WO2024204528A1

WO2024204528A1 - Method for controlling plant disease, composition for controlling plant disease, and use of sodium chloride

Info

Publication number: WO2024204528A1
Application number: PCT/JP2024/012617
Authority: WO
Inventors: 慎一吉田; 暢彦道順; 仁工藤
Original assignee: 株式会社カネカ; 三井物産株式会社
Priority date: 2023-03-30
Filing date: 2024-03-28
Publication date: 2024-10-03

Abstract

One purpose of the present invention is to provide a method for controlling plant disease by suppressing a reduction in bacteriophage activity.　An embodiment of the present invention is a method for controlling plant disease, the method comprising a step for bringing a plant into contact with a composition for controlling plant disease, the composition containing more than 0 mass % but less than 0.9 mass % sodium chloride and a bacteriophage exhibiting bacteriolytic activity against bacteria.

Description

Method for controlling plant diseases, composition for controlling plant diseases, and use of sodium chloride

The present invention relates to a method for controlling plant diseases, a composition for controlling plant diseases, and the use of sodium chloride.

In order to control harmful bacteria that cause various plant diseases in agricultural crops, agricultural chemical compounds such as copper agents and antibiotics have been used. However, the use of agricultural chemical compounds has many problems, such as chemical damage and the possibility of causing a breakdown in the balance of the bacterial flora, so in recent years, a method that applies bacteriophages as a new control method has attracted attention. Bacteriophages (also referred to as "phages" in this specification) are a general term for viruses that infect only bacteria. Many phages, also called lytic phages, specifically adsorb to the host target bacteria, inject their own DNA, and self-amplify using the translation mechanism of the bacteria. Furthermore, they disperse the amplified phages by lysing the bacteria, and repeatedly infect new target bacteria. Biopesticides that use the lytic activity of such bacteriophages to control harmful bacteria that cause plant diseases are being developed and researched.

For example, Patent Document 1 discloses a method for preventing or reducing symptoms or disease caused by Xylella fastidiosa or Xanthomonas in a plant, the method comprising contacting the plant with a virulent bacteriophage that contains X. fastidiosa and/or Xanthomonas in its host range.

Furthermore, Non-Patent Document 1 discloses that spraying an aqueous solution containing 3.0% or more by mass of sodium chloride on the leaves can have a detrimental effect on plant growth, causing leaves to brown and fall off.

Special Publication No. 2015-534983

Unlike conventional copper agents and antibiotics, bacteriophages are a type of virus and are natural products, so no reported chemical damage has been reported to date. In addition, because they have a relatively high degree of host specificity, they only target specific genera and species of bacteria, and their impact on the balance of the bacterial flora is extremely limited. They are also harmless to not only humans and other animals, but also plants, making them highly safe. For this reason, control methods using bacteriophages have attracted a great deal of attention in recent years.

The above biopesticides can be applied to plants to be controlled by spraying them on the leaves of the plants, for example. However, the inventors' research has revealed that exposure to ultraviolet light, etc., reduces the activity of the bacteriophage over time.

Therefore, one of the objects of the present invention is to provide a method for controlling plant diseases in which the decrease in bacteriophage activity is suppressed.

The inventors conducted extensive research to solve the above problems and discovered that the use of a plant disease control composition containing sodium chloride at a specific concentration suppresses the decline in bacteriophage activity, leading to the present invention.

