JP7495050B2 - Soil treatment agent for agricultural and horticultural fungal disease inhibitors - Google Patents

Description

The present invention relates to an agricultural and horticultural plant disease suppression composition and a method for cultivating agricultural and horticultural plants using the same. More specifically, the present invention relates to a composition that contains a bacterium of the genus Bacillus, which induces disease resistance in plants and suppresses plant diseases, and a method for cultivating agricultural and horticultural plants in which the soil in which the plants are to be planted is treated with the composition.

Plant cultivation techniques are necessary in a variety of fields, including agriculture, tourism, disaster prevention, and education. During cultivation, irrigation and fertilization are carried out, and at the same time, appropriate treatments are carried out to eliminate pests and prevent diseases.
If appropriate disease control is not carried out, problems such as leaf spot development and leaf drop will occur in the leaves, browning of petals and poor flowering in the flowers, and reduced yield and effects on taste in the fruit will occur.
Major diseases of agricultural and horticultural plants include powdery mildew and black spot. For example, powdery mildew develops on cucumbers and eggplants, which are popular in home gardening, and affects the quality and yield of the fruit. Damage can be suppressed by spraying the leaves with Benica Green V Spray or Pancho TF granular water dispersible agent (both manufactured by Sumitomo Chemical Garden Products Co., Ltd.), which are available on the market, but foliar sprays require a sufficient amount of the agent to be applied evenly to the leaves, which takes time and effort.

As a measure against insect damage, for example, the insecticides Ortran Granules (manufactured by Sumitomo Chemical Gardening Co., Ltd.) and Dantotsu (registered trademark) Granules (manufactured by Sumitomo Chemical Co., Ltd.) are commercially available. When the base of a plant is treated with the agent, the active ingredient of the granules dissolves in the moisture of the soil. The plant's roots absorb the moisture in the soil, and the active ingredient is distributed throughout the plant. The worker only needs to spray the agent at the base of the plant, which is a significant labor saving compared to the foliar sprays mentioned above.
Labor-saving is also required for fungicides. A technology has been proposed to induce disease resistance in plants (e.g., rice) using chemically synthesized products (JP Patent Publication 7-184659; Patent Document 1), and Oryzemate (registered trademark) granules (manufactured by Meiji Seika Pharma Co., Ltd.) that improve plant disease resistance are on the market, but many users are concerned about the toxicity and environmental pollution of insecticides and fungicides synthesized by chemical reactions, and therefore, particularly in the case of home gardening, technologies that utilize natural substances and microorganisms are required.

A technique for suppressing disease damage by foliar treatment with Bacillus amyloliquefaciens is known, which relies on antagonism between pathogenic and non-pathogenic bacteria (Japanese Patent No. 3482462; Patent Document 2).
When disease resistance is induced in a plant, the concentration of salicylic acid in the plant body increases. It is known that an increase in the concentration of salicylic acid activates the transcription of genes encoding proteins that are effective in disease resistance, such as PR1 and PR5, among PR proteins (PR: Pathogenesis Related), and produces antibacterial substances (Cell Engineering Special Issue, Plant Cell Engineering Series 19, New Edition: Plant Disease Resistance from the Molecular Level, Shujunsha; Non-Patent Document 2).
A method for controlling bacterial wilt disease utilizing the disease resistance induction effect of Bacillus thuringiensis in tomato is known (Microbes Environ., Vol. 28, No. 1, 128-134, 2013; Non-Patent Document 1). This method is different from the present invention, which is directed to the control of plant diseases transmitted through above-ground parts, in that it targets the control of plant diseases transmitted through soil by treating soil with a microbial preparation.

Japanese Patent Application Laid-Open No. 7-184659 Japanese Patent No. 3482462

Microbes Environment. , Vol. 28, No. 1,128-134,2013 Cell Engineering Special Issue Plant Cell Engineering Series 19 New Edition Plant Disease Resistance from the Molecular Level Shujunsha

The objective of the present invention is to provide a composition for suppressing agricultural and horticultural plant diseases that contains naturally occurring substances as active ingredients, and a method for cultivating agricultural and horticultural plants using the composition.

As a result of extensive research, the inventors discovered that treating soil with Bacillus bacteria can induce resistance in plants and protect them from diseases that infect above-ground parts, thus completing the present invention.

