JP3132195B2 - New microorganism and plant disease control agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel microorganism belonging to Bacillus subtilis and a plant disease controlling agent derived from the microorganism.

[0002]

BACKGROUND OF THE INVENTION It is known that disease in agricultural and horticultural crops is often caused by filamentous fungi or bacteria present in the soil in which the crops are grown. Conventionally, as a means of controlling plant diseases caused by such pathogenic fungi and pathogenic bacteria, a chemical method of fumigation disinfection of soil using a soil disinfectant such as a chlorpicrin agent or a methyl bromide agent, Ecological methods such as rotation with non-host crops and breeding methods using disease resistant varieties and rootstocks are generally performed.

[0003] However, chemical methods such as soil fumigation disinfecting the soil are extremely toxic to the soil fungicide and may harm the health of workers and nearby residents. Is also destroyed, and there is a risk that the invasion of new pathogens may cause serious damage.

[0004] Rotation is an effective control method experimentally, but under the current intensive agricultural production system, it is difficult to select an effective rotation crop due to the difficulty of selecting a crop that is economically feasible. In addition, it is not an effective control method for diseases having a wide host range. The cultivation of resistant varieties and resistant rootstocks requires a great deal of labor and a long period of time, and is effective only for the control of specific diseases and does not guarantee resistance to new pathogens that enter from outside the region have.

[0005] For the above reasons, there is a demand for an effective control method which is an alternative to these means. As a control method which is highly safe and does not pollute the environment, a biological control method utilizing a specific antagonistic microorganism which exists naturally. Has been studied. In other words, a method for controlling a living organism that suppresses the growth of pathogenic bacteria and infection of the plants by applying microorganisms having an antagonistic action to the pathogenic bacteria to plants and soil has been developed. For example, as a controlling method using a microorganism of the genus Pseudomonas, JP-A-60-186230 and JP-A-61-19568.
6, JP-A-62-123104, JP-A-62-1484
13, JP-A-63-22005, JP-A-64-1657
9, JP-A-2-35075, JP-A-2-35076, JP-A-2-46283, JP-A-2-59504, JP-A-2-59504
149507 and Japanese Patent Application Laid-Open No. 2-211861 are disclosed. As a control method using a microorganism of the genus Bacillus, JP-A-63-273470, JP-A-2-48509,
JP-A-2-209803, JP-A-3-128988, JP-A-4-117278 and the like disclose the control methods using microorganisms of the genus Fusarium in JP-A-64-90107,
-165506 and the like are disclosed. However, very few are effective in controlling plant diseases.

[0006]

Under such circumstances, the present inventors have proposed, as a method to replace the controlling means,
After isolating various microorganisms from compost and searching for effective microorganisms for controlling plant diseases, they belonged to Bacillus subtilis, and iturin and surfactin
It has been found that a specific strain producing n) has a very large effect on controlling various plant diseases, thereby completing a plant disease controlling agent.

[0007] Iturin is produced by a certain strain belonging to the genus Bacillus and has already been reported as a component having an antibacterial action against plant pathogenic bacteria (Tetrahedron Letters 23,
No. 30, 3065-3068, 1982). On the other hand, surfactin is a surfactant produced by certain strains of the genus Bacillus (BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICAT).
IONS 31, 488-494, 1968), but no antibacterial action against plant pathogens is known so far. In the process of studying the inhibitory effect of iturin and surfactin on the growth of phytopathogenic bacteria, the present inventors showed that surfactin alone does not exhibit antibacterial activity against phytopathogenic bacteria. Has been found to be dramatically enhanced. When trying to control plant diseases with these antibacterial substances, it is extremely advantageous if microorganisms producing antibacterial substances can be directly applied to plants without isolating the antibacterial substances from the microorganisms. Further research has led to the discovery of an agent for controlling plant diseases caused by microorganisms that produce iturin and surfactin.

[0008]

The present invention relates to a plant disease control agent comprising Bacillus subtilis SD142 strain and a culture thereof and / or a microbial cell as an active ingredient.

Hereinafter, the present invention will be described in detail. The Bacillus subtilis SD142 strain used in the present invention was isolated from compost. This strain has the following bacteriological properties.

Bacteriological properties (a) Morphology (1) Bacterial shape: rod-like (2) Bacterial size: 0.7-0.9 × 1.5-3.0μ
m (3) Polymorphism: none (4) Motility: yes (5) Spore presence: yes Spore shape: oval or cylindrical (6) Gram stain: positive (7) Acid-fast: negative (b ) Growth situation Gravy agar plate culture: colonies are circular and 1 to 1 in diameter
2mm, peripheral shape is wavy, viscous, and glossless. (c) Physiological properties (1) Nitrate reduction: positive (2) VP test: positive (3) Indole formation: negative (4) Citric acid use: positive (5) Succinic acid use: negative (6) Use of propionic acid: negative (7) Use of tartaric acid: negative (8) Urease: negative (9) Oxidase: positive (10) Catalase: positive (11) Growth range: pH5-9 Temperature 20-50 ° C (12) 10% NaCl medium: growth (13) Anaerobic culture: negative (14) Egg yolk reaction: negative (15) Starch hydrolysis: positive (16) Arginine degradation: positive (17) Tyrosine degradation: negative (18) Gelatin Liquefaction: Positive (19) Esculin degradation: Positive (20) OF test: Oxidative (21) Acid production from glucose: Negative

[0011] The above-mentioned features are
Of Systematic Bacteriology (Bergeys M
anual of Systematic Bacteriology) and identified this strain as Bacillus subtilis (Bacillus
subtilis) and identified as Bacillus subtilis S
The strain was named strain D142 (hereinafter abbreviated as SD142).
The strain was deposited with the Research Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology under the deposit number of microorganism, "Microbial Deposit No. 13204".

