JP7514142B2 - Plant disease control agent and plant disease control method - Google Patents

Description

NPMD NITE P-03227 NPMD NITE P-03228

The present invention relates to a plant disease control agent and a plant disease control method.

Traditionally, chemically synthesized pesticides have been used to minimize damage caused by pests and disease-causing bacteria and to increase crop productivity, as they are both effective and economical. However, in recent years, there has been growing interest in biological pesticides, which use living organisms or substances derived from living organisms as active ingredients, from the perspective of their impact on the environment and on humans and livestock.

Biological pesticides are broadly divided into four types: natural enemy insects, natural enemy nematodes, microorganisms (viruses, bacteria, fungi, protozoans, etc.), and biologically produced substances (pheromones, hormones, acidic toxins, extracts, etc.). Among these, microbial pesticides are pesticides that use microorganisms to control crop diseases, and are expected to reduce the burden on the environment compared to chemically synthesized pesticides, and to suppress the emergence of bacteria and pests that are resistant to chemically synthesized pesticides.

For example, Bacillus subtilis does not have the ability to directly attack pathogens that cause plant diseases, but because it is antagonistic to certain pathogens, it is registered as an agricultural chemical to control gray mold and powdery mildew on vegetables.

As a technology for controlling plant diseases using Bacillus bacteria, Patent Documents 4 and 5 disclose a plant disease control agent that contains a specific strain of Bacillus subtilis as an active ingredient.

Japanese Patent Application Laid-Open No. 5-51305 Japanese Patent Application Laid-Open No. 6-133763

The present invention has been made in consideration of the above circumstances, and provides a novel Bacillus bacterium having excellent plant disease control activity, a plant disease control agent containing the Bacillus bacterium, and a plant disease control method using the plant disease control agent.

That is, the present invention includes the following aspects.
(1) A plant disease control agent comprising, as an active ingredient, one or more types of Bacillus subtilis cells or cultures selected from the group consisting of Bacillus sp. TTCC2111 strain (NITE P-03227) and Bacillus sp. TTCC2122 strain (NITE P-03228).
(2) The plant disease control agent according to claim 1, wherein the plant disease is selected from the group consisting of gray mold, green mold, blue mold and powdery mildew.
(3) A method for controlling a plant disease, comprising applying the plant disease control agent according to claim 1 or 2 to a target plant.
(4) Bacillus sp. TTCC2111 strain (NITE P-03227).
(5) Bacillus sp. TTCC2122 strain (NITE P-03228).

The plant disease control agent and plant disease control method of the above aspect can effectively control plant diseases.

1 is a graph showing the disease incidence rate of mandarin oranges in Example 3. 1 is a graph showing the average number of spores per colony of Erysiphe pea powdery mildew in Example 4. 1 is a graph showing the germination rate of conidia of Erysiphe pea powdery mildew in Example 4. 1 is a graph showing the number of lesions per ^cm2 of primula petals in Example 5. 1 is a graph showing the severity of cucumber powdery mildew in Example 6. 1 is a graph showing the disease severity of cucumber powdery mildew in Example 7. 1 is a graph showing the disease severity of cucumber powdery mildew in Example 8. 1 is a graph showing the diameter of lesions on cucumber leaves in Example 10. 1 is a graph showing the diameter of lesions on begonia leaves in Example 10.

<Plant disease control agents>
A plant disease control agent according to one embodiment of the present invention contains, as an active ingredient, one or more types of Bacillus subtilis cells or cultures selected from the group consisting of Bacillus sp. TTCC2111 strain (NITE P-03227) (hereinafter, sometimes simply referred to as "TTCC2111 strain") and Bacillus sp. TTCC2122 strain (NITE P-03228) (hereinafter, sometimes simply referred to as "TTCC2122 strain").

In the present invention, "containing Bacillus subtilis cells or a culture as an active ingredient" means containing Bacillus subtilis cells or a culture as an ingredient that has the effect of controlling plant diseases.

The plant disease control agent of this embodiment can efficiently suppress the proliferation and activity of a wide range of pathogenic bacteria that cause plant diseases.
The plant disease control agent of the present embodiment is not toxic or pathogenic to plants and has a high control effect against plant diseases. In addition, since it is a microbial pesticide that uses bacteria present in nature as an active ingredient, it is highly safe and has a small impact on the environment compared to chemical pesticides. Even if it is applied, there are no problems such as danger to humans and livestock, environmental pollution, or residues in agricultural crops. Therefore, it is possible to provide consumers with agricultural crops that are safe and free of chemical damage. In addition, the incidence of resistant bacteria of the target pathogen is very low compared to chemical pesticides.

As shown in the examples described later, the Bacillus sp. TTCC2111 strain was screened for Bacillus subtilis having a growth-inhibiting effect against Botrytis cinerea, and the Bacillus sp. TTCC2122 strain was screened for Bacillus subtilis having a growth-inhibiting effect against Penicillium digitatum, and they were selected as Bacillus subtilis having a growth-inhibiting effect against Botrytis cinerea and Bacillus subtilis having a growth-inhibiting effect against Penicillium digitatum, respectively.

Bacillus sp. TTCC2111 strain and Bacillus sp. TTCC2122 strain (hereinafter collectively referred to as "Bacillus subtilis of this embodiment") are newly isolated and identified Bacillus subtilis, and are very useful novel Bacillus subtilis that have the effect of controlling plant diseases. Therefore, the inventors deposited these Bacillus subtilis as new strains at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on May 28, 2020. The accession numbers are NITE P-03227 for Bacillus sp. TTCC2111 strain and NITE P-03228 for Bacillus sp. TTCC2122 strain.