Thus, an embodiment of the present invention is as follows.
[1] A method for controlling a plant disease, comprising a step of contacting a target plant with a plant disease control composition containing sodium chloride in an amount greater than 0 mass% and less than 0.9 mass%, and a bacteriophage that exhibits lytic activity against bacteria.
[2] The plant disease control method according to [1], wherein the step is a step of foliar spraying of a plant disease control composition to a target plant.
[3] The method for controlling a plant disease according to [1] or [2], comprising a step of mixing sodium chloride with a bacteriophage that exhibits lytic activity against bacteria to prepare a plant disease control composition.
[4] The plant disease control method according to any one of [1] to [3], wherein the plant disease control composition further contains a protecting agent.
[5] The plant disease control method according to [4], wherein the protective agent contains skim milk.
[6] The plant disease control method according to any one of [1] to [5], wherein the plant disease control composition further contains a spreading agent.
[7] The plant disease control method according to [6], wherein the wetting agent comprises an organic silicone wetting agent.
[8] The plant disease control method according to any one of [1] to [7], wherein the plant disease control composition further contains at least one component selected from an agriculturally acceptable carrier and an agriculturally acceptable solvent.
[9] A plant disease control composition comprising more than 0 mass% and less than 0.9 mass% sodium chloride, and a bacteriophage exhibiting lytic activity against bacteria.
[10] The plant disease control composition according to [9], further comprising a protecting agent.
[11] The plant disease control composition according to [10], wherein the protecting agent comprises skim milk.
[12] The plant disease control composition according to any one of [9] to [11], further comprising a spreading agent.
[13] The plant disease control composition according to [12], wherein the wetting agent comprises an organic silicone wetting agent.
[14] The plant disease control composition according to any one of [9] to [13], further comprising at least one component selected from an agriculturally acceptable carrier and an agriculturally acceptable solvent.
[15] Use of sodium chloride in a plant disease control composition containing a bacteriophage exhibiting lytic activity against bacteria, in order to improve the residual activity of the bacteriophage exhibiting lytic activity against bacteria in a target plant.
[16] Use of sodium chloride in the production of a plant disease control composition containing a bacteriophage exhibiting lytic activity against bacteria.
This specification includes the disclosure of Japanese Patent Application No. 2023-054780, which is the priority basis of this application.

One aspect of the present invention provides a method for controlling plant diseases in which the decrease in bacteriophage activity is suppressed.

Figure 1 is a graph showing the results of stabilization evaluation 1 of the embodiment, with the horizontal axis representing sodium chloride concentration and the vertical axis representing residual activity one day after leaving the sample (day 1) (Residual Activity Ratio vs. Day 0).

1. Definitions Terms used in this specification are defined below.

In this specification, "plant disease" refers to a general term for illnesses that occur in plants. Known plant diseases include diseases caused by infectious pathogens such as viruses, bacteria, fungi, actinomycetes, viroids, phytoplasmas, nematodes, mites, or insects, as well as diseases caused by non-infectious pathogens such as a lack or excess of nutrients or water, or chemical damage. In this specification, plant disease refers to diseases caused by bacteria, i.e., plant pathogenic bacteria, unless otherwise specified.

In this specification, "control" refers to prevention or treatment (eradication) (from the Japan Pesticide Manufacturers Association website). Therefore, in this specification, "plant disease control" refers to the prevention of plant diseases, particularly target bacteria, or the treatment of plant diseases caused by target bacteria.

In this specification, the term "target plant" refers to a plant to which the plant disease control composition of the present invention is applied. This plant corresponds to a plant that has developed a specific plant disease due to infection with the target bacterium, or a plant that is at risk of infection with the target bacterium.

2. Plant disease control composition 2-1. Overview One aspect of the present invention is a plant disease control composition. The plant disease control composition of the present invention contains sodium chloride of more than 0 mass % and less than 0.9 mass %, and a bacteriophage that exhibits lytic activity against bacteria.

The plant disease control composition of the present invention suppresses the decrease in bacteriophage activity, exhibits bacteriolytic activity against target bacteria that may be pathogenic bacteria for plant diseases, and suppresses the decrease in bacteriophage activity over time after contact with a plant compared to conventional compositions. By using the plant disease control composition of the present invention for plant disease control, it is possible to provide a sustainable pesticide against bacterial plant diseases that is safe for the human body, does not cause chemical damage to the environment, and is capable of specifically preventing or treating the target plant disease.

In addition, when the term "plant disease control composition" is used in this specification, it refers to a composition used for plant disease control purposes.