The fact that SD142 produces iturin and surfactin means that the supernatant obtained by removing the cells from the culture solution of SD142 is adjusted to pH 2 and the components that precipitate are extracted with methanol, and the components separated by TLC are separated. It can be confirmed by mass spectrum, infrared absorption spectrum and amino acid analysis.

The medium that can be used in the present invention may be any medium as long as the strain can be grown by culturing. For example, as a carbon source used in the medium, glucose, saccharose, starch, saccharified starch solution, saccharides such as molasses, organic acids such as citric acid, alcohols such as glycerin, and nitrogen sources include ammonia, ammonium sulfate, phosphorus ammonium, Ammonium salts such as salt and nitrates and nitrates are appropriately used. Examples of the inorganic salt include salts such as phosphoric acid, potassium, magnesium, and manganese, such as potassium dihydrogen phosphate, potassium chloride, calcium chloride, magnesium sulfate, manganese sulfate, and ferrous sulfate. As trace organic nutrients, vitamins, amino acids, nucleic acid-related substances, and the like are not particularly required for the growth of bacteria, but they may be added, or organic substances such as peptone, meat extract, yeast extract, and soybean meal may be added. Further, various additives such as an antifoaming agent can be added as needed.

The culture in the method of the present invention can be carried out under aerobic conditions, for example, by aeration stirring, shaking culture, solid culture, or the like. The culture conditions are not particularly limited, but the temperature is 25 to 45 ° C, the pH is 5.0 to 8.0,
The culture time is suitably in the range of 15 to 60 hours.

The SD142 cultured as described above can be used without being separated from the culture, and the cells can be separated and used by a usual method, for example, a treatment such as membrane separation or centrifugation. . Furthermore, it is also possible to use dried cells obtained by drying the cultured or isolated cells by freeze-drying, spray-drying, or the like, or to formulate them together with various additives according to a conventional method of pesticide preparations. It can also be used. For example, granules, emulsions, wettable powders, flowables and the like can be mentioned.

Various diseases of agricultural and horticultural crops can be controlled by applying the thus-prepared culture of SD142 or the isolated cells to plants or soil. Control agent of the present invention, Alternaria (Alternaria),
Cerospora, Phytophthor
a), Botrytis, Rhizoctoni
a), Pyricularia, cochliobolus
(Cochliobolus), Fusarium, Phisium (Pht
hium), Verticilium, Gibberella (G
ibberella), Xanthomonas, Pseudomonas, Agrobacte
rium), Erwinia (Erwinia) and the like, and has a remarkable effect on controlling diseases caused by pathogens. In particular, bacterial wilt fungus, seedling wilt fungus, soft rot fungus, epidemic wilt fungus, wheat wilt fungus, various Fusarium wilt fungus, wilt wilt fungus, root rot, wilt fungus, cruciferae, cruciferous root knot It is effective against germs, crested wilt fungus, white silk rot, turf large patch and the like. The method of applying the pesticidal agent of the present invention is appropriately selected depending on the above-mentioned use form (preparation, etc.), crops, diseases and the like.
For example, ground liquid spraying, ground solid spraying, airborne spraying,
Methods include spraying solid air in the air, water surface application, in-house application, soil application, surface treatment (seed disinfection, etc.) and nursery box application.

Since the application rate varies depending on the type of disease, applied crops, dosage form, etc., it cannot be specified unconditionally.
When the cells are mixed with the soil, the mixing amount is suitably about 10 ⁵ to 10 ⁹ cells per 1 g of the soil, and the mixing time is preferably before sowing or seedling.

[0017]

The present invention will be described in more detail with reference to the following examples. Example 1 Glucose 2%, polypeptone 1%, potassium phosphate 0.1%.
100m medium containing 1% and 0.05% magnesium sulfate
1 into a 500 ml Sakaguchi flask, inoculate one platinum loop of SD142, and incubate 2 at 35 ° C. and 120 rpm.
A culture solution was obtained by shaking culture for 4 hours. An 8 mm diameter paper disc impregnated with this culture solution was placed on a sucrose-potato agar plate medium mixed with various plant pathogens, and cultured at 25 ° C. One week later, the size of the blocking circle formed around the paper disk was examined. Table 1 shows the results
As shown in Table 2, SD142 inhibited the growth of a great variety of plant pathogens.