The Bacillus subtilis bacteria of this embodiment are preferably viable bacteria. Note that "viable bacteria" here refers to a state in which the bacteria are capable of metabolism, division, proliferation, etc. Therefore, spores of Bacillus subtilis are also included in viable bacteria as long as they have the ability to germinate. However, the plant disease control agent of this embodiment may contain dead Bacillus subtilis bacteria.

The Bacillus subtilis cells of this embodiment can be used as a bacterial powder. The method for preparing the bacterial powder is not particularly limited and can be appropriately selected from methods commonly used for preparing Bacillus subtilis bacterial powder, such as freeze-drying (lyophilization), spray-drying, and drum-drying. Since the bacterial powder can be obtained in the form of live bacteria, it is preferable to prepare the bacterial powder by the freeze-drying method.

The culture of Bacillus subtilis in this embodiment is not particularly limited as long as it is obtained by culturing Bacillus subtilis in a medium, and may be a culture solution obtained by growing the cells in a medium, a supernatant of the culture solution (a culture solution from which solid components such as cells have been removed by centrifugation or the like), a concentrate of the culture solution, a culture solution subjected to a cell crushing treatment or the like, or a culture solution from which intracellular components have been extracted. That is, the plant disease control agent of this embodiment may contain, as an active ingredient, a metabolite of Bacillus subtilis in this embodiment or a preparation thereof (e.g., a dried product, a concentrate, an extract, etc.). The centrifugation treatment, the concentration method, the cell crushing treatment, the extraction method, the fermentation method, etc. can be appropriately selected from known methods and carried out by ordinary methods.

The medium for culturing the Bacillus subtilis in this embodiment is not particularly limited as long as it is a medium in which each Bacillus subtilis can grow, and can be appropriately selected from media commonly used in culturing Bacillus subtilis and modified media thereof. Specific examples include Tryptic Soy Agar plate medium and Potato Sucrose Broth (PSB) medium.

The culture format is not particularly limited and can be appropriately determined taking into consideration the culture scale, the dosage form of the plant disease control agent, etc. For example, the culture may be applied to an agar plate medium or in a liquid medium. Examples of culture formats in liquid medium include stationary culture and batch culture. For example, in the case of subculture, it is preferable to culture on an agar plate medium or to culture stationarily or batch culture in an appropriate liquid medium, because this is simple.

The culture conditions for the Bacillus subtilis of this embodiment are not particularly limited, and the Bacillus subtilis can be cultured under conditions generally used for culturing Bacillus subtilis. For example, the culture temperature is preferably 20°C or higher and 40°C or lower, and more preferably 25°C or higher and 37°C or lower. The pH of the medium is preferably 3.5 or higher and 8.0 or lower, and more preferably 4.0 or higher and 7.0 or lower. In addition, the Bacillus subtilis of this embodiment is preferably cultured under aerobic conditions, like other Bacillus subtilis.

The Bacillus subtilis fungus cells or cultures that are the active ingredient of the plant disease control agent of this embodiment may consist of the fungus cells or cultures of only one type of Bacillus subtilis, either the TTCC2111 strain or the TTCC2122 strain, or may be a mixture of the fungus cells or cultures of these two types of Bacillus subtilis. Of these, it is preferable to use the fungus cells or cultures of the TTCC2122 strain alone as the active ingredient, as this is expected to be effective against a wider range of plant diseases.

The plant disease control agent of this embodiment may consist only of the fungus or culture of Bacillus subtilis of this embodiment, or may be a plant disease control composition (hereinafter sometimes simply referred to as the "composition of this embodiment") that further contains other substances according to the desired formulation to the extent that the control effect against plant diseases by the fungus or culture of Bacillus subtilis of this embodiment is not inhibited.

Other substances include carriers, surfactants, dispersants, adjuvants, and protective agents that are acceptable for use in pesticide formulations.

"Carrier acceptable for agricultural chemical formulations" refers to a substance that facilitates the application of a plant disease control agent, maintains the survival of Bacillus subtilis and antagonistic or suppressive effects against plant pathogens, and/or controls the rate of action of the plant disease control agent, and is non-hazardous or has low toxicity to the environment, such as soil or water quality, and/or animals (especially humans).

Specific examples of such carriers include porous solid carriers such as talc, bentonite, kaolin, clay, diatomaceous earth, white carbon, vermiculite, hydrated lime, ammonium sulfate, silica sand, and urea; and liquid carriers such as water, isopropyl alcohol, methylnaphthalene, xylene, cyclohexanone, and alkylene glycol.

Examples of surfactants and dispersants include dinaphthyl methanesulfonate, alcohol sulfate, lignin sulfonate, alkylaryl sulfonate, polyoxyethylene glycol ether, polyoxyethylene sorbitan monoalkylate, polyoxyethylene alkylaryl ether, etc.

Examples of the adjuvant include carboxymethylcellulose, polyethylene glycol, propylene glycol, gum arabic, and xanthan gum.
Examples of the protective agent include skim milk and pH buffers.

The composition of this embodiment may further contain other active ingredients having pharmacological actions, specifically herbicides, fungicides, insecticides, fertilizers (e.g., urea, ammonium nitrate, superphosphates), etc., to the extent that they do not affect the active ingredient Bacillus subtilis.

