TWI571511B - Strains belonging to bacillus, microbial formulation, and method for cultivation of plant - Google Patents

Description

Bacillus strain, microbial preparation and plant cultivation method

The present invention relates to novel microorganisms useful for controlling plant diseases, controlling nematodes, and promoting plant growth. More specifically, it relates to microorganisms belonging to Bacillus amyloliquefaciens which are close relatives disclosed in the literature, and exhibits more excellent plant disease control action, nematode control effect and/or plant growth promoting effect. a novel microorganism of the genus Bacillus sp. AT-332 and a strain of Bacillus sp. AT-79, and a plant disease control agent using the microorganisms and cultures of the microorganisms, Nematode control agent and plant growth promoter.

The main plant diseases and nematode control methods are chemical pesticides. Until now, chemical pesticides have made it possible to produce stable crops. However, in recent years, due to the continuous use of chemical pesticides, the environmental impacts and the emergence of drug-resistant bacteria have become difficult, and existing chemical pesticides have been difficult to prevent, and bacterial diseases and the like have become increasingly difficult. Therefore, regarding the control methods other than chemical pesticides, attention has been paid to the use of biological control techniques for microorganisms isolated from nature, and several microbial pesticides have been produced. However, existing microbial pesticides are When compared with chemical pesticides, it has the disadvantage of being unstable or applying less disease. Based on this situation, it is desired to develop a novel microbial pesticide having a new applicable disease and exhibiting a stable control effect.

For the use of microbial plant disease control agents, Talaromyces flavus, Pseudomonas fluorescens, Erwinia carotovora, Trichoderma viride A (Trichoderma atroviride) agent, a Bacillus simplex agent, a Bacillus subtilis agent, or the like is registered as a microbial pesticide.

As for the nematode control agent using microorganisms, a Pasteuria penetrans agent and a Monacrosporium phymatophagum agent have been registered as microbial pesticides.

In the specification of Japanese Patent No. 2955655 (Patent Document 1), a plant disease controlling agent using bacteria belonging to Bacillus amyloliquefaciens has been disclosed. The active ingredient of this plant disease control agent is the production of microorganisms, and the bacteria themselves are not used as pesticides. Further, the object to be controlled is a disease caused by a filamentous fungus, and there is no description of the prevention of a disease caused by bacteria. In Japanese Patent Laid-Open Publication No. 2009-247302 (Patent Document 2), it is disclosed that a filamentous fungal disease and a bacterial disease can be simultaneously prevented, and the microorganism body is effective as a microbial pesticide, but no case concerning a nematode is described. Control.

In the specification of Japanese Patent No. 3471815 (Patent Document 3, WO98/050422), it is disclosed that a plant disease controlling agent for a Bacillus bacterium which is useful for a wide range of plant diseases and is effective against corn rootworm is used. However, there is no record about the control of nematodes. Further, in Japanese Patent No. 4071036 (Patent Document 4, US 2004/265292), it is disclosed that it can be used for plant disease control and pest control. The Bacillus sp. D747 strain, but did not record the control of nematodes.

In the specification of Japanese Patent No. 3471811 (Patent Document 5, WO 96/032840), a nematode controlling agent using a bacterium of the genus Bacillus has been disclosed. The active ingredient of this nematode control agent is a bacterium or a spore of a strain of Bacillus firmus which has anti-nematode activity, but does not describe the control of plant diseases. Further, Japanese Patent No. 4,359,653 (Patent Document 6, WO1997/012980) discloses a method for controlling nematodes by a nematode-resistant toxin produced by a novel strain of Bacillus thuringiensis, but does not describe a plant. Disease prevention.

On the other hand, in agriculture, chemical fertilizers are important agricultural materials that affect crop yields, but 30 to 50% of the chemical fertilizer components used are not used by crops and spread to the environment, becoming the river's superiority. Reasons for water pollution of groundwater and groundwater. Moreover, the production of chemical fertilizers uses a large amount of fossil fuels, and the price increase of fossil fuels complements the cost of chemical fertilizers. Further, nitrogen oxides (NO _x ) which are nitrogen fertilizer decomposition products are considered to have a greenhouse effect of about 300 times that of carbon dioxide, and concerns about global warming have become a problem. In addition, it is expected that there will be a shortage of food in the future due to the increase in the world population, and the use of materials for improving crop productivity is indispensable, so the necessity of replacing existing chemical fertilizers with more environmentally friendly materials is necessary. improve.

In contrast to such a situation, research on the use of soil microorganisms to increase crop yields is mainly carried out on a wide range of Rhizobium bacteria (Rhizobium), Pseudomonas bacteria, and Bacillus bacteria, but because of its low effect, Therefore, it is rarely used.

As described above, no overall effect on plant diseases has been found, and it is also possible to use Bacillus bacteria which are resistant to nematode control and have a plant growth promoting effect.

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] Japanese Patent No. 2955655

[Patent Document 2] Japanese Patent Laid-Open Publication No. 2009-247302

[Patent Document 3] Japanese Patent No. 3471815

[Patent Document 4] Japanese Patent No. 4071036

[Patent Document 5] Japanese Patent No. 3471811

[Patent Document 6] Japanese Patent No. 4359653

It is an object of the present invention to provide a novel microorganism isolated from nature which has an effect of suppressing the onset of most plant diseases, nematode control, and/or growth promoting action of plants.

Another object of the present invention is to provide a plant disease control agent, a nematode control agent, and a plant growth promoter which are used as an effective bacteria and which can be used as a biological pesticide (microbial preparation).

The inventors of the present invention have succeeded in separating a new strain belonging to the genus Bacillus from the natural world in view of the above-mentioned problems, and have been effective in inhibiting the growth of most plant diseases, nematode control effects, and plant growth promoting effects. The present invention has been completed.

The present invention relates to the following strains 1 to 4, the microbial preparations of 5 to 8, and the cultivation methods of the plants of 9.

A strain characterized by having 16S rDNA represented by a nucleotide sequence of SEQ ID NO: 2 or 3.

2. The strain according to the above 1, wherein the culture of the strain body and/or the strain exhibits the effects of the plant disease control action, the nematode control effect, and/or the plant growth promoting action.

3. The strain according to the above 1 or 2 which is a strain of Bacillus sp. AT-332 and which has 16S rDNA represented by the nucleotide sequence of SEQ ID NO: 2.

4. The strain according to the above 1 or 2, which is a strain of Bacillus sp. AT-79, and which has 16S rDNA represented by the nucleotide sequence of SEQ ID NO: 3.

A microbial preparation containing a culture of the strain and/or strain according to any one of the above 1 to 4 as an active ingredient.

6. The microbial preparation according to the above 5, which is a plant disease control agent.

7. The microbial preparation according to the above 5, which is a nematode control agent.

8. The microbial preparation according to the above 5, which is a plant growth promoter.

A plant cultivation method, which comprises treating a plant with the microorganism preparation according to any one of the above 5 to 8.

The Bacillus sp. AT-332 strain and the AT-79 strain of the present invention are present in plants such as roots, stems, leaves, seeds, fruits, etc. by cultivating the culture (including the bacterium) or the culture-separating bacterium thereof. It can inhibit the occurrence of various plant diseases in a wide range, and can prevent nematodes, and can promote the growth of useful plants.

Fig. 1 shows a molecular phylogenetic tree using the 16S rDNA base sequence of the Bacillus sp. AT-332 strain and the AT-79 strain. In the figure, the number near the branch of the branch is the bootstrap, and the line at the bottom left represents the scale bar.

Figure 2 (a) shows the plant growth of the AT-332 strain in the basic test (no treatment). Long promotion effect photo.

Fig. 2(b) is a photograph showing the plant growth promoting effect of the AT-332 strain in the basic test (Example 12).

Fig. 2(c) is a photograph showing the plant growth promoting effect of the AT-332 strain in the basic test (Comparative Example 12).