[0018]

[Table 1]

Example 2 (Production example of bacterial cells) 100 ml of a medium having the following composition, adjusted to pH 7, and sterilized by high pressure and heat was put into a 500 ml Sakaguchi flask, and one platinum loop of SD142 was inoculated. 35
Preculture was performed at 120 ° C. for 10 hours at 10 ° C. 15 L of a medium having the same composition was placed in a 30 L fermenter, inoculated with 100 ml of a precultured bacterial solution, and cultured at 35 ° C. for 30 hours under aerobic conditions to obtain a culture solution. Further, the obtained culture solution was
Viable cells were obtained by centrifugation at 000 rpm for 10 minutes.

Addition amount of medium component (g / l) グ ル コ ー ス glucose 10 polypeptone 30 KH ₂ PO ₄ 1 MgSO ₄ .7H ₂ O 0.5─────────────────────────

Example 3 (Example of controlling tomato seedling damping off) Tomato seedling damping off Rhizoctonia cultivar cultured on a vermiculite bran medium for 2 weeks.
Contaminated soil was prepared by mixing Rhizoctonia solani with a high pressure heat sterilized soil at a ratio of 5%. Diameter 9c
About 250 g of contaminated soil was packed in a pot of m, and the culture solution of SD142 obtained in Example 2 or the separated viable cells were mixed with the contaminated soil so that the number of bacteria per gram of soil was about 10 ⁷ . 1 tomato (variety: Momotaro) seeds per pot
Five seeds were sowed and cultivated in a 25 ° C constant temperature bath. Plant height 1
At 5 cm or 5 or more true leaves, the onset of disease was investigated. Tested in 2 replicates of each treatment group, 3 died, 2 wilted, 1 showed lesions,
The disease incidence index was determined by taking healthy ones as 0, and the disease incidence and the control rate were calculated by the following equations.

[0022]

(Equation 1)

(Equation 2)

As shown in Table 2, by treating the soil with the microorganisms of the present invention, the incidence of tomato seedling blight was significantly reduced as compared to the untreated plot, and an extremely high control effect was obtained. Was.

[0024]

[Table 2]

Example 4 (Example of controlling cucumber wilt disease) Fusarium oxysporum f sp.cu
cumerinum) was cultured in a bran medium for 3 weeks, and then mixed with a high-pressure heat sterilized soil at a ratio of 1% to prepare a contaminated soil. About 600g filling contaminated soil in a pot having a diameter of 15cm, the dried cells of the viable cells of lyophilized SD142 obtained by Example 2 of the test sections so that the number of viable bacteria per soil 1g is about 10 ⁸ Mixed into contaminated soil. Twenty cucumber seeds were sown in each pot and covered with 100 ml of sterilized soil. After cultivation in a greenhouse for about 3 weeks, the disease status was investigated. The test was performed in three repetitions of each treatment plot, and the ratio of the number of withered strains to the total number of strains was defined as the diseased strain rate, and the control rate was calculated based on the ratio.
As shown in Table 3, by treating the soil with the microorganism according to the present invention, the incidence of cucumber wilt was significantly reduced as compared with the untreated group, and an extremely high control effect was obtained.

[0026]

[Table 3]

Example 5 (Example of controlling melon root rot) A pot having a diameter of 9 cm is filled with about 250 g of soil contaminated with melon root rot fungus, and the culture solution of SD142 obtained in Example 2 or separated viable cells are isolated. Was mixed so that the number of bacteria per gram of soil was about 10 ⁷ . Pre-cultivated melon (variety: Melody No.2)
One seedling was planted per pot and cultivated in a greenhouse for one month. The test was performed in 9 repetitions of each treatment section, and the disease incidence and the control rate were calculated in the same manner as in Example 3. As shown in Table 4, by treating the soil with the microorganism according to the present invention, the incidence of melon root rot was significantly reduced as compared with the untreated plot, and an extremely high control effect was obtained.

[0028]

[Table 4]

Reference Example The effect of the combination use of iturin and surfactin was examined. Sucrose containing predetermined concentrations of iturin and surfactin
A pre-cultured plant pathogen was inoculated in the center of a petri dish on a potato plate medium and cultured at 25 ° C. Assuming that the growth area of the plant pathogenic bacteria on the medium containing no iturin and surfactin was 100, the area ratio (%) of the pathogenic bacteria growing on the test medium was measured, and the mycelial growth inhibition rate of the pathogenic bacteria was calculated. As shown in Table 5, the antibacterial action of iturin was remarkably enhanced by the presence of surfactin.

[0030]

[Table 5]

[0031]

As described above, the novel microorganism of the present invention has an extremely excellent effect on the control of plant diseases, especially on the control of plant diseases derived from soil diseases.

Claims (2)

(57) [Claims] 1. A novel microorganism belonging to Bacillus subtilis, Bacillus subtilis strain SD142 (Bacillus sub.
btilis SD142) (Microfabrication laboratory No. 13204). 2. A plant disease controlling agent comprising a culture of Bacillus subtilis SD142 strain and / or a microbial cell as an active ingredient.