JP3237240B2 - 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 plant disease control agent for controlling and controlling the disease of agricultural and horticultural crops and cultivating healthy crops.

[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.

On the other hand, it has been reported that antibacterial substances produced by microorganisms are effective for controlling various plant diseases. For example, iturin produced by certain strains belonging to the genus Bacillus is known to inhibit the growth of various phytopathogenic fungi (Japanese Unexamined Patent Publication Nos. 59-212416 and 59-124416).
1-289005, JP-A-61-2899898). However, it is not an effective and effective control method against plant diseases. That is, it cannot be said that it has reached a practical level.

[0007]

SUMMARY OF THE INVENTION As described above, the present inventor has studied on an iturin-like substance which is excellent in efficacy in order to develop a plant disease controlling agent which is highly safe and does not pollute the environment. .

In the course of studying the inhibitory effect of the iturin-like substance on the growth of phytopathogenic bacteria, the present inventors have found that the surfactin-based peptide alone does not exhibit antibacterial activity against phytopathogenic bacteria, but surprisingly, The present invention has been found to significantly enhance the antibacterial activity of iturin-based peptides against plant pathogenic bacteria by acting on them, and to show excellent effects in controlling various diseases such as cucumber vine scab and tomato seedling blight, and completed the present invention. I came to.

[0009]

Means for Solving the Problems The present invention provides an iturin-based peptide and a surfactin-based peptide at least one each.
It relates to a plant disease controlling agent containing at least one species.

Hereinafter, the present invention will be described in detail. The iturin-based peptide is a peptide-based substance represented by the following general formula (1) having an antibacterial activity against plant pathogenic bacteria (for example, Tetrahedron Letters 23 , No30, 3065 to 306).
8, 1982).

[0011]

Embedded imageOn the other hand, surfactin-based peptides have the following general formula:
A peptide-based substance represented by (2) (for example, BIOC
HEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONSThree
1488-494, 1968) has antibacterial activity against plant pathogenic bacteria
Is not known to date.

[0012]

Embedded image R and R 'in the formula are preferably straight-chain or branched alkyl groups having 3 to 6 carbon atoms. For example, R is CH ₃ CH ₂ C
H ₂ —, CH ₃ CH ₂ CH (CH ₃ ) —, CH ₃ CH _{2
CH 2 CH 2 CH 2 -,} CH 3 CH (CH 3) CH 2 C
H ₂ —, CH ₃ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ —,
R ′ is CH ₃ CH (CH ₃ ) CH ₂ —, CH ₃ C
_{H 2 CH 2 CH 2 -,} CH 3 CH 2 CH 2 - including without being limited thereto. The iturin-based peptide and the surfactin-based peptide, which are essential components of the control agent of the present invention, can be used to culture microorganisms having the ability to produce these substances, for example, by culturing a strain belonging to Bacillus subtilis and separating it from the culture solution. However, by using a strain that produces both substances, both substances can be produced more efficiently.

[0013] The composition of the medium and the cultivation conditions cannot be specified unconditionally depending on the strain to be used, but may be determined according to the production of general antibiotics. For example, as a carbon source used in the medium, glucose, saccharose, starch, starch and the like are used. As a nitrogen source such as a saccharified solution, sugars such as molasses, organic acids such as citric acid, and alcohols such as glycerin, ammonium salts and nitrates such as ammonia, ammonium sulphate, ammonium phosphate, ammonium salt and ammonium nitrate 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 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 may be added as needed. In general, the culture may be carried out by aeration and stirring under aerobic conditions. After completion of the culture, the method of isolating iturin or surfactin from the culture may be performed according to the method of separating a normal fermentation product from the culture. For example, a precipitation method using an acid, an extraction method using various organic solvents, a separation method using chromatography, and the like are appropriately combined.

The mixing ratio of the iturin peptide and the surfactin peptide cannot be unequivocally defined because it varies depending on the crop or disease to be applied, but it is 1: 9 to 9: 1, preferably 1: 2 to 2: 1. .

When these components are used as a plant disease controlling agent, they can be used as they are without adding other components, and various additives such as a solid carrier and an emulsifier can be added in accordance with a conventional method of agricultural chemical formulation. It can also be used in the form of granules, emulsions, wettable powders and the like together with the product.