2-2. Composition 2-2-1. Constituents The plant disease control composition of the present invention contains, as an essential component, a bacteriophage that exhibits bacteriolytic activity against bacteria as an active ingredient. The plant disease control composition of the present invention also contains sodium chloride as an essential component in a range of more than 0 mass% and less than 0.9 mass%. By containing sodium chloride in a predetermined range in the plant disease control composition, it is possible to suppress a decrease in the activity of the bacteriophage, which is an active ingredient, after application of the plant disease control composition to a plant. The plant disease control composition of the present invention may further contain a protective agent. The plant disease control composition of the present invention may further contain a spreading agent. The plant disease control composition of the present invention may further contain at least one component selected from an agriculturally acceptable carrier and an agriculturally acceptable solvent. Furthermore, the plant disease control composition of the present invention may further contain other components as necessary. Each of the components will be specifically described below.

(1) Bacteriophage The plant disease control composition of the present invention contains a bacteriophage exhibiting lytic activity against bacteria as an essential active ingredient. In the plant disease control composition, the target bacterium of the present invention is lysed by this active ingredient, so that plant diseases caused by the target bacterium can be prevented or treated. The bacteriophage may be used alone or in combination of two or more kinds.

In the present invention, the bacteriophage is not particularly limited as long as it has lytic activity against the target bacterium. Many bacteriophages are specific to a particular genus, species, or strain of bacteria. The term "bacteriophage" is synonymous with the term "phage."

Bacteriophages include, but are not limited to, bacteriophages belonging to any of the following virus families: Myoviridae, Siphoviridae, Podoviridae, Autographiviridae, Ackermannviridae, Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, or Tectiviridae. The bacteriophage may be a lytic bacteriophage. A lytic bacteriophage is one that does not enter the lysogenic pathway but instead follows the lytic pathway by completing a lytic cycle.

Also, the bacteriophage of the present invention can be from the Caudovirales order of phages. Caudovirales is an order of tailed bacteriophages that have a double-stranded DNA (dsDNA) genome. Each virion of the Caudovirales order has an icosahedral head that contains the virion genome and a flexible tail. The Caudovirales order includes, for example, the following bacteriophage families: Myoviridae (long contractile tails), Siphoviridae (long non-contractile tails), Podoviridae (short non-contractile tails), Autographiviridae, and Ackermannviridae.

When used for plant disease control, the amount of active ingredient contained per unit amount in the plant disease control composition depends on various conditions such as the formulation, type of plant pathogenic bacteria, type of target plant, application location, and application method. It is preferable that the phage, which is the active ingredient, is contained in an amount sufficient to contact and infect the plant pathogenic bacteria that have infected the target plant. Therefore, each condition should be taken into consideration and determined within the scope of common technical knowledge in the field so that the bacteriophage contained in the plant disease control composition of the present invention is in an effective amount against the target bacteria after application.

The concentration of the bacteriophage in the plant disease control composition of the present invention is not particularly limited and can be appropriately set depending on the type and severity of the disease to be treated, the application method, and the formulation. The concentration of the bacteriophage is, for example, preferably 1×10 ³ PFU/mL or more, preferably in the range of 1×10 ⁵ to 1×10 ¹⁵ PFU/mL, more preferably in the range of 1×10 ⁷ to 1×10 ¹³ PFU/mL, and can be, for example, in the range of 1×10 ⁷ to 1×10 ¹¹ PFU/mL, or 1×10 ⁸ to 1×10 ¹⁰ PFU/mL.

The plant disease control composition of the present invention can contain a combination of two or more phages as active ingredients. For example, even if the target bacteria is the same, if the phages recognize different cell surface receptors, a synergistic or complementary effect of bacteriolytic activity can be expected by combining the phages. Furthermore, even if the target bacteria are different, bacteriolytic activity can be exerted against different bacteria, and control activity against a wide range of pathogenic bacteria can be expected.