Fig. 2(d) is a photograph showing the plant growth promoting effect of the AT-332 strain in the basic test (Comparative Example 13).

Fig. 3 is a graph showing the growth promoting effect of the AT-332 and AT-79 strains on the cabbage in the pot experiment (Example 13).

The inventors of the present invention have re-developed microbial pesticides and/or microbial fertilizers which have a very broad antibacterial spectrum and exhibit anti-nematode activity and have a plant growth promoting effect on various plant diseases, and are aimed at various plants from various plants. , soil, etc. screening microorganisms. As a result, a useful knowledge is obtained that the strains isolated from the soil taken in Ibaraki Prefecture, Japan, exhibit a wide range of antibacterial activity against various plant pathogenic bacteria, and also exhibit high nematicidal activity against nematodes, and have plants. Growth promotion effect.

It is understood from the bacteriological properties described later that the two newly isolated strains (AT-332 strain and AT-79 strain) are gram-positive bacilli which are motility and grow under aerobic conditions to form spores. Also, both the catalase reaction and the oxidase reaction were positive. Further, based on the results of identification of a base sequence of about 1500 bp on the 5'-end side of 16S rDNA, the two strains were identified as novel strains of Bacillus belonging to Bacillus amyloliquefaciens. Its performance "shows the effect on a wide range of plant diseases, and also shows a high control effect on nematodes, and The AT-332 strain and the AT-79 strain are regarded as novel strains, and are named as Bacillus species belonging to Bacillus amyloliquefaciens: AT-332. Strains and AT-79 strains.

The novel strain AT-332 strain and the AT-79 strain of the present invention are authorized institutions of Japan: the authorized microbiological center for the evaluation of technical institutions of the independent administrative legal person product (address: 292-0818, Kishozu, Chiba Prefecture, Japan) 2-5-8) Hosting with Bacillus sp. AT-332 and AT-79 (original storage day (trustee day): May 2, 2011, trustee number: NITE BP-1095 and NITE BP-1094). In addition, another application for biomaterials was filed in Taiwan, and experiments have been carried out to confirm survival. The Taiwan registered numbers of AT-332 strain and AT-79 strain are BCRC 910571 and BCRC 910570, respectively.

The bacteriological properties of Bacillus sp. AT-332 (registered number of Japan NITE BP-1095, registered number of Taiwan BCRC 910571) are as follows. Here, the bacteriological properties are determined by reference to the following documents.

PRIEST (FG), GOODFELLOW (M.), SHUTE (LA) and BERKELEY (RCW): Bacillus amyloliquefaciens sp. nov., nom. rev. Int. J. Syst. Bacteriol., 1987, 37, 69-71. Bergey's Manual of Systematic Bacteriology, Second Edition volume 3.

(1) Morphological properties

Morphology: Bacillus

Size: 0.8 to 0.9 μm wide and 1.5 to 2.0 μm long

Sportiness: +

Epiphytic state of flagella: Zhou Mao

The presence or absence of spores: + (quasi-end)

(2) Culture properties

Medium: nutrient agar medium (30 ° C)

Shape: round

Uplifted state: flat

Periphery: Whole

Surface shape: smooth

Viscosity: Viscosity

Transparency: opaque

Hue: cream

Gloss: dull

Pigment production: no production

(3) Physiological properties

Gram staining: +

Reduction of nitrate:-

Denitrification reaction:-

MR test:-

VP Quiz: +

吲哚 generation:-

Hydrogen sulfide generation:-

Hydrolysis of starch: +

Use of citric acid: -(Koser)+(Christensen)

Utilization of inorganic nitrogen sources: - (nitrate)

+(ammonium salt)

Urease:-

Oxidase: +

Catalase: +

Growth range pH5: + pH8: + pH9: +

Growth temperature 37 ° C: + 45 ° C: + 50 ° C: + 55 ° C: -

Growth in anaerobic conditions:-

OF test (oxidation / fermentation): -/-

Produce acid/gas from sugar:

L-arabinose: +/-

D-glucose: +/-

D-Fructose: +/-

Maltose: +/-

Lactose: -/-

D-sorbose (sorbitose): +/-

Inositol: +/-

D-xylose: +/-

D-mannose: +/-

D-galactose: -/-

Sucrose: +/-

Trehalose: +/-

D-mannitol: +/-

Glycerin: +/-

--galactosidase activity:-

Arginine dihydrolase activity:-

Amino acid decarboxylase activity:-

Tryptophan acid deaminase activity:-

Gelatinase activity: +

The bacteriological properties of Bacillus sp. AT-79 (registered number in Japan NITE BP-1094, registered in Taiwan as BCRC 910570) are as follows.

(1) Morphological properties

Morphology: Bacillus

Size: 0.8 to 0.9 μm wide and 1.5 to 2.0 μm long

Sportiness: +

Epiphytic state of flagella: Zhou Mao

The presence or absence of spores: + (quasi-end)

(2) Culture properties

Medium: nutrient agar medium (30 ° C)

Shape: round

Uplifted state: flat

Periphery: Whole

Surface shape: smooth

Viscosity: Viscosity

Transparency: opaque

Hue: cream

Gloss: dull

Pigment production: no production

(3) Physiological properties

Gram staining: +

Reduction of nitrate:-

Denitrification reaction:-

MR test:-

VP Quiz: +

吲哚 generation:-

Hydrogen sulfide generation:-

Hydrolysis of starch: +

Use of citric acid: -(Koser)+(Christensen)

Utilization of inorganic nitrogen sources: - (nitrate) + (ammonium salt)

Urease:-

Oxidase: +

Catalase: +

Growth range pH5: + pH8: + pH9: +

Growth temperature 37 ° C: +

45°C: +

50 ° C: +

55°C:-

Growth in anaerobic conditions:-

OF test (oxidation / fermentation): -/-

Produce acid/gas from sugar:

L-arabinose: +/-

D-glucose: +/-

D-Fructose: +/-

Maltose: +/-

Lactose: -/-

D-sorbose: +/-

Inositol: +/-

D-xylose: +/-

D-mannose: +/-

D-galactose: -/-

Sucrose: +/-

Trehalose: +/-

D-mannitol: +/-

Glycerin: +/-

--galactosidase activity:-

Arginine dihydrolase activity:-

Amino acid decarboxylase activity:-

Tryptophan acid deaminase activity:-

Gelatinase activity: +

The base sequence of the 16S rDNA on the 5' end side of the Bacillus sp. AT-332 strain of the present invention is shown in SEQ ID NO: 2, and the base sequence of the 16S rDNA on the 5' end side of the Bacillus sp. AT-79 strain. As shown in the serial number 3.

The sequence numbers 2 and 3 are different only in the bases at bases 444 and 1242. The base number 444 is in the sequence number 2 is guanine (g), the sequence number 3 is adenine (a); the number 1242 base is in the sequence number 2 is adenine (a), and the sequence number 3 is Guanine (g).

Therefore, the microorganism of the present invention is the 5' end side 16S of the nucleotide sequence having the sequence number 1 including the sequence number 2 and the sequence number 3 described above (that is, the bases of the numbers 444 and 1242 are shown as r). rDNA, and this can be considered a feature.

In the present invention, the analysis of the 16S rDNA base sequence is carried out by the following method.

DNA extraction was carried out using InstaGene Matrix (manufactured by BIO RAD, Inc., California (CA), USA), PCR was carried out using PrimeSTAR HS DNA Polymerase (manufactured by Takara Bio Co., Ltd.), and cycle sequencing was performed using BigDye Terminator v3. .1 Cycle Sequencing Kit (Applied Biosystems, Inc., California (CA), USA). The primer used (Nakagawa Kyoho et al.: The base sequence determination method of the 16S rRNA gene by the gene analysis method, the Japanese Society of Radiation Fungi, the classification and identification of the radiobacteria, 88-117pp. Japan Society for Academic Affairs, 2001) It is 9F, 339F, 785F, 1099F, 536R, 802R, 1242R and 1541R. The sequencing was carried out using an ABI PRISM 3100 Genetic Analyzer System (Applied Biosystems, Inc., California (CA), USA).