The method for applying the pesticidal composition of the present invention is appropriately selected depending on the above-mentioned use form (preparation, etc.), crops and diseases, etc.
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.

The iturin-based peptide or surfactin-based peptide can be used without being separated from the culture medium, and a microorganism producing both active ingredients can be directly applied to a plant or soil, and can be used on the plant or soil. It is also possible to produce both components in it.

Examples of such microorganisms include, for example, Bacillus subtilus SD142 strain (Microtechnical Laboratory No. 13204) (hereinafter abbreviated as SD142). SD142 was isolated from compost and exhibited the following mycological properties.

Bacteriological properties (a) Form (1) Bacterial shape: rod-shaped (2) Bacterial size: 0.7-0.9 × 1.5-3.0 μm
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

[0020] The controlling agent of the present invention is Alternaria (Alterna).
ria), Cerospora, Phytoph
thora), Botrytis, Rhizoctonia
onia), Pyricularia, Cochliobolus, Fusarium, Pisium
hthium), Verticilium, Gibberella
(Gibberella), Xanthomonas (Xanthomonas), Pseudomonas (Pseudomonas), Agrobacterium (Agrobac
terium), Erwinia and the like, and has a remarkable effect on controlling diseases caused by pathogenic bacteria such as Erwinia.

In particular, soil wilt fungi such as bacterial wilt, seedling wilt, soft rot, wilt, wheat wilt, various fusarium wilts, wilt wilt, root rot, sheath wilt, crucifer, rape It is effective against clubroot fungi, crested wilt fungus, white silkworm fungus, turf large patch and the like.

[0022]

The present invention will be described in more detail with reference to the following examples. Example 1 The combined effect of an iturin-based peptide and a surfactin-based peptide (hereinafter sometimes simply referred to as iturin and surfactin, respectively) was examined. A sucrose-potato plate medium containing predetermined concentrations of iturin and surfactin was inoculated with a pre-cultured plant pathogen at the center of a petri dish,
Cultured at 25 ° C. The growth area of phytopathogenic bacteria on medium without iturin and surfactin is defined as 100.
Measure the area ratio (%) where the pathogenic bacteria grew on the test medium,
The mycelial growth inhibition rate of the pathogenic bacteria was calculated.

As shown in Table 1, the antibacterial action of iturin was significantly enhanced by the presence of surfactin.

[0024]

[Table 1]

Example 2 (Example of controlling tomato seedling damping-off) Tomato seedling damping-off Rhizoctonia cultivated on a vermiculite-brass 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
The pot m was filled with about 250 g of contaminated soil, and iturin and surfactin were dissolved in distilled water and mixed with the contaminated soil to a predetermined concentration. 15 seeds of tomato (variety: Momotaro) were sown in each pot and cultivated in a thermostat at 25 ° C. The disease status was examined at a height of 15 cm or 5 or more true leaves. The disease index was determined by assuming 3 withered, 2 withered, 2 with diseased spots and 0 with healthy lesions, and calculated the disease incidence and control rate by the following formulas.

[0026]

(Equation 1)

(Equation 2) As shown in Table 2, the coexistence of surfactin significantly enhanced the disease control effect of iturin.

[0027]

[Table 2]

Example 3 (Example of controlling cucumber gray mold) Cucumber seeds were planted in a pot having a diameter of 9 cm and cultivated in a greenhouse. Iturin and surfactin were dissolved at a predetermined concentration in distilled water, and about 10 ml per strain was sprayed on the seedlings 10 days later. After air-drying, the leaf surface of the cucumber gray mold fungus Botrytis cinerea (Botryt
is cinerea) and a relative humidity of 90
%, Cultivated at 25 ° C. for further 5 days, and measured the diameter of the lesion. The control rate was calculated by the following equation.

[0029]

(Equation 3) As shown in Table 3, the coexistence of surfactin significantly enhanced the disease control effect of iturin.