That is, the present invention relates to the following agricultural and horticultural plant disease suppression compositions [1] to [6] and agricultural and horticultural plant cultivation methods [7] to [10].
[1] A composition for suppressing agricultural and horticultural plant diseases, comprising a bacterium of the genus Bacillus as an active ingredient.
[2] The agricultural and horticultural plant disease suppression composition according to the above item 1, wherein the Bacillus bacterium is Bacillus thuringiensis.
[3] The agricultural and horticultural plant disease suppression composition according to the above item 1 or 2, wherein the Bacillus bacterium induces disease resistance in a plant body.
[4] The agricultural and horticultural plant disease suppression composition according to any one of the above items 1 to 3, wherein the agricultural and horticultural plant disease is a disease caused by a filamentous fungus.
[5] The agricultural and horticultural plant disease suppression composition according to the above item 4, wherein the agricultural and horticultural plant disease caused by a filamentous fungus is powdery mildew and black spot.
[6] The composition for suppressing agricultural and horticultural plant diseases according to any one of items 1 to 5 above, wherein the agricultural and horticultural plant is a rose, a cucumber, an eggplant or a strawberry.
[7] A method for cultivating an agricultural or horticultural plant, comprising treating soil for cultivating the agricultural or horticultural plant with the agricultural or horticultural plant disease suppression composition according to any one of items 1 to 6 above.
[8] The method for cultivating an agricultural or horticultural plant according to item 7, wherein the agricultural or horticultural plant disease suppression composition is sprayed or mixed into soil for cultivating the agricultural or horticultural plant.
[9] The method for cultivating an agricultural or horticultural plant according to the above item 7 or 8, wherein the agricultural or horticultural plant is a rose, a cucumber, an eggplant or a strawberry.
[10] The method for cultivating an agricultural or horticultural plant according to any one of items 7 to 9, wherein the agricultural or horticultural plant disease suppression composition in an amount equivalent to 1.3×10 ¹⁰ to 1.3×10 ¹² CFU of Bacillus bacteria per plant is sprayed or mixed into soil.

The agricultural and horticultural plant disease suppression composition of the present invention, which contains Bacillus bacteria as an active ingredient for suppressing agricultural and horticultural plant diseases, is an agricultural chemical that contains a naturally occurring substance as an active ingredient, which is in demand in home gardening, and allows consumers to cultivate agricultural and horticultural plants without worrying about toxicity or environmental pollution. In addition, agricultural and horticultural plants can be cultivated using soil treatment techniques that contribute to reducing the labor required by users.

1 is a graph showing the results of Test Example 1 (expression levels of PR1 and PR2).

The active ingredient of the agricultural and horticultural plant disease suppression composition according to the present invention is a bacterium of the genus Bacillus.
The genus Bacillus is a type of eubacteria that is classified into the order Bacillales, class Bacilli, phylum Firmicutes, and family Bacillaceae. Examples of species include Bacillus subtilis, Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis. Bacillus is a very common type of bacteria that is widely distributed in nature.
In the agricultural chemical industry, Bacillus subtilis is used as a disease control agent utilizing its antagonistic action against pathogenic bacteria (e.g., Botkiller (registered trademark) wettable powder; Idemitsu Kosan Co., Ltd.), and Bacillus thuringiensis is used as an insecticide because it contains a crystalline protein toxin that kills lepidoptera that ingest it (e.g., Zentari (registered trademark) wettable powder; Sumitomo Chemical Co., Ltd. and others).

The agricultural and horticultural plant disease suppression composition of the present invention exhibits disease suppression effects on agricultural and horticultural plants by treating soil with the active ingredient (Bacillus bacteria). It is believed that the composition of the present invention exerts its effect by inducing disease resistance in plants by the Bacillus bacteria.
The amount of the agricultural and horticultural plant disease suppression composition used for soil treatment is not particularly limited as long as it is an amount that exhibits an agricultural and horticultural plant disease suppression effect, but is usually an amount that results in a bacterial concentration of the Bacillus bacteria of 1.3 x ¹⁰ to 1.3 x ¹⁰ CFU per plant. If the concentration is too low, the medicinal effect will not be obtained, and if it is too high, mass production will be difficult in terms of cost.
In the present invention, the bacteria (bacterial culture) can be used as is, but in consideration of workability, it is preferable to formulate the bacteria into granules, dusts, wettable powders, etc., in the same manner as ordinary pesticides.
For example, in the case of granules or powders, surfactants, binders, colorants, preservatives, antioxidants, bulking agents, etc. can be mixed as desired.