Use BLAST (ALTSCHUL, (S.F.) et al., etc.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acid Res. 1997.25, 3389-3402) searches for the homology of the international base sequence database (GenBank/DDBJ/EMBL), and the result is AT-332. The 16S rDNA base sequence of the strain and the AT-79 strain showed high homology to the 16S rDNA derived from Bacillus, and the 16S rDNA of the Bacillus amyloliquefaciens BCRC11601 strain showed a homology rate of 99.9%. The highest homology. On the other hand, the homology of the international base sequence database (GenBank/DDBJ/EMBL) was searched, and as a result, the AT-332 strain and the AT-79 strain did not detect the 16S rDNA base sequence derived from the genus Bacillus. A completely identical 16S rDNA base sequence.

In the present invention, molecular system analysis is carried out in the following manner.

The 16S rDNA of the reference strain derived from the putative close relatives was obtained from the international base sequence library (GenBank/DDBJ/EMBL) using the nucleotide sequence of the 16S rDNA obtained above, and the molecular phylogenetic tree analysis was performed.

The source strain of 16S rDNA used to estimate the molecular phylogenetic tree is as follows.

. Bacillus subtilis IAM12118T (AB042061)

. Bacillus subtilis subsp.spi zizenii NBRC101239T (AB325584)

. Bacillus mojavensis IFO15718 T (AB021191)

. Bacillus vallismortis DSM11031 T (AB021198)

. Bacillus amyloliquefaciens BCRC11601 T (EE433406)

. Bacillus atrophaeus J CM9070 T (AB021181)

. Bacillus aerophilus 28K T (AJ831844)

. Bacillus sonorensis BCRC17416 T (EF433411)

. Bacillus licheniformis DSM13 T (AE017333)

. Bacillus altitudinis 41KF2b T (AJ831842)

. Bacillus cereus ATCC14579 T (NC_004722) BSL2

The T at the end of the plant name is the reference strain indicating the variety. BSL indicates a biosafety level (marker level 2 or higher). The registration number is indicated in parentheses.

The resulting molecular phylogenetic tree is shown in Figure 1.

The number near the branch of the branch is the bootstrap, and the line at the bottom left represents the scale bar.

Since the AT-332 strain and the AT-79 strain do not have the property of reducing nitrate as described above, the bacteriological properties as described in the Bergey's Manual are not completely identical to those of the liquefied Bacillus licheniformis. In addition, although the results of 16S rDNA analysis can be considered that AT-332 strain and AT-79 strain are close relatives of Bacillus amyloliquefaciens, but can not be determined as liquefied Bacillus amyloliquefaciens, it is determined that AT-332 strain and AT-79 strain are Bacillus A new strain of the genus.

The Bacillus sp. AT-332 strain and the AT-79 strain of the present invention can be cultured by a known means such as static culture or liquid culture on a solid medium, and the types and culture conditions of the available medium, etc. There is no particular limitation as long as it is a bacterium that can survive the bacterium. For example, a general medium such as a meat extract medium may be mentioned, and examples thereof include a medium containing glucose, peptone, and yeast extract. Also, in addition to the liquid medium, A solid medium such as a slant medium to which an amaranth is added and a plate medium can also be used.

As far as the carbon source of the medium is concerned, all the carbon sources which can be used as a nutrient source can be utilized. Specific examples thereof include sugars such as glucose, galactose, lactose, sucrose, maltose, malt extract, molasses, glycoside, and starch hydrolysate, and examples thereof include AT-332 strain and AT-79 strain. A variety of synthetic or natural carbon sources that can be utilized.

As for the nitrogen source of the culture medium, the organic nitrogen-containing substance such as peptone, meat extract, yeast extract, soybean meal, or corn steep liquor is similarly used, and the strain can be utilized. A variety of synthetic or natural products.

Further, according to the conventional method of microbial culture, inorganic salts such as salt and phosphate, salts of metals such as calcium, magnesium, and iron, and micronutrient sources such as vitamins and amino acids may be added as necessary.

The culture can be carried out under the conditions of aerobic conditions such as shaking culture and aeration culture. The culture temperature is 20 to 40 ° C, preferably 25 to 35 ° C; the pH is 5 to 8, ideally 6 to 7; and the culture period is 1 to 4 days, preferably 2 to 3 days.

The culture containing the Bacillus sp. AT-332 strain and the AT-79 strain of the present invention has a property of suppressing various plant diseases, controlling nematodes, and promoting the growth of useful plants.

a cultured product obtained by mixing a culture containing the Bacillus sp. AT-332 strain and the AT-79 strain of the present invention with a strain of the AT-79 strain, a mixture of the culture and other components, or the cultured body after centrifugation Plants such as roots, stems, leaves, seeds, fruits, etc., which are cultured and isolated, such as a cultured cell, a mixture of cultured and isolated cells and other components, and a diluted substance diluted with a liquid or a solid are present. It can inhibit various plant diseases and prevent nematodes in the soil or its cultivated soil.

Regarding the Bacillus sp. AT-332 strain and the AT-79 strain of the present invention, only To be able to survive, whether it is the state of vegetative cells, the state of spores, or the state of coexistence, can be used as a plant disease control agent, nematode control agent and plant growth promoter. In addition, it can be used in a state in which the medium components are mixed during the culture, or in a state in which the components other than the bacterial cells are removed by washing with distilled water or the like.

The Bacillus sp. AT-332 strain and the AT-79 strain of the present invention can prevent Oomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes from being administered according to the application form. Plant diseases caused by fungi and bacteria, as well as Ditylenchus dipsaci, Ditylenchus destructor, Pratylenchus sp., Meloidogyne sp., and cystic nematodes Plant parasitic nematodes such as Heterodera sp. and Globodera spp. At the same time, it can promote the growth of grains, vegetables, fruits, flowers and beans.

The pathogenic bacteria of the Bacillus sp. AT-332 strain and the AT-79 strain which can be prevented by the present invention include, for example, rice Pyrethia oryzae, Cochliobolus miyabeanus, and Rhizoctonia solani, Gibberella fujikuroi; wheat powdery mildew (Erysiphe graminis f.sp. hordei, wheat white powdery mildew (Erysiphe graminis f.sp. Tritici)), Rust fungus (Puccinia striiformis, Puccinia graminis, Puccinia recondita f.sp.tritici, Puccinia hordei), Gibberella zeae, net Pyrenophora teres, snow rot (Typhula incarnata), Typhula ishikariensis, Sclerotinia borealis, Micronectriella nivalis )), naked black 穂 Ustilago nuda, T. sphaeroides (Tilletia caries, Tilletia foetida), Tapesia yallundae, Rhynchosporium secalis f.sp .hordei), Septoria tritici, Leptosphaeria nodorum; Diaporthe citri, Elsinoe fawcettii, Phytophthora citrophthora, Penicillium Digitatum), Penicillium italicum; apple mulberry mali, Valsa ceratosperma, podosphaera leucotricha, Alternaria alternata apple pathotype, Ventoa Inaequalis), Gymnosporangium yamadae, Botryophaeria berengeriana f.sp.piricola, Zygophiala jamaicensis, Gloeodes pomigena, Mycosphaerella pomi, anthracnose Glomerella cingulata), Diplocarpon mali; Pit's black spot disease (Venturia na Shicola), Alternaria alternata japanese pear pathotype, Physalospora piricola, Gymnosporangium asiaticum; Monilinia fructicola, Cladosporium carpophilum, fruit rot (Phomopsis sp.); Pseudocercospora vitis, Marssonina viticola, Elsinoe ampelina, Glomerella cingulata, Uncinula necator, rust fungus Phakopsora ampelopsidis), Phomopsis sp.; Phyllactinia kakicola, Colletotrichum gloeosporioides, Cercospora kaki, Mycosphaerella nawae; Cladosporium carpophilum; gray star disease of cherry Monilinia fructicola; Sphaerotheca fuliginea, Didymella bryoniae, Colletototrichum lagenarium; Tomato Alternaria solani, Cladosporium fulvum; Eggplant Phomopsis vexans, Erysiphe cichoracearum; Alternaria japonica, Alternaria brassicae, Alternaria alternata (Alternaria brassicicola)), Cercosporella brassicae; Puccinia allii; Pythium ultimum, Pythium zingiberis; Strawberry Sphaerotheca humuli, Glomerella cingulata Soybean (Cercospora kikuchii), Elsinoe glycines, Diaporthe phaseolorum var.sojae; Cercospora canescens, Uromyces phaseoli var.azukicola; Kidney Bean Anthracnose (Colletotrichum lindemuthianum); black rot fungus of groundnut (Cerc Osporidium personatum), Cercospora arachidicola, Sphaceloma arachidis; Erysiphe pisi of pea; Alternaria solani of potato; Exobasidium reticulatum, white star pathogen (Elsinoe leucospila), Pestalotiopsis theae (Pestalotiopsis longiseta); Tobacco Alternaria longipes, Erysiphe cichoracearum, Colletotrichum gloeosporioides; Beet's brown spot pathogen (Cercospora beticola); turf Curvularia geniculata, Ceratobasidium spp.; Diplocarpon rosae, Sphaerotheca pannosa; Septoria obesa, white rust Puccinia horiana; Botrytis cinerea, Sclerotinia sclerotiorum, and the like of various crops; however, it is not limited thereto.