[0030]

[Table 3]

Example 4 100 ml of a medium having the following composition, adjusted to pH 7, and sterilized by high pressure heat and heat was placed in a 500 ml Sakaguchi flask.
One loopful of SD142 was inoculated at 35 ° C. and 120 rpm.
Pre-cultured for 10 hours under the following conditions. 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 3,000 rpm at 1 rpm.
Centrifugation was performed for 0 minutes to obtain a culture supernatant and viable cells.

Addition amount of medium components (g / l) ───────────────────────── glucose 10 polypeptone 30 KH ₂ PO ₄ 1 MgSO ₄ .7H ₂ O 0.5───────────────────────── The pH of the culture supernatant was adjusted to 2 by adding hydrochloric acid, and the precipitate was extracted with methanol. did. This extract was fractionated using a solvent of methanol / chloroform with a column of 30 mm in diameter and 25 cm in length packed with Kieselgel 60 (manufactured by Merck). The active fraction was analyzed by TLC (Merck Art.5554 Kieselgel6).
0 F ₂₅₄ ). Infrared absorption spectrum, mass spectrometry, amino acid analysis, NM
Confirmed by R analysis. In addition, the inactive fraction was purified by TLC and subjected to mass spectrometry, amino acid analysis and infrared absorption spectrum analysis to confirm that it was surfactin.
The contents of iturin and surfactin in the medium were 22
5 ppm and 307 ppm.

Example 5 An 8 mm diameter paper disc impregnated with the culture solution obtained in Example 4 was mixed with sucrose containing various plant pathogenic bacteria.
It was placed on a potato agar plate medium and cultured at 25 ° C.
One week later, the size of the blocking circle formed around the paper disk was examined.

As shown in Table 4, the culture solution containing iturin and surfactin inhibited the growth of a great variety of plant pathogens.

[0035]

[Table 4]

Example 6 (Example of controlling tomato seedling damping off) Tomato seedling damping off Rhizoctonia cultivar cultured on 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 filled in a pot of m, and the culture solution of SD142 obtained in Example 4 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. The test was conducted in two repetitions of each treatment section. At the time when the height of the plant was 15 cm or the true leaf was 5 or more, the onset of disease was investigated in the same manner as in Example 2.

As shown in Table 5, the incidence of tomato seedling wilt was significantly reduced by treating the soil with a culture or cells of a microorganism that produces iturin and surfactin, as compared to the untreated plot. However, an extremely high control effect was obtained.

[0038]

[Table 5]

Example 7 (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. Approximately 600 g of contaminated soil was filled in a pot having a diameter of 15 cm, 20 cucumber seeds were sown in each pot, and 100 ml of sterilized soil was covered with soil. SD1 obtained according to Example 4
The dried cells obtained by freeze-drying the 42 living cells were mixed so that the number of living cells per gram of soil was about 108. About 3 in the greenhouse
After cultivation for a week, the disease development 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 6, the treatment of microorganisms that produce iturin and surfactin on the soil significantly reduced the incidence of cucumber vine wilt disease, and showed a very high control effect. Obtained.

[0041]

[Table 6]

Example 8 (Example of Control of Melon Root Rot) A pot of 9 cm in diameter was filled with about 250 g of soil contaminated with melon root rot fungus, and the culture solution of SD142 obtained in Example 4 or separated viable cells were isolated. Was mixed so that the number of bacteria per gram of soil was about 107. 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 2.

As shown in Table 7, the incidence of melon root rot was significantly reduced by treating the soil with a culture or cells of a microorganism that produces iturin and surfactin, as compared to the untreated group. An extremely high control effect was obtained.

[0043]

[Table 7]

[0045]

As described above, an iturin-based peptide which did not show a sufficient plant disease control effect by itself, exhibits an extremely excellent control effect when used in combination with a surfactic peptide having no antibacterial activity. Thus, the plant disease controlling agent of the present invention can effectively control a wide range of plant diseases, particularly soil diseases, and can provide a plant disease controlling agent which is highly safe and does not pollute the environment.

Continuation of the front page (56) References JP-A-5-85911 (JP, A) JP-A-2-240004 (JP, A) JP-A-6-133763 (JP, A) JP-A-59-212416 (JP) , A) JP-A-61-289005 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A01N 63/02

Claims (1)

(57) [Claims] 1. A plant disease control agent comprising at least one kind of iturin peptide and at least one surfactin peptide.