The plant disease control composition of the present invention may contain, as an active ingredient, any one or more of the following bacteriophages (i) to (vi), but is not limited thereto:
(i) a phage having a genomic DNA sequence consisting of the nucleotide sequence shown in SEQ ID NO:1 or a nucleotide sequence having 90% or more, 95% or more, or 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO:1;
(ii) a phage having a genomic DNA sequence consisting of the base sequence shown in SEQ ID NO: 2 or a base sequence having 90% or more, 95% or more, or 99% or more sequence identity to the base sequence shown in SEQ ID NO: 2;
(iii) a phage having a genomic DNA sequence consisting of the base sequence shown in SEQ ID NO: 3 or a base sequence having 90% or more, 95% or more, or 99% or more sequence identity to the base sequence shown in SEQ ID NO: 3;
(iv) a phage having a genomic DNA sequence consisting of the base sequence shown in SEQ ID NO: 4 or a base sequence having 90% or more, 95% or more, or 99% or more sequence identity to the base sequence shown in SEQ ID NO: 4;
(v) a phage having a genomic DNA sequence consisting of the nucleotide sequence shown in SEQ ID NO:5 or a nucleotide sequence having 90% or more, 95% or more, or 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO:5;
(vi) A phage having a genomic DNA sequence consisting of the base sequence shown in SEQ ID NO:6, or a base sequence having 90% or more, 95% or more, or 99% or more sequence identity to the base sequence shown in SEQ ID NO:6.

The plant disease control composition of the present invention may contain, but is not limited to, for example, a combination of the bacteriophages (i), (ii), (iv) and (v) above, or a combination of the bacteriophages (i), (ii), (v) and (vi) above as an active ingredient.

(2) Sodium chloride The plant disease control composition of the present invention contains sodium chloride as an essential component. In the plant disease control composition of the present invention, sodium chloride is contained in an amount of more than 0 mass% and less than 0.9 mass%, preferably 0.05 to 0.8 mass%, more preferably 0.15 to 0.75 mass%, and even more preferably 0.20 to 0.70 mass%, so that the decrease in bacteriophage activity can be suppressed. In addition, within the above range, the damage to plants caused by sodium chloride, i.e., the disadvantages caused by salt damage, is sufficiently small compared to the advantages brought about by the above effect, so that the plant disease control composition of the present invention can be used for plants. The fact that the decrease in bacteriophage activity can be suppressed by a specific amount of sodium chloride is an effect that could not be predicted from conventional knowledge. The reason why the above effect is exerted is unclear, but the present inventors predicted that it is because of the effect of reducing the risk of bacteriophage association and aggregation due to electrostatic interaction. The inventors believe that if the sodium chloride concentration is too high, the risk of bacteriophage association and aggregation due to hydrophobic interactions increases, and that the above concentration range can be specifically used to inhibit bacteriophage association and aggregation.

There are no particular limitations on the sodium chloride, and salt prepared for edible or medical use, industrial sodium chloride, etc. can be used as appropriate.

(3) Protective Agent The plant disease control composition of the present invention may contain a protective agent as an optional component. The protective agent is expected to have an effect of reducing damage caused by ultraviolet rays to the phage. In addition, by using the protective agent, the decrease in activity of the bacteriophage can be further suppressed. As the protective agent, a protein-based protective agent containing a protein as a main component is preferable. Examples of the protective agent include skim milks, caseins, gelatins, etc. As the protective agent, it is particularly preferable to include skim milks, for example, skim milk. The protective agent may be used alone or in combination of two or more types.

When the plant disease control composition contains a protective agent, the content of the protective agent in the plant disease control composition is preferably 10% by mass or less, preferably 5% by mass or less, preferably 2% by mass or less, preferably 1% by mass or less, and preferably 0.5% by mass or less. When the plant disease control composition contains a protective agent, the content of the protective agent in the plant disease control composition is preferably 0.01% by mass or more, preferably 0.03% by mass or more, preferably 0.05% by mass or more, preferably 0.07% by mass or more, and preferably 0.09% by mass or more. The content of the protective agent in the plant disease control composition may be, for example, 0.01% by mass to 10% by mass, 0.05% by mass to 5% by mass, or 0.05% by mass to 0.5% by mass.

(4) Spreading Agent The plant disease control composition of the present invention may contain a spreading agent as an optional component. The spreading agent has the effect of improving the physicochemical properties of the plant disease control composition, such as wetting, emulsifying, dispersing, penetrating, adhering, defoaming, and spreading properties, of the plant disease control composition on the plant body. In addition, the use of the spreading agent can further suppress the decrease in the activity of the bacteriophage. A surfactant can generally be used as the main component of the spreading agent. One type of spreading agent may be used alone, or two or more types may be used in combination.