The plant disease controlling agent of the present invention also contains a post-harvest disease controlling agent for preventing spoilage of preserved crops after harvesting, particularly for preventing spoilage of fruits and the like. The type of the crop to which the post-harvest disease preventing agent of the present invention is applied is not limited, and examples thereof include strawberries, grapes, figs, citrus, peach, cantaloupe, watermelon, apple, pear, banana, pineapple, etc., cucumber, Tomatoes, cabbage, cabbage, onions, onions, carrots, white radishes, ginger, green peppers, eggplant, pumpkin, bean sprouts and other vegetables. The type of the mold which causes the disease after harvesting is not limited, and examples thereof include Botrytis cinerea, Colletotrichum gloeosporioides, and Alternaria alternata.

In the case of the nematode which can be prevented by the Bacillus sp. AT-332 strain and the AT-79 strain of the present invention, for example, plant parasitic nematodes such as Meloidogyne hapla and Nematokia nematode are described. (Meloidogyne incognita), Meloidogyne javanica and other Meloidogyne species; Globodera rostochiensis and other Globodera species forming cystic nematodes; Heterodera avenae, Heterodera glycines, Heterodera schachtii, Heterodera trifolii and other Heterodera species; seed tumor nematodes Anguina genus; Aphelenchoides of stem and leaf nematodes; Belonolaimus longicaudatus and other genus Belonolaimus; and nematodes Pine wood nematode (Bursaphelenchus xylophilus) and other pine nematodes (Bursaphelenchus) variety; Cryptocaryon species, Criconema species, Criconemella species, Criconemoides species, Mesocriconema species; Stem and bulb nematode potato spoilage nematodes (Ditylenchus Destructor), Ditylenchus dipsaci and other species of Ditylenchus; the species of the genus Dolichodorus of the awl nematodes; the Helicotylenchus multicinctus of the spiral nematode And other Helicotylenchus varieties; sheath and sheathoid nematodes of the genus Hemicycliophora and Hemicriconemoides; Hirschmanniella; Hoplolaimus species of the genus Spiroptera; Nacobbus genus of the pseudo-root nodule; Longidorus elongatus and other Longidorus species; root rot Nematodes (Pratylenchus neglectus), Northern root rot nematodes (Pratylenchus penetrans) Pratylenchus curvitatus, Pratylenchus goodeyi, and other species of the genus Pratylenchus; Radopholus similis and other genus Radopholus Variety; Rotylenchus robustus and other Rotylenchus species; Scutellonema species; Stubby root nematodes (original burr nematodes) Trichodorus primitivus) and other Trichodorus species; Paratrichodorus genus; Tylenchorhynchus claytoni, Tylenchorhynchus dubius, and other dwarf nematodes Genus (Tylenchorhynchus) variety; citrus nematode citrus nematode (Tylenchulus) variety; the nematode genus Xiphinema variety, etc.; but is not limited thereto.

The Bacillus sp. AT-332 strain and the AT-79 strain of the present invention are particularly useful for controlling the genus Meloidogyne, the genus Globodera, the species of the genus Heterodera, and the genus Rhizoctonia. (Pratylenchus) variety, Radopholus cultivar, Rotylenchus genus and Tylenchulus genus, especially suitable for repelling the genus Meloidogyne and the genus Pratylenchus , the species of the genus Globodera and the species of the genus Heterodera.

Examples of the Bacillus sp. AT-332 strain and the AT-79 strain of the present invention which can promote growth include, for example, cereals such as rice, wheat, corn; vegetables such as carrots, cucumbers, and white radishes. Pumpkin, lettuce, eggplant, tomato, cabbage, potato, cabbage, chrysanthemum, small pine, green pepper, onion, onion, ginger, garlic, strawberry; mushrooms, such as mushrooms; fruit trees, such as persimmons, pears, citrus, grapes, apples , peaches; flowers, such as chrysanthemums, tulips, roses; beans, such as soybeans, sesame, peanuts, etc.

The plant disease control agent, the nematode control agent, and the plant growth promoter of the present invention are Bacillus sp. AT-332 strain and AT-79 strain which are capable of controlling plant diseases and nematodes and having plant growth promoting effects as described above. Effective bacteria.

In the plant disease control agent, the nematode control agent, and the plant growth promoter of the present invention, the AT-332 strain or the AT-79 strain may be used singly or in combination. Also, individual variants can be used. The mutant system refers to those having the bacteriological characteristics of the above-mentioned AT-332 strain and the AT-79 strain and having a plant disease control effect, a nematode control effect, and a plant growth promoting effect, and can utilize a natural mutant strain, by ultraviolet rays or chemistry. Mutants, cell fusion strains, and recombinant strains caused by the mutant.

The plant disease control agent, the nematode control agent and the plant growth promoter of the present invention are added to the plant body at a concentration of 10 ⁵ to 10 ¹⁰ /ml when the bacteria of the AT-332 strain and the AT-79 strain are used. Ideal.

Further, when a culture of the AT-332 strain and/or the AT-79 strain is used, the amount thereof can be appropriately determined in accordance with the case of the above-mentioned bacteria.

The microbial preparation (plant disease control agent, nematode control agent and plant growth promoter) of the present invention may be used alone with the cells and/or cultures of the AT-332 strain and the AT-79 strain, or may be an inert liquid or The solid carrier is diluted and, if necessary, a surfactant, a dispersing agent, or another adjuvant is added to prepare a drug. Specific examples of the preparation include a granule, a powder, a wettable powder, a suspension preparation, an emulsion, and the like.

Examples of the carrier include, for example, talc, bentonite, kaolin, clay, diatomaceous earth, white carbon, vermiculite, slaked lime, ammonium sulfate, cerium, urea, a porous solid carrier, water, isopropyl alcohol, A liquid carrier such as methylnaphthalene, xylene, cyclohexanone or alkylene glycol. Examples of the surfactant and the dispersant include, for example, dinaphthylmethanesulfonate, alcohol sulfate, lignosulfonate, alkylarylsulfonate, polyoxyethylene glycol ether, polyoxygen. A polyoxyethylene sorbitan alkylate or a polyoxyethylene aryl aryl ether. Examples of the adjuvant include carboxymethylcellulose, polyethylene glycol, propylene glycol, gum arabic, xanthan gum, and the like, and examples of the protective agent include skim milk powder, pH buffering agent, and the like. In this case, the amount of the cells of the AT-332 strain and the AT-79 strain and/or the amount of the culture thereof, and the application period and the applicable amount can be appropriately determined in accordance with the above-mentioned conditions.