The dosage form is not particularly limited as long as it can maintain the active ingredient Bacillus subtilis in a viable state, and can be, for example, in a liquid state, a solid state, or a combination thereof.
Examples of liquid forms include water dispersible granules, wettable powders, water-soluble agents, suspension preparations, emulsions, etc. Examples of solvents for suspending the bacterial cells in liquid forms include water (including sterilized water, deionized water, and ultrapure water), physiological saline, buffer solutions (including phosphate buffer solutions and carbonate buffer solutions), and culture media for the bacteria.
Examples of the solid form include granules, powders, gels, and the like.

The content of the Bacillus subtilis fungus or culture per a given amount of the plant disease control agent of this embodiment varies depending on various conditions such as the type of each Bacillus subtilis or its combination, the type of the target plant to be applied, the formulation, and the application method. Usually, it is preferable that the Bacillus subtilis is contained in an amount sufficient for exerting an antagonistic or suppressive effect against plant pathogens when the plant disease control agent of this embodiment is applied. This content may be determined by taking into consideration each condition so that the Bacillus subtilis fungus or culture is a desired (abundant) amount per a given area of the target plant to be treated after application within the scope of technical common knowledge in the field. The content of Bacillus subtilis in the plant disease control agent of this embodiment can be, for example, in the range of about 10 ⁹ cfu/g or more and 10 ¹¹ cfu/g or less in terms of fungus concentration. Alternatively, when the plant disease control agent of the present embodiment is in a liquid state, the content of Bacillus subtilis can be, for example, in terms of cell concentration, in the range of about ¹⁰⁶ cells/mL to ¹⁰¹⁰ cells/mL. In this case, the agent can be diluted 2-fold to 1000-fold with water, physiological saline, a buffer solution, or the like at the time of application, as necessary.

<Plant disease control method>
A plant disease control method according to one embodiment of the present invention includes applying the above-mentioned plant disease control agent to a target plant (hereinafter, sometimes referred to as the "application step").

In this specification, the term "target plant" refers to a plant to which a plant disease control agent is applied. The target plant may be of any type, so long as it is a plant that can develop a disease due to infection with a pathogen. The target plant may be either an angiosperm or a gymnosperm. The angiosperm may be either a dicotyledonous plant or a monocotyledonous plant.
Examples of monocotyledonous plants include plants of the Poaceae family.
Examples of dicotyledonous plants include plants of the Convolvulaceae family, the Rosaceae family, the Apiaceae family, the Solanaceae family, the Liliaceae family, the Fabaceae family, the Cucurbitaceae family, and the Brassicaceae family.

Specific target plants include, for example, peas (Pisum sativum), cucumbers (Cucumis sativus), peppers (Capsicum annuum Group), tomatoes (Solanum lycopersicum), eggplants (Solanum melongena), strawberries (Fragaria ananassa), mandarin oranges (Citrus unshiu), begonias, and primulas.

[Application process]
In the application step, the above-mentioned plant disease control agent is applied to a target plant.
The plant disease control agent may be directly applied as it is, or may be diluted in a solvent such as water before use. Specific examples of the application method of the plant disease control agent include a method of directly spraying the plant, a method of spraying the soil, a method of directly applying the agent to the seeds of the plant, and a method of adding the agent to water or fertilizer added to the plant or soil. The application amount and number of applications of the plant disease control agent can be appropriately adjusted depending on the target disease, the target crop, the application method, the tendency of occurrence, the degree of damage, the environmental conditions, the formulation used, and the like. The application time of the plant disease control agent is not particularly limited, and the agent may be applied for the purpose of exerting a control effect before the occurrence of the disease, or may be applied for the purpose of suppressing the growth of pathogens and treating the disease after the occurrence of the disease.

The plant disease control agent of the present embodiment has an excellent control effect against a wide variety of bacteria and fungi. Examples of plant pathogens that can be controlled by the plant disease control agent of the present embodiment include black spot disease fungus (Diaporthe citri), common scab disease fungus (Elsinoe fawcettii), brown rot disease fungus (Phytophthora citrophthora), green mold disease fungus (Penicillium digitatum), and blue mold disease fungus (Penicillium italicum);
"Cucurbits" powdery mildew (Sphaerotheca fuliginea), vine wilt (Didymella bryoniae), anthracnose (Colletotrichum lagenarium);
"Tomato" leaf spot fungus (Alternaria solani), leaf mold fungus (Cladosporium fulvum), powdery mildew fungus (Oidium neolycopersici, Oidiopsis sp., Leveillula taurica);
Brown spot disease fungus (Phomopsis vexans) and powdery mildew fungus (Erysiphe cichoracearum) of eggplant;
"Strawberry" powdery mildew (Sphaerotheca humuli), anthracnose (Glomerella cingulata);
Powdery mildew fungus (Oidiopsis sicula) on bell pepper;
"Pea" powdery mildew (Erysiphe pisi);
"Various crops" include, but are not limited to, Botrytis cinerea.

The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[Example 1]
(Selection of natto bacteria that control green mold disease)
1. Preparation of natto bacteria culture solution As the natto bacteria, the natto bacteria 720 strain owned by Takano Foods Co., Ltd. was used. The natto bacteria culture solution was prepared using the following method. First, each strain was cultured on a Trypticase Soy Agar (TSA) plate medium at 37°C for 16 hours, suspended in a Trypticase Soy Broth (TSB) medium with a platinum loop, and cultured with shaking at 28°C and 105 rpm for 24 hours. After culturing, the natto bacteria were collected by centrifugation (3000rcf, 8 minutes, 22°C), and the supernatant was removed and then suspended in sterilized water. This operation was repeated three times to remove the medium components, and the culture solution concentration was adjusted to _OD600 = 1 using a spectrophotometer and used for the experiment.