The spreading agent preferably contains an organic silicone-based spreading agent. By containing an organic silicone-based surfactant, the decrease in the activity of the bacteriophage can be further suppressed.

An organic silicone-based spreading agent (also called an organic silicone-based surfactant) refers mainly to silicone oils to which hydrophilicity has been imparted by the introduction of organic functional groups such as polyether groups, for example, silicone oils having polyether groups. In organic silicone-based surfactants, various organic groups are known to be introduced other than the aforementioned polyether groups, and they can also be used as long as they are compatible with the purpose of this disclosure. An example of an organic silicone-based surfactant to which a polyether group has been introduced is a compound represented by the following structural formula (I):

[In formula (I),
_R1 is represented by the following general formula (II):
-R ₂ -O-(C ₂ H ₄ O) _x -(C ₃ H ₆ O) _y -R ₃ (II)
(in formula (II), _R2 is an unsubstituted or substituted alkylene group having 2 to 6 carbon atoms, _R3 is a hydrogen atom, an unsubstituted or substituted alkyl group having 1 to 6 carbon atoms, or an acetyl group ( _-COCH3 ), x is an integer of 0 to 15, and y is an integer of 0 to 10),
Me is a methyl group;
m is an integer from 0 to 10;
and n is an integer from 1 to 10.

Preferably, in structural formula (I),
_R1 is represented by the following general formula (III):
-R ₂ -O-(C ₂ H ₄ O) _x -R ₃ (III)
(wherein _R2 is a propylene group, _R3 is a hydrogen atom or a methyl group, and x is an integer of 0 to 15),
Me is a methyl group;
m is an integer from 0 to 3;
n is 1.

The organic silicone surfactant is preferably at least one selected from the group consisting of trisiloxane ethoxylate, polyoxyethylene methyl polysiloxane, polyoxyalkylene methyl polysiloxane, and polyether polymethyl siloxane copolymer. Among these, trisiloxane ethoxylate, polyoxyethylene methyl polysiloxane, or trisiloxane ethoxylate is preferred, with trisiloxane ethoxylate being more preferred.

Specific examples of organic silicone surfactants include the following (product names): Trisiloxane ethoxylates include Silwet (registered trademark) L-77, Silwet (registered trademark) 408, and Silwet (registered trademark) 440 (manufactured by Momentive performance materials). Polyoxyethylene methyl polysiloxanes include Makupika (registered trademark) (manufactured by Ishihara Sangyo Kaisha). Polyoxyalkylene methyl polysiloxanes include KF-640 (manufactured by Shin-Etsu Chemical Co., Ltd.). Polyether polymethyl siloxane copolymers include Break-Thru (registered trademark) (manufactured by Evonik Goldschmidt Chemical Corporation) and Break-Thru (manufactured by Sankei Chemical Co., Ltd.). Some commercially available organic silicone surfactants further contain other ingredients.

A more preferred example of a trisiloxane ethoxylate is "Silwet (registered trademark) L-77," which is a polyalkylene oxide-modified heptamethyltrisiloxane.

The content of the organic silicone-based spreading agent in the composition is not particularly limited, and may be appropriately selected in consideration of various conditions such as the type of plant pathogenic bacteria, the type of target plant, the application location, and the application method. When the plant disease control composition contains an organic silicone-based spreading agent, the content of the organic silicone-based spreading agent in the plant disease control composition is preferably 1% by mass or less, preferably 0.5% by mass or less, preferably 0.3% by mass or less, preferably 0.2% by mass or less, and preferably 0.15% by mass or less. When the plant disease control composition contains an organic silicone-based spreading agent, the content of the organic silicone-based spreading agent in the plant disease control composition is preferably 0.01% by mass or more, preferably 0.015% by mass or more, preferably 0.02% by mass or more, preferably 0.025% by mass or more, and preferably 0.03% by mass or more. The content of the organic silicone-based spreading agent in the plant disease control composition may be, for example, 0.01% by mass to 1% by mass, 0.01% by mass to 0.5% by mass, or 0.01% by mass to 0.15% by mass.