As the surfactant, a nonionic surfactant, an anionic surfactant, or the like can be used.
Examples of nonionic surfactants include polyoxyalkylene allyl phenyl ethers, polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene allyl phenyl ethers, polyoxyethylene styryl phenyl ethers, polyoxyethylene alkyl phenyl ether formaldehyde condensates, polyoxyethylene-polyoxypropylene block polymers, polyoxyethylene-polyoxypropylene block polymer alkyl phenyl ethers, sorbitan fatty acid esters (e.g., sorbitan monooleate, sorbitan laurate), polyoxyethylene fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene castor oil ethers, and polyethylene glycol fatty acid esters.

Examples of anionic surfactants include sodium, calcium, or ammonium salts of alkyl sulfate, polyoxyethylene alkyl ether sulfate, polyoxyethylene alkyl phenyl ether sulfate, polyoxyethylene benzyl (or styryl) phenyl ether sulfate, or polyoxyethylene-polyoxypropylene block polymer sulfate; sodium, calcium, ammonium, or alkanolamine salts of alkyl sulfonate, dialkyl sulfosuccinate, alkyl benzene sulfonate (e.g., calcium dodecyl benzene sulfonate, etc.), mono- or di-alkyl naphthalene sulfonate, naphthalene sulfonate formaldehyde condensate, lignin sulfonate, polyoxyethylene alkyl phenyl ether sulfonate, or polyoxyethylene alkyl ether sulfosuccinate; and sodium or calcium salts of polyoxyethylene alkyl ether phosphate, polyoxyethylene mono- or di-alkyl phenyl ether phosphate, polyoxyethylene benzyl (or styryl) phenyl ether phosphate, or polyoxyethylene-polyoxypropylene block polymer phosphate.

Examples of binders include polyvinyl alcohol, polyacrylamide, carboxymethyl cellulose, sodium alginate, starch cyclodextrin, dextrin, and lignin sulfonate.

Examples of colorants include zinc oxide, cuprous oxide, watching red, chlorine method titanium oxide pigment, oil furnace black, yellow lead, oxysulfide phosphor, cadmium yellow, cadmium red, fluorescent pigment, graphite, black iron oxide, ultrafine calcium carbonate, cobalt blue, cobalt green, cobalt purple, chalk powder, Prussian blue, thermal black, chromium oxide, titanium oxide (athanase), titanium oxide (rutile), copper oxide, disazo yellow, red iron oxide, granulated carbon black, brown iron oxide, channel black, ultrafine titanium oxide, iron black, natural graphite powder, copper phthalocyanine blue, copper phthalocyanine green, vanadate phosphor, fine titanium oxide, fast yellow 10G, red iron oxide, and molybdenum red.

Examples of preservatives include Preventol (registered trademark) D2 (chemical name: benzyl alcohol mono (poly) hemiformal), PROXEL (registered trademark) GXL (S) (chemical name: 1,2-benzisothiazolin-3-one, 20%), Biohope (registered trademark) and Biohope L (chemical name: organic nitrogen sulfur compound, organic bromine compound), Bestside (registered trademark) -750 (chemical name: isothiazolin-based compound, 2.5 to 6.0%), 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, 2-bromo-2-nitropropane-1,3-diol, sodium benzoate, potassium sorbate, sodium dehydroacetate, parachlorometaxylenol, and 2,6-dimethylphenol.

Examples of antioxidants include tetrakis[methylene-3-(3,5-di-t-butyl-4-hydroxyphenyl)propionate]methane (Tominox (registered trademark) TT, API Corporation, product name/IRGANOX (registered trademark) 1010 or IRGANOX 1010EDS, Ciba Japan, product name), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, mixed tocopherols, α-tocopherol, ethoxyquin, and ascorbic acid.

Examples of bulking agents include clay, calcium carbonate, talc, bentonite, silica sand, silica stone, zeolite, and diatomaceous earth.

In addition, in the case of extrusion granulation, it may be necessary to add water. The water that can be used may be tap water, ion-exchanged water, distilled water, filtered water, sterilized water, groundwater, etc.