2. Preparation of conidial suspension Penicillium digitatum, a green mold pathogen in citrus, was used as the IUPd2 strain stored at the Ibaraki University Faculty of Agriculture. Conidial formation was induced by culturing the fungus on a Potato Sucrose Agar (PSA) plate medium for 3 days, irradiating it with a black light blue lamp for 3 days, and then placing it in the dark for 3 days. The conidia on the medium were suspended in sterilized water, and then filtered with sterilized tissue paper to remove the mycelium. The conidia were collected by centrifugation (1700rcf, 5 minutes, 22°C), and the supernatant was removed and then resuspended in sterilized water. This process was repeated three times to wash the conidia. The conidial suspension was prepared to a concentration of 1.0 x 10 ⁵ conidia/mL using a hemocytometer and used in the experiment.

3. Confrontation culture test 100 μL of P. digitatum conidial suspension was dropped onto a PSA plate medium and spread with a conlarger stick, after which a paper disk (diameter 5 mm) soaked in Bacillus subtilis natto culture solution was placed in the center of the plate medium. After culturing this in a dark place at 25°C for 3 days, the diameter of the inhibition zone formed around the paper disk was measured. The test was repeated twice.

As a result of the dual culture test, all the tested strains showed an inhibitory effect on the mycelial growth of P. digitatum, and among them, the TTCC2122 strain showed a stronger inhibitory effect than the other strains.
The TTCC2122 strain, which has been confirmed to have an excellent inhibitory effect, was deposited at the National Institute of Advanced Industrial Science and Technology (Address: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on May 28, 2020 under the accession number NITE P-03228.

[Example 2]
(Selection of natto bacteria that control gray mold)
1. Preparation of Bacillus subtilis natto culture solution Using the same method as in "1." of Example 1, Bacillus subtilis natto culture solutions were prepared for 720 species of Bacillus subtilis natto.

2. Preparation of conidial suspension The gray mold fungus Botrytis cinerea used was the TV335 strain stored at the Faculty of Agriculture, Ibaraki University. A conidial suspension was prepared using the same method as in "2." of Example 1.

3. Confrontation culture test 100 μL of B. cinerea conidial suspension was dropped onto a PSA plate medium and spread with a cone-large stick, after which a paper disk (diameter 5 mm) soaked in Bacillus subtilis natto culture solution was placed in the center of the plate medium. After culturing this in a dark place at 25°C for 3 days, the diameter of the inhibition zone formed around the paper disk was measured. The test was repeated twice.

As a result of the dual culture test, all the tested strains showed an inhibitory effect on the mycelial growth of B. cinerea, and among them, the TTCC2111 strain showed a stronger inhibitory effect than the other strains.
The TTCC2111 strain, which has been confirmed to have an excellent inhibitory effect, was deposited at the National Institute of Advanced Industrial Science and Technology (Address: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on May 28, 2020 under the accession number NITE P-03227.

[Example 3]
(Test to confirm improvement of preservation quality of mandarin oranges during storage)
A culture solution (cell concentration: 1×10 ⁸ cells/mL) of the TTCC2122 strain was prepared using the same method as in "1." of Example 1. For reference, a culture solution (cell concentration: 1×10 ⁸ cells/mL) was also prepared for the Bacillus subtilis natto No. 2 strain held at Ibaraki University. A conidial suspension (1.0×10 ⁵ conidia/mL) of P. digitatum, the green mold pathogen of citrus, was also prepared using the same method as in "2." of Example 1. A conidial suspension (1.0×10 ⁵ conidia/mL) of P. italicum, the blue mold pathogen of citrus (preserved at the Faculty of Agriculture, Ibaraki University, IUPi1 strain), was also prepared.

Next, four wounds of 1 mm depth and 3 mm width were made on the surface of the mandarin orange, and 15 μl of the culture solution of the TTCC2122 strain was dropped per wound. 16 hours after the treatment, 15 μl of a conidial suspension (1.0×10 ⁵ conidia/mL) of P. digitatum and P. italicum (stored at the Faculty of Agriculture, Ibaraki University, IUPi1 strain) was dropped and inoculated into each wound. Four days after the inoculation of the conidia, the surface of the mandarin orange was observed, and the ratio (percentage) of the wounds on which mold was observed to the wounds on the mandarin orange subjected to the inoculation test was calculated as the disease incidence rate (%). The results are shown in FIG. 1. In FIG. 1, the "control" group is mandarin oranges inoculated with P. digitatum or P. italicum without being subjected to the dropwise treatment of the culture solution of the TTCC2122 strain.

As shown in Figure 1, in the wounds treated with the culture solution of the TTCC2122 strain, the disease incidence rate was significantly suppressed regardless of whether the green mold pathogen (P. digitatum) or blue mold pathogen (P. italicum) was inoculated. This suppressive effect was also significant in comparison with the Bacillus subtilis natto No. 2 strain used as a reference.

[Example 4]
(Test to confirm the therapeutic and control effects of Bacillus subtilis natto culture solution against pea powdery mildew)
The TTCC2111 and TTCC2122 strains were cultured on a Trypticase Soy Agar (TSA) plate medium at 37°C for 16 hours, and then suspended in Potato Sucrose Broth (PSB) medium using a platinum loop. These were cultured at 28°C and 105 rpm for 24 hours with shaking to prepare culture solutions of the TTCC2111 strain and the TTCC2122 strain (each bacterial cell concentration: 1 x ¹⁰⁸ cells/mL).

