa Hikari No. 3 type p) grown in an oven for the stage of unfolding the third leaf in a plastic pot of 6 cm in diameter. As a comparative example, the Impression wettable powder diluent (produced by SDS Biotech KK) with the 250-fold dilution rate was similarly tested in the same way. The following day, the spore suspension of Colletorichum lagenarium from the cucumber was sprayed and inoculated. After keeping the pots in a humid environment at 22 ° C for 24 hours, the pots were allowed to remain in the greenhouse for seven days and the rate of the diseased area on the first and second leaves was investigated with eyes to thereby determine the preventive value. The preventive value (%) was calculated based on the rate of the diseased area in the untreated region. As can be seen from the results in Table 2, by the treatment with the microbiological agent of the present invention, the incidence of Colletorichum lagenarium from cucumber was very low compared to the untreated region, and significantly high control effects were obtained. two
    Example 3 and Comparative Example 3: Test for the effects on Phytophthora infestans on tomatoes
    [00202] Sufficient doses of wettable powders in Formulation Examples 1 and 3 with a 250-fold dilution rate were sprayed on tomatoes (variety: Sugar block) grown in a greenhouse for the fifth stage of deployment leaf in a plastic pot of 6 cm in diameter. As a comparative example, the Impression wettable powder diluent (produced by SDS Biotech KK) with the 250-fold dilution rate was similarly tested in the same way. The following day, the suspension of the phytophthora infestans zoospores in the tomato was sprayed and inoculated. After keeping the pots in a humid environment at 22 ° C for 16 hours, the pots were allowed to remain in the greenhouse for three days and the rate of the diseased area on the third, fourth and fifth leaves was investigated with eyes to thereby determine the preventive value. The preventive value (%) was calculated based on the rate of the diseased area in the untreated region. As can be seen from the results in Table 3, by treatment with the microbiological agent of the present invention, the incidence of Phytophthora infestans in tomatoes was very low compared to the untreated region, and significantly high control effects were obtained. Table 3
    Example 4 and Comparative Example 4: Test for the effects on Pseudoperonospora cubensis on cucumber
    [00203] Sufficient doses of the wettable powders in Formulation Examples 1 and 3 with the 250-fold dilution rate were sprayed on a cucumber spray (variety: Hikari No. 3 type p) grown in a greenhouse for the unfolding of the third leaf in a plastic pot of 6 cm in diameter. As a comparative example, the Impression wettable powder diluent (produced by SDS Biotech KK) with the 250-fold dilution rate was similarly tested in the same way. The following day, the zoospore suspension of Pseudoperonospora cubensis in the cucumber was sprayed and inoculated. After keeping the pots in a humidity environment at 22 ° C for 18 hours, the pots were allowed to remain in the greenhouse for three days and the rate of the diseased area on the first and second leaves was investigated with eyes to thereby determine the value preventive. The preventive value (%) was calculated based on the rate of the diseased area in the untreated region. As can be seen from the results in Table 4, by treatment with the microbiological agent of the present invention, the incidence of Pseudoperonospora cubensis in the cucumber was very low compared to the untreated region, and significantly high control effects were obtained. Table 4
    Example 5 and Comparative Example 5: Test for the effects on AltenariaAlternaria mali on apple
    [00204] Apple leaves (variety: Orin) were collected and sufficient doses of wettable powders in Formulation Examples 1 and 3 at the 250-fold dilution rate were sprayed by a spray gun on the back side of the leaves . As a comparative example, the Impression wettable powder diluent (produced by SDS Biotech KK) with the 250-fold dilution rate was similarly tested in the same way. After spraying, the leaves were air-dried and the spore suspension of Altenaria Alternaria mali on the apple was also sprayed and inoculated. After the leaves were left to stand at 20 ° C in a humid condition for four days, the rate of the diseased area was investigated with eyes to thereby determine the preventive value. The preventive value (%) was calculated based on the rate of the diseased area in the untreated region. As can be seen from the results in Table 5, by treatment with the microbiological agent of the present invention, the incidence of Altenaria Alternaria mali in the apple was very low compared to the untreated region, and significantly high control effects were obtained. Table 5
    Example 6 and Comparative Example 6: Test for effects on cucumber Sphaerotheca fuliginea (Field test)