Microbial preparation of the invention (plant disease control agent, nematode control agent and plant formation) The long accelerator may optionally contain an active ingredient other than the active ingredient of the present invention, such as an insecticide, another bactericide, a herbicide, a plant growth regulator, a fertilizer, or the like. Further, the plant disease controlling agent, the nematode controlling agent and the plant growth promoting agent of the present invention may contain other strains of the AT-332 strain and/or the AT-79 strain.

As the fungicide component, for example, Bitertanol, Bromuconazole, Cyproconazole, Difenoconazole, Diniconazole, and Enilconazole can be cited. ), Epoxiconazole, Fluquinconazole, Fenbuconazole, Flusilazole, Flutriafol, Hexaconazole, Imibenconazole, Ipconazole, Metconazole, Myclobutanil, Penconazole, Propiconon, Prothioconazole, simeconazole, and trimethoate (Triadimefon), Triadimenol, Tebuconazole, Tetraconazole, Triticonazole, Prochloraz, Pefurazoate, Imazalil, Safford Trip (Triflumizole), Cyazofamid, Benomyl, Carbendazim, Thiabendazole, fuberidazole, ethaboxam, and itide ( Etridiazole), fumaric acid imidazole (oxpoco) Nazole fumarate), Hymexazol, Azoxystrobin, dimoxystrobin, enestroburin, fluoxastrobin, Kresoxim-methyl, Metominostrobin, oressastrobin, picoxystrobin, Pyraclostrobin, Trifloxystrobin, carboxin, Benalaxyl , Boscalid, bixafen, fenhexamid, blessing Flutolanil, furametpyr, mepronil, metalaxyl, mefenoxam, ofurace, oxadixyl, jiabaoxin (Oxycarboxin), penthiopyrad, thifluzamide, tiadinil, Dimethomorph, flumorph, flumetover, fluoride Fluopicolide, Carpropamid, diclocymet, mandipropamid, Fluazinam, Pyrifenox, bupirimate, Cyprodinil, Fenarimol, ferimzone, Mepanipyrim, Nuarimol, Pyrimethanil, Triforine, mix Fenpiclonil, Fludioxonil, Aldimorph, dodemorph, Fenpropimorph, Tridemorph, Fenpropidin, Iprodione, Procymidone, Vinclozolin, Famoxadone, Fenamidone, Octetidone (octhi) Linone), Probenazole, anilazine, diclomezine, Pyroquilon, proquinazid, Tricyclazole, tetradendron (captafol) ), Captan, Dazomet, Folpet, fenoxanil, Quinoxyfen, Amisulbrom, Manzeb, Manganese Maneb, metam, Metiram, Ferbam, Propineb, thiuram, Zineb, Fumi Ziram, Diethofencarb, Iprovalicarb, Benthiavalicarb-isopropyl, Propamocarb hydrochloride, Thiophanate methyl, Pyribencarb, Bordeaux mixture, alkaline copper chloride, basic sulfur Copper acid, copper hydroxide, copper octahydroxyquinolate, dodine, iminoctadine albesilate, iminoctadine triacetate, gazatine, Kasugamycin, streptomycin, Polyoxin, Oxytetracycline, validamycin A, Binapacryl, Dinocap ), Dinobuton, Dithianon, Isoprothiolane, Edifenphos, Iprobenfos, Fosetyl, Fossex Aluminum Fosetyl-aluminium), Pyrazophos, Tolclofos-Methyl, Chlorothalonil, Dichlofluanid, Flusulfamide, Hexachlorobenzene, Heat Fthalide, Pencycuron, Quintozene, cyflufenamid, Cymoxanil, dimethirimol, Ethirimol, Frosting (furalaxyl), Metrafenone, Spiroxamine, Amobam, sulfur, lime Yellow mixture, echlomezole, potassium bicarbonate, calcium bicarbonate, thiadiazine, tecloftalam, triazine, copper nonylphenol sulfonate, hydroxyisoxazole Hydroxyisoxazole), fluoromide, polycarbamate, methasulfocarb, EDDP, IBP, Tolfenpyrad, fluopyram, ididini Isotianil) and Isopyrazam, but are not limited thereto.

Examples of the insecticide component include, for example, Acetamiprid, Pymetrozine, Fenitrothion, Acephate, Carbaryl, and Nadine ( Methomyl), Cartap, cyhalothrin, Ethofenprox, Teflubenzuron, flubendiles Flubendiamide, Flufenoxuron, Tebufenozide, Fenpyroximate, Pyridaben, Imidacloprid, Buprofezin, BPMC, MIPC, Malathion, Methidathion, Fenthion, Diazinon, oxydeprofos, vamidothion, Ethiofencarb, Bigap (Pirimicarb), Permethrin, Cypermethrin, Bifenthrin, Halfenprox, Silafluofen, nitenpyram, Chlorfluazuron ), Methoxyfenozide, Tebufenpyrad, Pyrimidifen, Kelthane, Propargite, Hexythiazox, Clofentezine , Spinosad, Milbemectin, BT (Bacillus thuringiensis), Indoxacarb, metaflumizone, chlorfenapyr, Fenpney ( Fipronil), Etoxazole, Acequinocyl, and pirimiphos-met Hyl), Arrinathrin, chinomethionat, chlorpyrifos, Abamectin, Emamectin benzoate, Fenbutatin oxide, Terbufos, Ethoprophos, Cadusafos, Phenamiphos, Fensulfothion, DSP, dichlofenthion, Fosthiazate, Oxamyl, Asamimidofos, fosthietan, Isazofos, thionazin, Benfuracarb, Spirodiclofen, ethiofencarb, valley speed Azinphos-methyl, Disulfoton, Methiocarb, Oxydemeton methyl, Parathion, Cyfluthrin, Betacitin (beta-) Cyfluthrin), butyl pyrimidine (tebupirimfos), spiromesifen, endosulfan, Amitraz, Tralomethrin, acetoprole, Ethiprole, Aizusong (Ethion), Trichlorfon, Methamidophos, Dichlorvos, Mevinphos, Monocrotophos, Dimethoate, Formetanate , Formothion, Mecarbam, Thiometon, Naled, Parathion-methyl, Cyanophos, Detergent (Diamidafos), albendazole, oxibendazole, fenbendazole, Oxfendazole, Propaphos, Sulprofos, Pu Prothiofos, Propenofos, Isofenphos, Temephos, Phinthoate, dimethylvinphos, Chlorfenvinphos, kill Tetrachlorovinphos, Phoxim, Isoxathion, Pyraclofos, chlorpyrifos, Pyridaphenthion, Phosalone, Phosmet, dioxabenzofos, quinalphos, Pyrethrins, Allethrin, Prallethrin, Resmethrin, Permethrin, Tefluthrin, Fenpropathrin, α-Cypermethrin, and Lambda-Cyhalothrin ), Deltamethrin, Fenvalerate, Esfenvalerate, Flucythrinate, Fluvalinate, cycloprothrin, Thiodicarb ), Aldicarb, alanycarb, Metolcarb, Xylylcarb, Propoxur, Fenoxycarb, Fenothiocarb, must Finnish (bifenazate), Carbofuran, Carbosulfan, sulfur, Pyrifluquinazon, furathiocarb, Diafenthiuron, Diflubenzuron , Hexaflumuron, Novaluron, Lufenuron, Chlorfluazuron, Tricyclohexyltin hydride, sodium oleate, potassium oleate, methope , hydroprene, Binapacryl, Chlorobenzilate, bromopropylate, Tetradifon, Bensultap, benzomate, Chromafenozide, halofenozide, Endosulfan, Diofenolan, Tolfenpyrad, triazamate, nicotine sulfate ), Thiaclogrid, Thiamethoxam, Clothianidin, Dinotefuran, Fluazinam, Pyriproxyfen, fluacrypyrim ), Hydramethylnon, Cyromazine, TPIC, Thiocyclam, Fenicide Fenazaquin, Polynactin complex, Azadirachtin, Rotenone, Hydroxypropyl Starch, Mesulfenfos, Phocarbcarb , aldoxycarb, metam-sodium, Morantel tartrate, Dazomet, Levamisole hydrochloride, trichlamide , but not limited to, pyridalyl, chlorantraniliprole, cyenopyrafen, and cyflumetofen.