Spreaders other than organic silicone-based spreaders include nonionic surfactants, anionic surfactants, cationic surfactants, or combinations thereof (excluding organic silicone-based spreaders). More specifically, for example, polyoxyethylene alkyl ether compounds, polyoxyethylene fatty acid ester compounds, lignin sulfonate compounds, naphthylmethanesulfonate compounds, alkylsulfosuccinate compounds, and tetraalkylammonium salt compounds can be mentioned. However, since spreaders (excluding organic silicone-based spreaders) may affect the bacteriolytic activity of phages, the content of spreaders (excluding organic silicone-based spreaders) in the plant disease control composition is preferably 1% by mass or less, more preferably 0.5% by mass or less, more preferably 0.1% by mass or less, more preferably 0.05% by mass or less, more preferably 0.01% by mass or less, and more preferably 0.001% by mass or less.

(5) At least one component selected from an agriculturally acceptable carrier and an agriculturally acceptable solvent The plant disease control composition of the present invention may contain at least one component selected from an agriculturally acceptable carrier and an agriculturally acceptable solvent. It is preferable that the at least one component selected from the agriculturally acceptable carrier and the agriculturally acceptable solvent does not inhibit or suppress the lytic activity of the phage against a target bacterium.

An agriculturally acceptable carrier is a substance that facilitates application of the composition, can maintain the viability and infectivity of the active ingredient, the phage, and/or can control the rate of action. An agriculturally acceptable carrier can be a substance that has no or very little harmful effects on the environment, such as soil and water quality, when applied outdoors, and further has no or very little harmful effects on animals, particularly humans.

Specific examples of agriculturally acceptable carriers include excipients. If desired, small amounts of wetting agents, emulsifiers, pH buffers, etc. may also be used. The carriers may be mixed in advance or immediately before application.

Examples of excipients include glucose, lactose, sucrose, gelatin, starch, malt, and wheat flour.

Specific examples of agriculturally acceptable solvents include water (including aqueous solutions), buffers, and liquid media. The solvent is preferably a sterile liquid.

(6) Other Active Ingredients In addition to the bacteriophage, the plant disease control composition of the present invention may contain one or more other active ingredients having different pharmacological actions, such as insecticides, nematicides, acaricides, herbicides, plant growth promoters, antibiotics, and biological pesticides.

2-2-2. Dosage form The plant disease control composition of the present invention may be in any form as long as it can maintain the infection site of the target bacterium on the target plant, the fixation to the target plant, and/or the ease of infection of the phage, which is the active ingredient, to the target bacterium. The plant disease control composition may be, for example, a liquid agent or wettable powder in a liquid state in which the bacteriophage is suspended in a suitable solution. For example, when the infection site of the target bacterium on the target plant is the leaves, flowers, fruits, stems, branches, or trunks of the aboveground parts, a liquid agent, wettable powder, or gel agent that spreads widely to the infection site and has high fixation is suitable, but is not limited thereto.

2-3. Preparation method The plant disease control composition of the present invention can be prepared, for example, by mixing sodium chloride with a bacteriophage that exhibits lytic activity against bacteria. When mixing, the amount of each component is within the above-mentioned range, for example, sodium chloride is used in an amount that is more than 0 mass% and less than 0.9 mass% in 100 mass% of the plant disease control composition. In addition to sodium chloride and bacteriophage, a protective agent may be added, a spreading agent may be added, or a protective agent and a spreading agent may be added. In addition, at least one component selected from an agriculturally acceptable carrier and an agriculturally acceptable solvent may be added. The bacteriophage may be provided as a liquid or solid bacteriophage. The bacteriophage contains a bacteriophage as an active ingredient and may have lytic activity by itself, but is used to prepare a plant disease control composition by mixing with sodium chloride and, if necessary, at least one component selected from a protective agent, a spreading agent, and an agriculturally acceptable carrier and an agriculturally acceptable solvent at or immediately before application to a plant. The lysis agent may be, for example, a liquid agent or wettable powder in which the bacteriophage is suspended in a suitable solution, or may be a solid powder, granule, or gel agent in which the bacteriophage is mixed with a carrier and solidified. The lysis agent may be provided in a state in which the bacteriophage is concentrated. For example, the lysis agent may be a culture of the bacteriophage as is or diluted, or may be prepared as a suspension by suspending the bacteriophage in a solvent such as water or a buffer solution.