From the pea leaves inoculated with powdery mildew two weeks before, leaves with many conidia were selected, and the conidia on the leaf surface were swept off with a paintbrush. The prepared culture solution of the TTCC2122 strain was then sprayed with a sprayer. The amount of culture solution sprayed was about 0.3 mL to 0.5 mL per pea leaf. For the control, sterilized water was sprayed instead of the culture solution. Two days after the spraying treatment, the pea leaves were immersed in a fixative and fixed. FAA (Formalin/Acetic acid/Alcohol) fixative was used as the fixative. The composition of the FAA fixative is formalin: acetic acid: ethanol = 1:1:1 by volume. Next, the bodies of the powdery mildew fungus on the fixed pea leaves were stained with methyl blue. Ten colonies per leaf were randomly selected, and the number of conidia formed per colony was counted. The results are shown in Figure 2.

As shown in Fig. 2, a decrease in the number of conidia formed was confirmed in the group sprayed with the culture solution of the TTCC2122 strain, compared to the control group. Furthermore, when the hyphae of the pea powdery mildew fungus on the leaves sprayed with the culture solution of the TTCC2122 strain were observed, stained areas resembling cell death were observed (not shown).
From the above, it was suggested that the culture solution of the TTCC2122 strain has a therapeutic effect against pea powdery mildew.

Next, the prepared culture solution of TTCC2111 strain and the culture solution of TTCC2122 strain were sprayed on the surface of one-week-old pea leaves. The amount of culture solution sprayed was about 0.3 mL to 0.5 mL per pea leaf. For the control, sterilized water was sprayed instead of the culture solution. The next day, the surface of the pea leaves was thoroughly dried, and then the conidia formed on the diseased pea leaves were placed on the entire treated leaves with a paintbrush for inoculation. Three days after inoculation, the inoculated leaves were fixed with FAA fixative, and the pea powdery mildew fungus bodies were stained with methyl blue. Those that formed germ tubes longer than the conidia were considered to have "germinated." The ratio (percentage) of the number of germinations to the number of conidia was calculated as the germination rate (%). The results are shown in Figure 3. Figure 3 shows the results of two independent tests performed on each group.

As shown in Fig. 3, a decrease in the germination rate was confirmed in the groups sprayed with the culture solution of the TTCC2111 strain and the culture solution of the TTCC2122 strain, compared to the control group. Furthermore, when observing the germ tubes of powdery mildew fungus in the leaves sprayed with the culture solution of the TTCC2122 strain, stained areas resembling cell death were observed (not shown).
From the above, it was suggested that the culture solutions of the TTCC2111 strain and the TTCC2122 strain have a control effect against pea powdery mildew.

[Example 5]
(Test to confirm the effect of natto culture solution on suppressing gray mold disease in primula petals)
Using the same method as in "1." of Example 1, a culture solution of TTCC2111 strain and a culture solution of TTCC2122 strain (each fungus cell concentration: 1 x 10 ⁸ cells/mL) were prepared. Next, the prepared culture solution of TTCC2111 strain and the culture solution of TTCC2122 strain were sprayed on the surface of Primula petals by spraying. The amount of each culture solution sprayed was about 0.3 mL to 0.5 mL per Primula petal. For the control, sterilized water was sprayed instead of the culture solution. 16 hours after the spraying treatment, a conidial suspension of Botrytis cinerea (1 x 10 ⁵ conidia/mL) was sprayed and inoculated. The number of lesions (pieces) per 1 cm ² of Primula petals formed 24 hours after inoculation was calculated. The results are shown in FIG. 4.

As shown in Figure 4, the group sprayed with culture solution from strain TTCC2111 and culture solution from strain TTCC2122 showed a disease suppression effect on gray mold. The effect was somewhat weak, but this was presumably due to the fact that the culture solution did not spread evenly due to the hydrophobic nature of the petal surface. When the infection behavior of the gray mold fungus was observed, it was confirmed that the extension of germinating hyphae was suppressed in the group sprayed with culture solution from strain TTCC2111 and culture solution from strain TTCC2122 (not shown). Therefore, it is believed that the disease suppression effect on gray mold can be improved by improving the composition of the culture solution and the treatment method.

[Example 6]
(Test to confirm the effect of Bacillus subtilis culture on suppressing cucumber powdery mildew)
Using the same method as in Example 4, culture solutions of TTCC2111 strain and TTCC2122 strain (each fungus concentration: 1 x ¹⁰⁸ cells/mL) were prepared. Next, the culture solutions were directly sprayed onto the cotyledons of 2-week-old cucumber seedlings. The amount of each culture solution sprayed was such that the entire cotyledon was covered with the culture solution. For the control, sterilized water was sprayed instead of the culture solution. 24 hours after the spraying treatment, spores of powdery mildew were placed on the entire treated leaf with a paintbrush to perform inoculation. The inoculated cucumber seedlings were cultivated at 25°C. Two weeks after the inoculation, the number and area of colonies generated on each cotyledon were investigated, and the disease index was judged according to the following criteria. The colony area was measured using imageJ software.

(standard)
0: No disease 1: 1 to 2 colonies observed 2: Colonies occupy less than 1/4 of the leaf area 3: Colonies occupy 1/4 to 1/2 of the leaf area 4: Colonies occupy 1/2 or more of the leaf area

Then, based on the determined disease index, the disease severity was calculated using the following formula. The results are shown in Figure 5. The number of leaves surveyed (N) for each group was 10.