The plant disease controlling agent, the nematode controlling agent and the plant growth promoting agent of the present invention can be applied directly in the original state or diluted with water or the like. Plant disease prevention The method of applying the herbicide, the nematode controlling agent, and the plant growth promoter is not particularly limited, and examples thereof include a method of directly spraying plants or pests, a method of spraying on soil, and water or fertilizer added to plants or soil. The method added, the method of covering the seed, and the like. In addition, since the application amount of the preparation varies depending on the target disease, the target pest, the target crop, the application method, the tendency to occur, the degree of the damage, the environmental conditions, the dosage form to be used, and the like, it is preferably adjusted as appropriate.

As described above, the Bacillus sp. AT-332 strain and the AT-79 strain of the present invention have a wide range of diseases and nematode control spectrum, can control the number of plant diseases and nematodes, and can promote the growth of useful plants. The plant disease control agent, the nematode control agent and the plant growth promoter of the present invention containing such strains have high environmental safety, and have control effects against several diseases and nematodes, so that they can be widely used without using other combined means. It can prevent diseases and nematodes, and can promote the growth of useful plants, and can be used as biological pesticides and/or biological fertilizers.

[Examples]

The present invention will be specifically described based on the following production examples, formulation examples, examples and comparative examples, but the present invention is not limited by these examples.

[Cultivation of AT-332 strain and AT-79 strain]

The AT-332 strain and the AT-79 strain were isolated from the soil containing the plant roots.

Specifically, in August 2009, the soil of Shougu City, Ibaraki Prefecture, Japan was taken, and 1 g of dried soil obtained by heat treatment (80 ° C, 10 minutes) was suspended in sterilized water. The suspension was diluted 10 ² to 10 ⁴ times, cultured in an ordinary broth medium (Yongyan Chemical Co., Ltd.) (28 ° C, 3 days), and the formed colonies were separated. The isolated colonies were placed on potato glucoam Agar medium to find strains which were effective against various plant pathogenic bacteria. Then, the strain was shown to be viable and cultured in potato dextrose liquid medium, and the strain showing activity against the second stage larva of the sweet potato root nodule was isolated from Bacillus sp. AT-332 strain and AT-79 strain.

The identification method of each strain, various analysis methods, results, and bacteriological properties are as described in [Embodiment].

Production Example 1: Cultivation and preparation of AT-332 strain

In the case of the pre-culture, a platinum ring of the preserved bacteria of the bacterium (AT-332 strain) of the present invention is sterilized in an ordinary broth medium (Essence Chemical Co., Ltd.) containing 60 ml per flask. After a 500 ml Erlenmeyer flask of a baffle, it was cultured for 1 day at 28 ° C with a rotary oscillating machine at a number of revolutions of 180 rpm.

60 ml of the culture obtained by the above preculture was incubated in a 5000 ml fermentor containing 2000 ml of LB medium (20 g of peptone, 10 g of yeast extract, 20 g of sodium chloride, and the balance of water), and then cultured in the main culture. For example, the culture was carried out at 35 ° C for 3 days at a rotation number of 500 rpm and a ventilation rate of 1 L/h.

From the above main culture, about 1800 g of the culture was obtained. The cell concentration was about 8.0 x 10 ⁹ CFU/ml.

About 1800 g of the obtained culture was frozen at -80 ° C, and then freeze-dried under reduced pressure and pulverized to obtain about 140 g of a dry powder. The cell concentration was about 1.0 x 10 ¹¹ CFU/g.

Production Example 2: Cultivation and preparation of AT-79 strain

In the case of the pre-culture, a platinum ring of the preserved bacteria of the bacterium (AT-79 strain) of the present invention is sterilized in an ordinary broth medium (Essence Chemical Co., Ltd.) containing 60 ml per flask. After the plate was placed in a 500 ml Erlenmeyer flask, it was incubated at 28 ° C for 1 day with a rotary oscillating machine at a number of revolutions of 180 rpm.

60 ml of the culture obtained by the above preculture was sterilized in 2000 ml. After 5,000 ml of fermentation tank of LB medium (20 g of pancreatic isatin, 10 g of yeast extract, 20 g of sodium chloride, and the balance of water), in the main culture, the culture was carried out at a rotation rate of 500 rpm and aeration rate of 1 L/h at 35 ° C. day.

From the above main culture, about 1700 g of the culture was obtained. The cell concentration was about 9.0 x 10 ⁹ CFU/ml.

About 1700 g of the obtained culture was frozen at -80 ° C, and then freeze-dried under reduced pressure and pulverized to obtain about 130 g of a dry powder. The cell concentration was about 1.1 x 10 ¹¹ CFU/g.

Next, a formulation example will be described. Further, "parts" means mass parts.

Formulation Example 1: Water and agent

60 parts of the dry powder obtained in Production Example 1, 25 parts of diatomaceous earth, 5 parts of white carbon, 8 parts of sodium lignin sulfonate, and 2 parts of sodium alkylnaphthalene sulfonate were mixed and pulverized to obtain a water and a solvent.

Formulation Example 2: granules

5 parts of the dry powder obtained in Production Example 1, 25 parts of bentonite, 66 parts of talc, 2 parts of sodium dodecylbenzenesulfonate, and 2 parts of sodium lignosulfonate were mixed and pulverized, and about 20 parts of water was added to knead. After kneading, the granules were granulated by a granulator, followed by drying and granulation to obtain granules.

Formulation Example 3: water and agent

60 parts of the dry powder obtained in Production Example 2, 25 parts of diatomaceous earth, 5 parts of white carbon, 8 parts of sodium lignin sulfonate, and 2 parts of sodium alkylnaphthalene sulfonate were mixed and pulverized to obtain a water and a solvent.

Formulation Example 4: granules

5 parts of the dry powder obtained in Production Example 2, 25 parts of bentonite, and 66 parts of talc. 2 parts of sodium dodecylbenzenesulfonate and 2 parts of sodium lignosulfonate are mixed and pulverized, about 20 parts of water is added, and kneaded by a kneading machine, then granulated by a granulator, followed by drying and granulation, and A granule is obtained.

Next, examples and comparative examples in which the effects of the plant disease controlling agent, the nematode controlling agent, and the plant growth promoting agent of the present invention were tested were described.

Example 1 and Comparative Example 1: Effect test on rice rice fever

In the greenhouse, rice (growth: koshihikari, 15 plants) which has grown to a 3-leaf stage in a plastic pot having a diameter of 6 cm, and 250-fold dilution of the water and the agent of Formulation Example 1 and Preparation Example 3 The liquid is sprayed in a sufficient amount with a spray gun. The Impression water and the agent (S.D.S Biotech Co., Ltd.) were diluted 250 times as a comparison agent and tested in the same manner. On the next day, a spore suspension of Pyricularia oryzae was spray-inoculated. The pot was kept in a humidity chamber at 22 ° C for 24 hours, and then placed in a greenhouse for 7 days, and the number of lesions in the inoculated leaves was examined to determine the price of the seed. The price control (%) is calculated based on the number of lesions in the non-treatment area. As a result, as shown in Table 1, by treating with the microorganism preparation of the present invention, the incidence of rice rice fever was significantly reduced as compared with the non-treated area, and an extremely high control effect was obtained.