The agricultural and horticultural disease suppression composition of the present invention is effective against diseases caused by filamentous fungi, including powdery mildew and black spot.
Target horticultural plants include grapes, wheat, peaches, strawberries, tomatoes, cucumbers, roses, mulberries, pears, persimmons, apples, sweet potatoes, etc., and among these, roses, cucumbers, eggplants, and strawberries are preferred.

In the present invention, a culture containing a bacterium of the genus Bacillus or a preparation prepared from the culture is applied to soil to suppress plant diseases. Specifically, the method includes applying the preparation to the surface of the soil after seeds germinate or around the base of planted seedlings, or mixing the preparation into the soil at the same time as sowing seeds or planting seedlings.

The present invention will now be described in more detail with reference to examples, comparative examples, and test examples, but the present invention is not limited to these examples. In the following examples, "parts" refers to parts by mass. The components used in the formulations of the examples and comparative examples are as follows.
(1) Bacillus thuringiensis Zentari Water Dispersible Granule Active Ingredient (Sumitomo Chemical Co., Ltd.)
(2) Sodium lignin sulfonate: New Calgen WG4 (manufactured by Takemoto Oil Co., Ltd.)
(3) Polyoxyalkylene allyl phenyl ether sulfate ammonium: New Calgen FS-700PG (manufactured by Takemoto Oil Co., Ltd.)
(4) Clay: for clay granules (manufactured by Shokozan Kogyosho Co., Ltd.)

Example 1:
The composition of Example 1 was prepared by mixing and stirring Bacillus thuringiensis Zentari active ingredient (the active ingredient of Zentari granular water dispersible sold by Sumitomo Chemical Co., Ltd.: manufactured by Sumitomo Chemical Co., Ltd., hereinafter referred to as BT active ingredient) with sterilized ion-exchanged water to a concentration of 3.0 x 10 ⁸ CFU/mL.
The BT bulk substance was diluted with sterilized water and cultured on a standard agar medium at 35° C. for 48 hours, whereupon 6.7×10 ¹⁰ CFU per gram were detected.

Example 2:
A test group treated with only BT technical substance at 1.3×10 ¹¹ CFU (2.0 g) was designated as Example 2.

Example 3:
A test group in which only BT technical substance was treated at 6.7×10 ¹⁰ CFU (1.0 g) was designated as Example 3.

Example 4:
Example 4 was a test group in which 3.4×10 ¹⁰ CFU (0.5 g) of BT technical substance alone was treated.

Example 5:
Example 5 was a test group in which 1.3×10 ¹⁰ CFU (0.2 g) of BT technical substance alone was treated.

Example 6:
Example 6 was a test group in which 2.6×10 ⁹ CFU (0.04 g) of BT technical substance alone was treated.

Example 7:
20 parts of BT bulk material, 2 parts of New Calgen WG4, 3 parts of New Calgen FS-700PG, and the remaining part of clay (total 100 parts) were mixed. 18 parts of tap water was added for granulation in an extrusion granulator. After granulation, the mixture was dried in a fluidized bed dryer (water was not included in the final composition) and treated in the test area designated as Example 7.

Comparative Example 1:
A composition consisting of sterile water alone was used as Comparative Example 1.

Comparative Example 2:
The test area that was not treated was used as Comparative Example 2.

Test Example 1:
Tomatoes were sown in 128-hole horticultural cell trays filled with sterilized horticultural soil (Nippi Pp horticultural soil, Nippon Hiryo Co., Ltd.) and grown for 3 weeks in a growth chamber (conditions: 25°C for 16 hours (light: fluorescent light on), 25°C for 8 hours (dark: fluorescent light off, appropriate irrigation with sterilized water).
The seedlings together with the soil were removed from the cell tray, placed in the liquid of Example 1 (BT bulk dilution solution) and Comparative Example 1 (sterile water), and maintained for 48 hours under the above-mentioned growth chamber conditions.
Thereafter, RNA was extracted from the test plants, and the gene expression state was confirmed by quantitative PCR. The results are shown in FIG.
As shown in Figure 1, induction of expression of the biodefense protein genes PR1 and PR5 was confirmed by treating one strain with 3.0 x ¹⁰⁸ in Example 1. It was confirmed that disease resistance was enhanced by treating Bacillus bacteria.