Disease severity = [Σ{(disease index) x (number of leaves)}/{4 x (number of leaves surveyed)}] x 100

As shown in Figure 5, the groups sprayed with the culture solution of the TTCC2111 strain and the culture solution of the TTCC2122 strain showed a disease-suppressing effect on cucumber powdery mildew. In particular, the group sprayed with the culture solution of the TTCC2122 strain showed a significant disease-suppressing effect on cucumber powdery mildew.

[Example 7]
(Test 2 to confirm the effect of Bacillus subtilis culture on the suppression of cucumber powdery mildew)
Using the same method as in Example 4, culture solutions of TTCC2111 strain and TTCC2122 strain (each with a bacterial cell concentration of 1 x ¹⁰⁸ cells/mL) were prepared. In addition, for reference, a 1000-fold dilution of a commercially available Bacillus preparation (recommended dilution rate, estimated bacterial cell concentration: 1 x ¹⁰⁸ cells/mL) was also prepared. Next, using these, a disease suppression effect confirmation test against cucumber powdery mildew was performed using the same method as in Example 6. The number of leaves (N) surveyed in each group was 10. The results are shown in Figure 6.

As shown in Figure 6, the groups sprayed with the culture solution of the TTCC2111 strain and the culture solution of the TTCC2122 strain showed an inhibitory effect on the onset of powdery mildew on cucumbers. On the other hand, no significant difference was observed compared to the control group in the group sprayed with a diluted solution of a commercially available Bacillus preparation, and the inhibitory effect on the onset of powdery mildew on cucumbers could not be confirmed.
In addition, leaves sprayed with culture fluid of TTCC2111 strain and culture fluid of TTCC2122 strain, and leaves sprayed with sterilized water (control group) were fixed with FAA fixative, and the mycelium of powdery mildew was stained with methyl blue. When the germ tubes of powdery mildew in these leaves were observed under an optical microscope, it was confirmed that conidial germination was suppressed in the groups sprayed with culture fluid of TTCC2111 strain and culture fluid of TTCC2122 strain (not shown).
From the above, it was suggested that the culture solutions of the TTCC2111 strain and the TTCC2122 strain have a disease suppressing effect against cucumber powdery mildew.

[Example 8]
(Test to confirm the inhibitory effect of Bacillus subtilis natto culture fluid and culture supernatant on cucumber powdery mildew)
Using the same method as in Example 4, the obtained culture solution was used as the culture solution of the TTCC2122 strain (containing bacteria, bacteria concentration: 1 x 10 ⁸ cells/mL), and the culture supernatant was used as the culture supernatant of the TTCC2122 strain (containing no bacteria). In addition, for reference, a 1000-fold dilution of a commercially available Bacillus preparation (recommended dilution rate, estimated bacteria concentration: 1 x 10 ⁸ cells/mL) was also prepared. Next, the culture solution (containing bacteria) or the culture supernatant (containing no bacteria) was directly sprayed onto the first true leaves of 2-week-old cucumber seedlings. The amount of each treatment solution sprayed was until the entire leaf was covered with the culture solution. For the control, sterilized water was sprayed instead of the culture solution. 24 hours and one week after the spraying treatment, powdery mildew spores were placed on the entire treated leaf with a paintbrush, and inoculation was performed. The inoculated cucumber seedlings were cultivated at 25°C. Two weeks after inoculation, the number and area of colonies developed on each cotyledon were examined, and the disease severity was calculated using the same method as in Example 6. The number of examined leaves (N) for each group was 10. The results are shown in Figure 7.

As shown in Figure 7, the group sprayed with the culture solution (containing fungus cells) of the TTCC2122 strain and the culture supernatant (not containing fungus cells) both showed an inhibitory effect on the onset of powdery mildew on cucumbers. When the powdery mildew fungus was inoculated one week after the spraying treatment, an inhibitory effect on the onset of powdery mildew on cucumbers was also observed, as in the case of inoculation 24 hours after the spraying treatment. In particular, when the powdery mildew fungus was inoculated one week after the spraying treatment, the group sprayed with the culture supernatant (not containing fungus cells) of the TTCC2122 strain showed a remarkable inhibitory effect on the onset of powdery mildew on cucumbers.

[Example 9]
(Test 3 to confirm the effect of Bacillus subtilis culture on the suppression of cucumber powdery mildew)
A culture solution of the TTCC2122 strain (cell concentration: 1 x ¹⁰⁸ cells/mL) was prepared using the same method as in Example 4. Sterile water was added to the prepared culture solution to prepare 5-fold, 10-fold and 20-fold diluted solutions. Using these, a disease suppression effect confirmation test against cucumber powdery mildew was carried out using the same method as in Example 6. The control value was calculated using the following formula. The results are shown in Table 1.

Control value = {1 - (average disease severity in spray-treated group) / (average disease severity in untreated (control) group)} x 100

As shown in Table 1, the control value was 35.5 or more in the groups sprayed with the culture solution (undiluted), 5-fold diluted solution, 10-fold diluted solution, and 20-fold diluted solution of TTCC2122 strain, demonstrating the effect of suppressing the onset of cucumber powdery mildew.