Example 2 and Comparative Example 2: Effect test on cucumber anthracnose

In the greenhouse, the first leaf and the second leaf of the 3-leaf stage cucumber (variety: Tokiwa light No. 3 P type) grown in a plastic pot having a diameter of 6 cm, Formulation Example 1 and The 250-fold dilution of the water and the agent of Formulation Example 3 was sprinkled with a spray gun in a sufficient amount. The Impression water and the agent (S.D.S Biotech Co., Ltd.) were diluted 250 times as a comparison agent and tested in the same manner. On the next day, the spore suspension of Colletorichum lagenarium was spray-inoculated. The pot was kept in a humid chamber at 22 ° C for 24 hours, and then placed in a greenhouse for 7 days, and the area ratio of the first leaf and the second leaf was visually examined to determine the price of the seed. The price control (%) is calculated based on the area ratio of the disease-free area. As a result, as shown in Table 2, by treating with the microbial preparation of the present invention, the incidence of cucumber anthracnose was significantly reduced as compared with the non-treated area, and an extremely high control effect was obtained.

Example 3 and Comparative Example 3: Effect test on tomato blight

In the greenhouse, for the 5-leaf stage tomato (variety: Sugar Lamp) grown in a plastic pot having a diameter of 6 cm, a 250-fold dilution of the water and the agent of Formulation Example 1 and Preparation Example 3 was sprayed with a spray gun in a sufficient amount. . The Impression water and the agent (S.D.S Biotech Co., Ltd.) were diluted 250 times as a comparison agent and tested in the same manner. On the next day, a suspension of zoospore of Phytophthora infestans was spray-inoculated. The pots were kept in a humidity chamber at 22 ° C for 16 hours, and then placed in a greenhouse for 3 days, and the incidence area ratios of the third, fourth, and fifth leaves were visually investigated to determine the price. The price control (%) is calculated based on the area ratio of the disease-free area. The results are shown in Table 3, and the tomato was treated by the microbial preparation of the present invention. The incidence of epidemics is significantly reduced compared to the untreated area, resulting in extremely high control effects.

Example 4 and Comparative Example 4: Effect test on cucumber bacillus

In the greenhouse, the cucumber of the 3-leaf stage (variety: light No. 3 P type) grown in a plastic pot having a diameter of 6 cm was sprayed with a spray gun by a 250-fold dilution of the water and the agent of the preparation example 1 and the preparation example 3. A sufficient amount. The Impression water and the agent (S.D.S Biotech Co., Ltd.) were diluted 250 times as a comparison agent and tested in the same manner. On the next day, the tourmaline suspension of Pseudoperonospora cubensis was spray-inoculated. The pot was kept in a humidity chamber at 22 ° C for 18 hours, and then placed in a greenhouse for 3 days, and the area ratio of the first and second leaves was visually investigated to determine the price of the seed. The price control (%) is calculated based on the area ratio of the disease-free area. As a result, as shown in Table 4, by treating with the microbial preparation of the present invention, the incidence of cucumber dew disease was significantly reduced as compared with the non-treated area, and an extremely high control effect was obtained.

Example 5 and Comparative Example 5: Effect test on apple spotted leaf disease

Take the leaves of apple (Wang Lin) and put the water of Formulation Example 1 and Formulation Example 3 on the back of the leaf. A 250-fold dilution of the agent was sprayed with a sufficient amount of the spray gun. The Impression water and the agent (S.D.S Biotech Co., Ltd.) were diluted 250 times as a comparison agent and tested in the same manner. After spreading, the leaves were air-dried and a spore suspension of Altenaria Alternaria mali was spray-inoculated. After being placed at 20 ° C for 4 days under high humidity, the area ratio of the disease was visually investigated to determine the price. The price control (%) is calculated based on the area ratio of the disease-free area. As a result, as shown in Table 5, by treating with the microbial preparation of the present invention, the incidence of apple spotted leaf disease was significantly reduced as compared with the non-treated area, and an extremely high control effect was obtained.

Example 6 and Comparative Example 6: Effect test on cucumber powdery mildew (field test)

Cucumbers were tested in a greenhouse maintained by the applicant company (test area: 4 m ² /zone, 10 plants/zone, 3 replicates). The onset is left to occur naturally. The water and the agent of Formulation Example 1 and Formulation Example 3 were diluted 500-fold, diluted 1000-fold, diluted 2000-fold, and sprinkled four times at intervals of 7 days, and the control price was calculated from the diseased area ratio of the leaves. The comparison agent is 500 times and 1000 times dilution of Impression water and agent (SDS Biotech Co., Ltd.), Botokiller water and agent (Icolight Development Co., Ltd.) 1000 times dilution, Botopika water and agent Company) 2000 times dilution, Ecoshot granule water and agent (Kumiai Chemical Industry Co., Ltd.) 1000-fold dilution, Morestan water and agent (Agro-kanesho Co., Ltd.) 3000-fold dilution. In addition, the area ratio of the untreated area was 47.4%. The price control (%) is calculated based on the area ratio of the disease-free area. As a result, as shown in Table 6, by treating with the microbial preparation of the present invention, the incidence of cucumber powdery mildew was significantly reduced as compared with the non-treated area, and an extremely high control effect was obtained. In addition, it is confirmed in the field that the existing commercial Bacillus subtilis agent (Impression water and agent (Patent Document 3), Botokiller water and agent, Botopika water and agent, Ecoshot granule water, and the like are used as a comparative agent. The agent (Patent Document 4)) has a remarkably high effect on the microbial preparation of the present invention. Further, when diluted by 500 times, a very high control effect similar to that of the morestan water and the agent of the chemical agent was exhibited.

Example 7 and Comparative Example 7: Effect test on eggplant gray mold (field test)

The eggplant was used for testing in the greenhouse owned by the applicant company (test area: 5.6 m ² / zone, 7 plants/zone, 3 replicates). The onset is left to occur naturally. The water and the agent of Formulation Example 1 and Formulation Example 3 were diluted 500-fold and 1000-fold, and were sprayed four times at intervals of 7 days, and the control price was calculated from the fruit-fruit incidence rate. The comparison agent was an Impression water and agent (SDS Biotech Co., Ltd.) 500-fold dilution, Botokiller water and agent (Icosu Kosei Co., Ltd.) 1000-fold dilution, Botopika water and agent (Icosu Corporation) 2000 Diluted dilution solution, Ecoshot granule water and agent (Kumiai Chemical Industry Co., Ltd.) 1000-fold dilution, Savier Flowable 20 (Syngenta Japan Co., Ltd.) 1500-fold dilution. In addition, the disease-free rate of the untreated area was 15%. The price control (%) is calculated based on the fruit rate of the untreated area. As a result, as shown in Table 7, by treating with the microbial preparation of the present invention, the incidence of gray mold of eggplant was significantly reduced as compared with the non-treated area, and an extremely high control effect was obtained. Moreover, it was confirmed in the field that the present invention is compared with the existing commercial Bacillus subtilis agent (Impression water and agent, Botokiller water and agent, Botopika water and agent, Ecoshot particle water and agent) used as a comparative agent. The microbial preparation has a significantly high effect. Further, at the time of 500-fold dilution, a very high control effect similar to that of the Savier Flowable 20 of the chemical agent was exhibited.

Example 8 and Comparative Example 8: Experiment on the control effect of rice seedling bacterial disease

Rice seedlings (variety: koshihikari) in a suspension of rice seedlings (Burkholderia plantarii) (1 × 10 ⁸ CFU/ml) obtained by shaking culture with PD liquid medium at 27 ° C for 52 hours Inoculation was carried out under reduced pressure for 1 hour to prepare rice seedlings infected with bacterial disease. In the 100-fold dilution of the water and the agent of the preparation example 1 and the preparation example 3, the rice seedlings infected with the rice seedling bacterial disease were immersed for 24 hours, and then the immersion treatment liquid was discarded and kept in a wet room at 32 ° C for 1 day. Promote germination. The Impression water and the agent (SDS Biotech Co., Ltd.) were diluted 100 times as a comparison agent and tested in the same manner. The germination seeds were planted in a plastic cup with a diameter of 6 cm which had been filled with the nursery soil. After 3 days of sowing, the seedlings were kept in the nursery bank at 30 ° C, and after 15 days in the wet room at 25 ° C, the whole seedlings were investigated for the onset of the disease. Seedling rate. The price control (%) is calculated based on the incidence rate of the untreated area. The seeding amount per cup is 3g (90 to 110) of dry rice. As a result, as shown in Table 8, by treating with the microbial preparation of the present invention, the incidence rate of the rice seedling bacterial disease was significantly reduced as compared with the non-treated area, and an extremely high control effect was obtained.