    [00205] The test was carried out in the greenhouse owned by the company using cucumbers (test region: 4 m2 / region; 10 plants / region; in triplicate). The disease was allowed to occur naturally. The wettable powder in Formulation Examples 1 and 3 with a 500-fold dilution rate, was sprayed 1,000 times and 2,000 times four times at seven-day intervals and the preventive value (%) was calculated from the disease area rate on the leaves. Impression wettable powder (SDS Biotech KK) with 500 times and 1,000 times dilution rate, Botokiller wettable powder (Idemitsu Kosan Co., Ltd.) with 1,000 times dilution rate, Botopika wettable powder (Idemitsu Kosan Co., Ltd.) with a 2000-fold dilution rate, Ecoshot water-dispersible granule (Kumiai Chemical Industry Co., Ltd.) with a 1,000-fold dilution rate and Morestan wetting powder (Agro-Kanesho Co ., Ltd.) with the 3,000-fold dilution rate were used as a comparative agent. The incidence of the disease in the untreated region was 47.4%. The preventive value (%) was calculated based on the incidence in the untreated region. As can be seen from the results in Table 6, by treatment with the microbiological agent of the present invention, the incidence of Sphaerotheca fuliginea from cucumber was greatly reduced compared to the untreated region, and significantly high control effects were obtained. A notably higher effect was well confirmed in the field when compared to conventional commercially available Bacillus subtilis agents (Impression wettable powder (Patent Document 3), Botokiller wettable powder, Botopica wettable powder, Ecoshot water dispersible granule (Document Patent Application 4)) used as a comparative agent. The microbiological agent of the present invention with a 500-fold dilution rate showed a very high effect equivalent to the wetting powder of Morestan which is a chemical agent. Table 6

    Example 7 and Comparative Example 7: Test for effects on eggplant Botrytis cinerea (Field test)
    [00206] The test was carried out in the greenhouse owned by the company using eggplants (test region: 5.6 m2 / region; 7 plants / region; in triplicate). The disease was allowed to occur naturally. The wettable powder in Formulation Examples 1 and 3 with a dilution rate of 500 times and 1,000 times was sprayed four times at intervals of seven days and the preventive value (%) was calculated from the incidence on the fruits. Impression wettable powder (SDS Biotech KK) with 500 times and 1,000 times dilution rate, Botokiller wettable powder (Idemitsu Kosan Co., Ltd.) with 1,000 times dilution rate, Botopika wettable powder (Idemitsu Kosan Co., Ltd.) with the 2,000-fold dilution rate, Ecoshot water-dispersible granule (Kumiai Chemical Industry Co., Ltd.) with the 1,000-fold dilution rate and Savior Flowable 20 (Syngenta Japan KK) with the 1,500-fold dilution rate was used as a comparative agent. The incidence of the disease in the untreated region was 15%. The preventive value (%) was calculated based on the incidence in the untreated region. As can be seen from the results in Table 7, by treatment with the microbiological agent of the present invention, the incidence of Botrytis cinerea was very low compared to the untreated region, and significantly high control effects were obtained. A notably higher effect was well confirmed in the field when compared to conventional commercially available Bacillus subtilis agents (Impression wettable powder, Botokiller wettable powder, Botopica wettable powder, Ecoshot water dispersible granule) used as a comparative agent. The microbiological agent of the present invention with a 500-fold dilution rate showed a very high equivalent effect for Savior Flowable 20 which is a chemical agent. Table 7
    Example 8 and Comparative Example 8: Test for effects on Burkholderia plantarii
    [00207] The seeded rice (variety: Koshihikari) was immersed to be inoculated into the suspension of Burkholderia plantarii (1 x 108 CFU / ml), which was obtained by stirring culture in liquid PD at 27 ° C for 52 hours for one hour under reduced pressure to prepare the seeds infected with Burkholderia plantarii. The seeds infected with Burkholderia plantarii were immersed in the wettable powder solution of Formulation Example 1 and Formulation Example 3 with a 100-fold dilution rate. After the solution was removed, the seeds were kept in an environment with humidity of 32 ° C for one day to stimulate germination. As a comparative agent, the Impression solution (SDS Biotech KK) with the 100-fold dilution rate was similarly tested in the same way. The seeds stimulated by germination were sown in a plastic cup with a diameter of 6 cm filled with soil for cultivation. The seedlings were kept in an environment to suspend the seedlings at 30 ° C for three days after sowing and in a humidity environment at 25 ° C for 15 days. Then, all seedlings were investigated for the presence of the disease to determine the rate of sick seedling. The preventive value (%) was calculated based on the rate of sick seedling in the untreated region. The amount of sowing per cup was 3 g of dry seed rice (90 to 110 grains). As can be seen from the results in Table 8, by treatment with the microbiological agent of the present invention, the rate of diseased seedlings of Burkholderia plantarii was very low compared to the untreated region, and significantly high control effects were obtained. Table 8
    Example 9 and Comparative Example 9: Test for the effect on Rhizoctonia solani.