[Example 10]
(Test to confirm the effect of natto culture on suppressing gray mold disease in cucumber and begonia)
A culture solution (cell concentration: 1×10 ⁸ cells/mL) of the TTCC2122 strain was prepared using the same method as in Example 4. Sterile water was added to the prepared culture solution to prepare 5-fold, 10-fold and 20-fold dilutions. For reference, a 1000-fold dilution (recommended dilution rate, estimated cell concentration: 1×10 ⁸ cells/mL) of a commercially available Bacillus preparation was also prepared. These were then sprayed onto the first true leaves of 3-week-old cucumbers and cut leaves of begonias using a sprayer. The amount of each solution sprayed was until the liquid dripped from the leaves. For the control, sterilized water was sprayed instead of the culture solution and dilution solution. After spraying, the cucumbers were kept in an artificial climate chamber at 25° C., and the treated leaves were cut off 24 hours after spraying. B. difficile, which had been cultured on PSA plate medium for 2 days, was then sprayed onto these treated leaves. A mycelium plug (diameter 5 mm) of A. cinerea was inoculated into two places on each treated leaf. The inoculated leaves were kept at 22°C in a plastic box covered with paper towels moistened with ion-exchanged water, and the lesion diameter was measured two days after inoculation. Three leaves were used for each treatment group in the test, and the same test was repeated three times. The results are shown in Figure 8 (lesion diameter on cucumber) and Figure 9 (lesion diameter on begonia).

As shown in Figure 8, the lesion diameter was reduced in the groups sprayed with the culture solution (undiluted), 5-fold diluted solution, 10-fold diluted solution, and 20-fold diluted solution of the TTCC2122 strain compared to the control group, demonstrating a disease-suppressing effect against cucumber gray mold. On the other hand, in the group sprayed with a diluted solution of a commercially available Bacillus preparation, no difference was observed in the lesion diameter compared to the control group, and a disease-suppressing effect against cucumber gray mold could not be confirmed. Also, the disease-suppressing effect against cucumber gray mold was particularly remarkable in the groups sprayed with the culture solution (undiluted), 5-fold diluted solution, and 10-fold diluted solution of the TTCC2122 strain.

As shown in Figure 9, the lesion diameter was reduced in the groups sprayed with the culture solution (undiluted), 5-fold diluted solution, 10-fold diluted solution, and 20-fold diluted solution of the TTCC2122 strain compared to the control group, demonstrating a disease-suppressing effect against begonia gray mold. On the other hand, in the group sprayed with a diluted solution of a commercially available Bacillus preparation, no difference was observed in the lesion diameter compared to the control group, and a disease-suppressing effect against begonia gray mold could not be confirmed. In addition, the disease-suppressing effect against begonia gray mold was remarkable in all groups sprayed with the culture solution (undiluted), 5-fold diluted solution, 10-fold diluted solution, and 20-fold diluted solution of the TTCC2122 strain.

[Example 11]
(Test to confirm the effect of Bacillus subtilis culture on suppressing strawberry powdery mildew)
A culture solution (cell concentration: 1×10 ⁸ cells/mL) of the TTCC2122 strain was prepared using the same method as in Example 4. Sterile water was added to the prepared culture solution to prepare 5-fold and 10-fold diluted solutions. These were sprayed onto the underside of leaflets of strawberry (variety: Roseberry Red) using a spray. Leaflets were cut from compound leaves of strawberry seedlings shortly after leaf-expansion (about 1 to 2 weeks) and used for the experiment. The amount of each solution sprayed was until the underside of the leaflets was covered with the treatment solution. For the control, sterilized water was sprayed instead of the culture solution and the dilution solution. 24 hours after the spray treatment, conidia were brushed off from the lesions of leaves infected with strawberry powdery mildew with a paintbrush, and the entire treated leaves were inoculated. The inoculated strawberry leaves were arranged in a plastic tray covered with moist filter paper, sealed, and maintained at 20° C. under fluorescent lighting (12 hours of lighting per day). Two weeks after the inoculation, the number and area of colonies developed on the leaves were examined under a stereomicroscope, and the disease severity was calculated in the same manner as in Example 6, and the control titer was calculated in the same manner as in Example 9. The results are shown in Table 2.

As shown in Table 2, the control value was 54.2 or more in the groups sprayed with the culture solution (undiluted), 5-fold diluted solution, and 10-fold diluted solution of TTCC2122 strain, demonstrating sufficient disease suppression effect against strawberry powdery mildew.

Furthermore, when the undersides of the leaves in each treatment group were observed under a stereomicroscope, colonies of powdery mildew fungi could be observed under the trichomes on the leaf surfaces of the groups sprayed with a 5-fold dilution and the groups sprayed with a 10-fold dilution, but it was clear that these had a lower mycelial density than the colonies seen in the group sprayed with sterilized water (control group) (not shown).

[Example 12]
(Test to confirm the effect of Bacillus subtilis culture on suppressing the development of powdery mildew on peppers)
A culture solution (cell concentration: 1×10 ⁸ cells/mL) of the TTCC2122 strain was prepared using the same method as in Example 4. Sterile water was added to the prepared culture solution to prepare 5-fold and 10-fold diluted solutions. These were sprayed onto the underside of sweet pepper leaves, which are susceptible to bell pepper powdery mildew, using a spray. The sweet pepper leaves were cut from seedlings 8 weeks after sowing and used in the experiment. The amount of each solution sprayed was such that the leaf surface was covered with the treatment solution. For the control, sterilized water was sprayed instead of the culture solution and the dilution solution. 24 hours after the spray treatment, conidia were scraped off from the lesions of the sweet pepper powdery mildew-infected leaves with a paintbrush and rubbed over the entire treated leaves for inoculation. The inoculated sweet pepper leaves were arranged in a plastic tray covered with moist filter paper, sealed, and maintained at 21° C. under fluorescent lighting (12 hours of lighting per day). Three weeks after inoculation, the number and area of colonies developed on the leaves were examined under a stereomicroscope, and the disease severity was calculated in the same manner as in Example 6, and the control value was calculated in the same manner as in Example 9. The results are shown in Table 3.