Test Example 2:
The eggplants were planted, and 13 days later, the compositions of Examples 2 to 6 were sprayed around the base of the plants. After the treatment, the plants were irrigated with sufficient water for 3 days. After 1 day from the treatment, powdery mildew fungus was inoculated by dusting method. The control effect was investigated 29 days after the treatment, and the results are shown in Table 1. Comparative Example 2 was also carried out at the same time as a control.

The disease severity and control value in Test Examples 2, 3, 4 and 6 were calculated as follows according to the method prescribed in the New Pesticide Practical Application Test Manual.
[Severity of disease]
The surveyed leaves were visually inspected and the severity of the disease was classified according to the following criteria.
0: No lesions observed.
1: A few lesions (several) are observed.
2: Lesions occupy less than 1/4 of the leaf area.
3: Lesions occupy less than 1/4 to 1/2 of the leaf area.
4: Lesions occupy more than 1/2 of the leaf area.

The above classification results were used to obtain the severity of disease for each iteration using the following formula (1).

効果は、防除価により下記のＡ～Ｄに評価される。新農薬実用化試験ではＣ以上で効果ありと認定される。
Ａ：９１以上
Ｂ：７１～９１
Ｃ：５１～７０
Ｄ：５０以下 [Control value]
Using an untreated plot as a control, the control value was calculated according to the following mathematical formula (2): The disease incidence used to calculate the control value was the average value of the replicates.

The effectiveness is rated as A to D according to the control value. In the new pesticide practical application test, a rating of C or higher is recognized as effective.
A: 91 or more B: 71 to 91
C: 51-70
D: 50 or less

The results in Table 1 show that in order to obtain efficacy against eggplant/powdery mildew, treatment with 1.3×10 ¹⁰ CFU/strain or more is necessary.

Test Example 3:
With an eye on industrial use, a test was conducted on a composition formulated into granules (Example 7) and compared with the BT technical substance itself (Example 2) and with no treatment (Comparative Example 2). The granules were prepared as follows.
Cucumbers were planted, and one day later, 2.0 g of the active ingredient of Example 2 and 10 g of the formulation (granules) of Example 7 were sprayed around the base of the plants. Five days after the treatment, powdery mildew fungus was inoculated by dusting method. The control effect was investigated 25 days after the treatment, and the results are shown in Table 2. Comparative Example 2 was also carried out at the same time as a control.

The results in Table 2 confirm that both the BT active ingredient and the granular formulation are effective against cucumber powdery mildew.

Test Example 4:
Tests were carried out on the test plots of Example 7 and Comparative Example 2 as follows.
Strawberries were planted, and 32 days later, 5 g of the granules of Example 7 were mixed into the soil and treated. One day and 14 days after the treatment, powdery mildew was inoculated by dusting method. The control effect was investigated 30 days after the treatment, and the results are shown in Table 3. Comparative Example 2 (untreated) was also carried out as a control.

The results in Table 3 confirm that the compound has efficacy against strawberry powdery mildew.

Test Example 5:
Tests were carried out on the test plots of Example 7 and Comparative Example 2 as follows.
Thirty-two days after planting, the base of the rose was treated with 5 g of the granules of Example 7. Black spot was allowed to develop naturally, and the control effect was investigated 14 days after the treatment, and the results are shown in Table 4. Comparative Example 2 (untreated) was also carried out as a control.

The results in Table 4 confirm that the compound has efficacy against rose black spot.

The agricultural and horticultural plant disease suppression composition of the present invention is an agricultural chemical that contains as an active ingredient a naturally occurring substance (Bacillus bacteria) that is in demand in home gardening, and allows users to cultivate agricultural and horticultural plants safely without the risk of environmental pollution.

Claims (4)

Soil treatment containing Bacillus bacteria as an active ingredient for suppressing fungal diseases of agricultural and horticultural plants
4. The soil treatment agent for suppressing a disease caused by a filamentous fungus in an agricultural or horticultural plant, wherein the disease caused by a filamentous fungus is powdery mildew or black spot . The soil treatment inhibitor for fungal diseases of horticultural plants according to claim 1, wherein the bacillus bacterium is Bacillus thuringiensis. The soil treatment inhibitor for fungal diseases in horticultural plants according to claim 1 or 2, in which the Bacillus bacterium induces disease resistance in plants. 4. The agent for suppressing a disease caused by a filamentous fungus in an agricultural or horticultural plant for soil treatment according to claim 1 , wherein the agricultural or horticultural plant is a rose, a cucumber, an eggplant, or a strawberry.

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