    [00208] 3 g of the Rhizoctonia solani culture product in a wheat bran medium were mixed in 500 ml of sterile soil to be filled in a plastic pot, and 1 g of the granular formulation of Formulation Example 2 and Formulation Example 4 was mixed in the soil, respectively. As a comparative agent, 84 mg of Impression wettable powder were similarly tested in the same way. Cucumbers (variety: Sagami-hanjiro) were sown and after growing the cucumbers at 23 ° C for one week, the germination rate was investigated. The control effect (% of the control title) was calculated based on the rate of diseased seeding in the untreated region. As can be seen from the results in Table 9, by treatment with the microbiological agent of the present invention, the rate of Rhizoctonia solani diseased sowing was greatly reduced compared to the untreated region, and significantly high control effects were obtained. Table 9
    Example 10 and Comparative Example 10: Activity against the second-stage larva of Meloidogyne sp. sweet potato
    [00209] Nematicidal activity in relation to the second-stage larva of Meloidogyne sp. of sweet potato hatched within 24 hours of the egg capsule collected from the eggplant roots (variety: Juryo). Each of the solutions of Formulation 1 and Formulation 3 with a 100-fold dilution rate (a Tween 20 solution with a 5,000-fold dilution rate) and an equivalent amount of Meloidogyne sp. sweet potato (approximately 50 earthworms) was added to a 24-hole microplate. As a comparative agent, the Impression solution (SDS Bioteck KK) with the 100-fold dilution rate was similarly tested in the same way. The plate was sealed and placed in an incubator at 28 ° C and a relative humidity of about 50%. After 72 hours, the mortality rate was investigated by observation using a stereoscopic microscope. At that time, immobile nematodes were considered to be dead. The nematicidal rate was calculated according to the expression described below. As can be seen from the results in Table 10, by treatment with the microbiological agent of the present invention, an extremely high nematicidal activity was obtained in relation to the second-stage larva of Meloidogyne sp. sweet potato. Expression 1 Nematicidal rate = (number of killed nematodes / number of tested nematodes) x 100 Table 10
    Example 11 and Comparative Example 11 (Test for the control effect against sweet potato Meloidogyne sp.
    [00210] In a 1 / 10,000 a-Wagner pot, each of the granular formulation of Formulation Example 2 and Formulation Example 4 was uniformly mixed in the soil infected with Meloidogyne sp. sweet potato at a rate of 40 kg / 10 and a small tomato (variety: Sugar block) was planted in this. As a comparative agent, Impression wettable powder (SDS Biotech K.K.) was similarly tested in the same manner at a rate of 3.3 kg / 10 a. One month after planting, the degree of damage to the roots (degree of root nodule) was classified and evaluated according to the criteria described below. The root nodule index was determined according to the expression as below to calculate the control titer. As can be seen from the results in Table 11, by treatment with the microbiological agent of the present invention, root damage caused by Meloidogyne sp. sweet potato were very low compared to the untreated region, and significantly high control effects were obtained.
    [00211] Degree of damage 0: No root nodule was observed. 1: The root nodules are hardly noticeable at first glance, however, some can be found. 2: Some root nodules are observed. 3: Moderate amount of root nodules is observed. 4: Several root nodules are observed throughout the rhizosphere. Expression 2 Root nodule index = (S (degree of damage x number of units) / the entire population investigated x 4) x 100 Preventive value = (1 - Root nodule index in the treated region / Root nodule index in the untreated region) x 100 Table 11
    Example 12 and Comparative Examples 12 to 13: Effect of promoting plant growth of strain AT-332 (basic test)
    [00212] A basic petri dish test was performed with respect to Arabidopsis thaliana to measure the effect of promoting plant growth of strain A-332. After immersing the Arabidopsis thaliana seeds in 1% sodium hypochlorite for 20 minutes, the seeds are immersed in a 70% ethanol solution for two minutes to sterilize the seed surface. After that, the seeds were washed with sterile distilled water to be used for the test. A Murashige and Skoog salt medium (pH 5.7) containing 0.8% agar was poured into a sterile dual-split petri dish and used for a test after being cooled.