As shown in Table 3, the control value was 38.6 or more in the group sprayed with the culture solution (undiluted) and 5-fold diluted solution of TTCC2122 strain, and the disease suppression effect on pepper powdery mildew was observed. On the other hand, the disease suppression effect on pepper powdery mildew was not observed in the group sprayed with the 10-fold diluted solution.

Furthermore, when the undersides of the leaves in each treatment group were observed under a stereomicroscope, no significant differences were found in the colonies formed in each treatment group (not shown). This was presumably because this fungus is endoparasitic, whereas other powdery mildew fungi are surface parasitic.

[Example 13]
(Test to confirm the effect of Bacillus subtilis culture on the suppression of tomato powdery mildew)
A culture solution (cell concentration: 1×10 ⁸ cells/mL) of the TTCC2122 strain was prepared using the same method as in Example 4. Sterile water was added to the prepared culture solution to prepare 5-fold and 10-fold diluted solutions. These were sprayed onto the primary leaves of 3-week-old tomatoes (variety: Ponterosa) using a sprayer. The amount of each solution was sprayed until the leaf surface was covered with the treatment solution. For the control, sterilized water was sprayed instead of the culture solution and the dilution solution. 24 hours after the spray treatment, conidia were scraped off from the lesions of the tomato powdery mildew-infected leaves with a paintbrush and rubbed onto the entire treated leaves for inoculation. The tomato seedlings after inoculation were cultivated at 25° C. Ten days after inoculation, the number and area of colonies occurring on the leaves were examined under a stereomicroscope, the disease severity was calculated using the same method as in Example 6, and the control value was calculated using the same method as in Example 9. The results are shown in Table 4.

As shown in Table 4, the control value was 36.4 or more in the groups sprayed with the culture solution (undiluted), 5-fold diluted solution, and 10-fold diluted solution of TTCC2122 strain, demonstrating the effect of suppressing the onset of tomato powdery mildew.

In addition, visual observation of the morphology of conidia and hyphae of tomato powdery mildew on the surface of inoculated leaves in each treatment group revealed that the group sprayed with culture solution (undiluted) of TTCC2122 strain showed a significant germination inhibitory effect compared to the control group (not shown). In addition, the group sprayed with a 5-fold diluted solution showed a lower density of hyphae forming colonies compared to the control group (not shown). No difference was observed between the group sprayed with a 10-fold diluted solution and the control group (not shown).

[Example 14]
(Test to confirm the effect of Bacillus subtilis culture solution on suppressing eggplant powdery mildew)
A culture solution (cell concentration: 1×10 ⁸ cells/mL) of the TTCC2122 strain was prepared using the same method as in Example 4. Sterile water was added to the prepared culture solution to prepare 5-fold and 10-fold diluted solutions. These were sprayed onto the primary leaves of 3-week-old eggplants (variety: Senryo 2-go) using a sprayer. The amount of each solution was sprayed until the leaf surface was covered with the treatment solution. For the control, sterilized water was sprayed instead of the culture solution and the dilution solution. 24 hours after the spray treatment, conidia were scraped off from the lesions of the eggplant powdery mildew-infected leaves with a paintbrush and rubbed over the entire treated leaves for inoculation. The inoculated eggplant seedlings were cultivated at 25° C. 10 days after inoculation, the number and area of colonies occurring on the leaves were investigated, the disease severity was calculated using the same method as in Example 6, and the control value was calculated using the same method as in Example 9. The results are shown in Table 5.

As shown in Table 5, the control value was 25.4 or more in the groups sprayed with the culture solution (undiluted), 5-fold diluted solution, and 10-fold diluted solution of TTCC2122 strain, demonstrating the effect of suppressing the onset of eggplant powdery mildew.

In addition, visual observation of the morphology of conidia and hyphae of eggplant powdery mildew on the surface of inoculated leaves in each treatment group revealed that the infection rate of eggplant powdery mildew was lower than that of other powdery mildew fungi, and colonies did not develop as well as other fungi. In addition, although the effect of suppressing eggplant powdery mildew was observed in the groups sprayed with the culture solution (undiluted), 5-fold diluted solution, and 10-fold diluted solution of TTCC2122 strain, it appeared to be lower than the effect of suppressing disease on powdery mildew fungi of other plant species that have been tested so far. This is presumably because, as mentioned above, eggplant powdery mildew has a lower infection rate than other powdery mildew fungi, making it difficult to see the difference from the control group.

From the above, it has become clear that strains TTCC2111 and TTCC2122 have a control effect against various plant diseases.

The plant disease control agent and plant disease control method of this embodiment can effectively control plant diseases.

Claims (5)

A plant disease control agent containing as an active ingredient one or more types of Bacillus subtilis cells or cultures selected from the group consisting of Bacillus sp. TTCC2111 strain (NITE P-03227) and Bacillus sp. TTCC2122 strain (NITE P-03228). The plant disease control agent according to claim 1, wherein the plant disease is selected from the group consisting of gray mold, green mold, blue mold, and powdery mildew. A method for controlling plant diseases, comprising applying the plant disease control agent according to claim 1 or 2 to a target plant. Bacillus sp. TTCC2111 strain (NITE P-03227). Bacillus sp. TTCC2122 strain (NITE P-03228).

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