Example 9 and Comparative Example 9: Effect test on cucumber seedling blight

3 g of the bran culture of the B. solani was mixed with 500 ml of the sterilized soil, and the mixture was filled in a plastic pot, and 1 g of the granules of the preparation example 2 and the preparation example 4 were mixed with the soil. Impression water and agent (S.D.S Biotech Co., Ltd.) 84 mg was used as a comparative agent and tested in the same manner. The cucumber was sown (phase mold half white), and after culturing at 23 ° C for 1 week, the germination rate was investigated. The control effect (% of control price) is calculated based on the incidence rate of the untreated area. Out. As a result, as shown in Table 9, by treating with the microbial preparation of the present invention, the incidence rate of cucumber seedling blight was significantly reduced as compared with the non-treated area, and an extremely high control effect was obtained.

Example 10 and Comparative Example 10: Activity against the second stage larvae of the sweet potato root nodule nematode

The nematicidal activity of the sweet potato root nodule nematode stage 2 larvae hatched by the egg yolk collected from the root of the eggplant (12) was tested within 24 hours. A 100-fold dilution of Formulation Example 1 and Formulation Example 3 (tween 20 5000-fold diluted solution) and an equivalent amount of Nodular nematode Phase 2 larvae suspension (about 50) were added to a 24-well microdisk. The Impression water and the agent (S.D.S Biotech Co., Ltd.) were diluted 100 times as a comparison agent, and tested in the same manner. The disc was sealed and placed in an incubator at 28 ° C and a relative humidity of approximately 50%. After 72 hours, the mortality was investigated by observation under a stereoscopic microscope. At this time, the immobile nematodes are regarded as death. The nematicidal rate is calculated by the following formula. The results are shown in Table 10. By treating with the microbial preparation of the present invention, extremely high nematicidal activity against the second stage larva of the sweet potato nodule nematode was obtained.

[Number 1] Nematode rate = (number of dead nematodes ÷ number of tested nematodes) × 100

Example 11 and Comparative Example 11: Control effect on the control of sweet potato root nodule nematodes

The granules of Formulation Example 2 and Formulation Example 4 were uniformly mixed into the contaminated soil of the sweet potato nodule nematode in a 1/10000 awa pot (Wagner pot) at a ratio of 40 kg/10 a, and a mini tomato (variety: Sugar Lamp) was planted. The Impression water and the agent (S.D.S Biotech Co., Ltd.) were used as a comparative agent at a ratio of 3.3 kg/10a, and were tested in the same manner. After one month of planting, the degree of damage of the roots (degree of nodules) was evaluated on the basis of the following criteria, and the nodule index was obtained by the following formula, and the price of the control was calculated. As a result, as shown in Table 11, by treating with the microbial preparation of the present invention, the root damage caused by the sweet potato root nodule nematode was significantly reduced as compared with the untreated area, and an extremely high control effect was obtained.

Victimity 0: no tumors at all

1: At first glance, it is not conspicuous, but you can see that there are a few tumors.

2: There are a few tumors in the scattered

3: I saw a moderate degree of tumor

4: Most tumors are seen in the root ring

Example 12 and Comparative Examples 12 to 13: Plant growth promoting effect of AT-332 (basic test)

A base petri dish test was carried out on Arabidopsis thaliana to measure the plant growth promoting effect of AT-332. The Arabidopsis seeds were immersed in a 1% sodium chlorite solution for 20 minutes, and then immersed in 70% ethanol for 2 minutes to sterilize the surface of the seeds. Thereafter, it was washed with sterilized distilled water and used in experiments. Murashige and Skoog salt medium (pH 5.7) containing 0.8% amaranth was injected into a sterilized dish divided into two parts, and after cooling, it was used in an experiment.

Inoculation of AT-332 (Example 12), Bacillus subtilis GB03 (Comparative Example 12), Bacillus subtilis MBI600 (Comparative Example 13) on the divided side placed on the culture dish Paper discs and germinated seeds of Arabidopsis inoculated in the remainder of the segmentation. The plate inoculated with bacteria and Arabidopsis was incubated at 22 ° C (lighting for 12 hours / shading for 12 hours) for 10 days, and the growth state of the plants was observed. The results are shown in the photographs of Figs. 2(a) to (d) together with the results of the control group (Fig. 2(a)) in which no bacteria were inoculated. Even compared to Bacillus subtilis GB03 (Comparative Example 12; (c)), Bacillus subtilis MBI600 (Comparative Example 13; (d)), which was actually used in the United States, AT-332 (Example 12; (b)) Significant plant growth promoting effects can also be seen.

Example 13: Plant growth promoting effect of AT-332 and AT-79 strains (pot experiment)

A pot experiment was conducted on cabbage seedlings to measure the plant growth promoting effect of AT-332 and AT-79 strains. First, the AT-332 and AT-79 strains were cultured in a liquid LB medium for 24 hours, and then the cells were collected by centrifugation. The recovered cells were suspended in a sterilized 0.85% aqueous sodium chloride solution to 1 × 10 ⁹ CFU/ml, and mixed in a pre-sterilized culture soil at a ratio of 40 ml per 1 kg of the mixture to prepare a treated soil. On the other hand, in the pre-sterilized culture soil, only the sterilized 0.85% sodium chloride aqueous solution was mixed at a ratio of 40 ml per 1 kg to prepare a non-treated soil. 100 g of each of the treated soil and the untreated soil was placed in a plastic pot (caliber 70 mm × height 68 mm), and seeds of cabbage (variety: Nozaki Cabbage No. 2) were sown therein. Thereafter, it was placed in a greenhouse set at 22 ° C, and the growth weight of the grown cabbage was measured 30 days later. The results are shown in Figure 3. It can be seen that AT-332 and AT-79 strains have obvious growth promoting effects on crops.

<110> SDS Biotech Co., Ltd.

<120> Bacillus sp. strain, microbial preparation, and plant cultivation method

<130> BOF-7708PCT

<150> PCT/JP2011/062109

<151> 2011-05-26

<160> 3

<170> PatentIn version 3.1

<210> 1

<211> 1472

<212> DNA

<213> Bacillus sp.AT-332/AT-79

<400> 1

<210> 2

<211> 1472

<212> DNA

<213> Bacillus sp.AT-332

<400> 2

<210> 3

<211> 1472

<212> DNA

<213> Bacillus sp.AT-79

<400> 3

The representative figure has no component symbols and the meaning of its representation.

Claims (8)

A strain belonging to the Bacillus sp. AT-332 strain of BCRC 910571 in Taiwan and having 16S rDNA represented by the nucleotide sequence of SEQ ID NO: 2. A strain belonging to the Bacillus sp. AT-79 strain of BCRC 910570 in Taiwan and having 16S rDNA represented by the nucleotide sequence of SEQ ID NO: 3. The strain of the strain body and/or the strain exhibiting the effects of the plant disease control action, the nematode control action, and/or the plant growth promoting action, as described in the first or second aspect of the patent application. A microbial preparation comprising the culture of the strain and/or the strain according to any one of claims 1 to 3 as an active ingredient. The microbial preparation as described in claim 4 of the patent application is a plant disease control agent. A microbial preparation as described in claim 4, which is a nematode control agent. The microbial preparation described in claim 4 is a plant growth promoter. A plant cultivation method for treating a plant with the microbial preparation according to any one of claims 4 to 7.