    [00213] AT-332 (Example 12), Bacillus subtilis GB03 (Comparative Example 12) and Bacillus subtilis MBI600 (Comparative Example 13) were inoculated respectively on a sterile paper disk placed in one of the split portions of the petri dish above, and the sprouted seeds of Arabidopsis thaliana were inoculated in the other portion of the petri dish. The plate inoculated with the bacteria and Arabidopsis thaliana was kept at 22 ° C (12 hours in the light / 12 hours being cut from the light) for ten days and the growth status of the plant was observed. The results are shown in photographs (a) to (d) in Figure 2 including the results of the control (Figure 2 (a)) where the bacteria were not inoculated. A notable effect of promoting plant growth was confirmed with AT-332 (Example 12; (b)) compared to Bacillus subtilis GB03 (Comparative Example 12; photo (c)) and Bacillus subtilis MBI600 (Comparative Example 13; photo (d)), which are actually sold and used on the United States market. Example 13: Effect of promoting plant growth of strains AT-332 and AT-79 (pot test)
    [00214] A pot test was carried out with respect to the Chinese cabbage seedlings to measure the plant growth promoting effect of the AT-332 and AT-79 strains. After cultivating the AT-332 and AT-79 strains in a liquid LB medium for 24 hours, the bacterial cells were collected by centrifugation. The bacteria cells collected were suspended in a 0.85% aqueous solution of sodium chloride to be contained in a concentration of 1 x 109 CFU / ml. 40 ml of the suspension was mixed with 1 kg of the previously sterilized culture soil to serve as the treated soil. On the other hand, 40 ml of 0.85% sodium chloride aqueous solution were mixed with 1 kg of the previously sterilized culture soil to serve as the untreated soil. 100 g of each treated soil and untreated soil were placed in a plastic pot (70 mm in diameter x 68 mm in height) respectively, and the Chinese cabbage seeds (variety: Nozaki Chinese Cabbage No. 2) were sown in the pot . Subsequently, the pots were placed in an oven maintained at 22 ° C and the fresh weight of the grown Chinese cabbage was measured after 30 days. The results are shown in Figure 3. An explicit effect of promoting culture growth of the AT-332 and AT-79 strains has been confirmed.
    权利要求:
    Claims (8)
    [0001]
    1. Bacillus sp. AT-332, characterized by the fact that it has the accession number NITE BP-1095 and contains 16S rDNA defined by SEQ ID NO: 2, and in which the bacteria is stabilized in a dry form and with white carbon.
    [0002]
    2. Bacteria from the strain of Bacillus sp. AT-79, characterized by the fact that it has the accession number NITE BP-1094 and contains 16S rDNA defined by SEQ ID NO: 3, and in which the bacteria is stabilized in a dry form and with white carbon.
    [0003]
    3. Bacteria according to claim 1 or 2, characterized by the fact that the bacterium per se shows an effect of controlling plant diseases, controlling nematodes and / or promoting plant growth.
    [0004]
    4. Microbiological agent, characterized by the fact that it contains: bacteria selected from the group consisting of: a) Bacillus sp. strain AT-332, in which it has the accession number NITE BP-1095 and contains 16S rDNA defined by SEQ ID NO: 2; and (b) Bacillus sp. strain AT-79, in which it has the accession number NITE BP-1094 and contains 16S rDNA defined by SEQ ID NO: 3; and / or culture of said bacterium; and an inert liquid or a solid vehicle: white carbon; and water.
    [0005]
    5. Microbiological agent according to claim 4, characterized by the fact that it is a plant disease control agent.
    [0006]
    6. Microbiological agent, according to claim 4, characterized by the fact that it is a nematode control agent.
    [0007]
    7. Microbiological agent, according to claim 4, characterized by the fact that it is a plant growth promoter.
    [0008]
    8. Method for growing the plants, characterized in that it consists of treating the plants with the microbiological agent as defined in any of claims 4 to 7, wherein the agent contains water as an inert liquid and has the bacteria in a concentration from 105 to 1010 units / ml.
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    同族专利:
    公开号 | 公开日
    US10219517B2|2019-03-05|
    WO2012161160A1|2012-11-29|
    NZ617974A|2015-11-27|
    US9504257B2|2016-11-29|
    CA2836726A1|2012-11-29|
    US20140179528A1|2014-06-26|
    PT2716748T|2017-03-21|
    EP2716748A1|2014-04-09|
    EP2716748A4|2014-10-22|
    CA2836726C|2019-10-22|
    CN103703120B|2015-04-15|
    BR112013030228A2|2016-12-13|
    IL229514A|2019-03-31|
    EP2716748B1|2016-12-21|
    CN103703120A|2014-04-02|
    ES2616911T3|2017-06-14|
    CL2013003374A1|2014-10-03|
    ZA201308830B|2015-04-29|
    MX348159B|2017-05-31|
    IL229514D0|2014-01-30|
    US20170027177A1|2017-02-02|
    AU2012259893B2|2015-09-24|
    MX2013013640A|2014-04-25|
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    法律状态:
    2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-07-02| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|
    2020-02-11| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|
    2020-09-15| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|
    2021-02-02| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-03-16| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 21/05/2012, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    JPPCT/JP2011/062109|2011-05-26|
    JP2011062109|2011-05-26|
    PCT/JP2012/062935|WO2012161160A1|2011-05-26|2012-05-21|Strain belonging to bacillus genus, microbiological agent, and plant cultivation method|
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