2-4. Application method The method of application of the plant disease control composition of the present invention is not particularly limited as long as the plant disease control composition can be brought into contact with the plant to be controlled, and examples of the method include a method of directly spraying or dispersing the composition on crops, a method of spraying the composition on soil, etc. The plant disease control composition may be directly applied as it is, or may be applied after diluting with water or the like as necessary.

The application rate of the plant disease control composition varies depending on the crop to which it is applied, the target disease, the application method, the tendency for occurrence, the extent of damage, environmental conditions, and the formulation used, so it is desirable to adjust it appropriately.

2-5. Target plants The target plant of the plant disease control composition of the present invention is not particularly limited as long as it is a plant that can develop a plant disease caused by bacteria. It may be either an angiosperm or a gymnosperm. Furthermore, it does not matter whether it is a herbaceous plant or a woody plant. Suitable specific examples of target plants include agriculturally important plants, such as crop plants such as cereals, vegetables, and fruits, and ornamental plants. Specifically, monocotyledonous plants include plants of the Poaceae family (e.g., rice, wheat, barley, corn, sugarcane, sorghum, sorghum, and turfgrass), plants of the Musaceae family (e.g., banana), plants of the Amaryllidaceae family (e.g., leeks, onions, garlic, and Chinese chives), and plants of the Liliaceae family (e.g., lilies and tulips). In addition, among dicotyledonous plants, Brassicaceae plants (e.g., broccoli, cabbage, radish, Chinese cabbage, rapeseed), Asteraceae plants (e.g., lettuce, burdock, chrysanthemum), Fabaceae plants (e.g., soybean, peanut, pea, kidney bean, lentil, chickpea, broad bean, licorice), Solanaceae plants (e.g., tomato, eggplant, potato, tobacco, bell pepper, capsicum, petunia), Rosaceae plants (e.g., strawberry, apple, pear, peach, loquat, almond, plum, rose, , plum, cherry), Cucurbitaceae plants (e.g., cucumber, gourd, pumpkin, melon, watermelon), Anacardiaceae plants (e.g., mango, pistachio, cashew nut), Lauraceae plants (e.g., avocado), Rutaceae plants (e.g., mandarin orange, grapefruit, lemon, yuzu), Convolvulaceae plants (e.g., sweet potato), Theaceae plants (e.g., tea plant), and Vitaceae plants (e.g., grape).

2-6. Target Plant Diseases The target plant diseases controlled by the plant disease control composition of the present invention include all plant diseases caused by bacteria, and are not particularly limited. As a mere example, the target plant diseases include plant diseases caused by bacteria of the genus Xanthomonas. For example, bacterial spot disease found in peaches, bacterial blight found in walnuts, bacterial pustule found in soybeans, angular leaf spot found in strawberries, bacterial blight found in tomatoes, peppers, lettuce, etc., black rot found in cabbage, Chinese cabbage, broccoli, etc., bacterial canker found in oranges and grapefruits, angular leaf spot found in cotton, and leaf blight found in rice. Other examples of such diseases include fire blight, which is caused by Erwinia bacteria and is seen in apples, bacterial soft rot, which is caused by Pectobacterium bacteria and is seen in onions, and ear soft rot, which is caused by Burkholderia bacteria and is seen in corn.

2-7. Effect The plant disease control composition of the present invention contains more than 0% by mass and less than 0.9% by mass of sodium chloride, which inhibits the decrease in bacteriophage activity after application and improves the plant disease control effect, making it useful for preventing and suppressing diseases caused by target bacteria. In addition, since phages are biological substances, no manifestation of phytotoxicity has been reported, and since they are highly specific, their impact on the bacterial flora balance is extremely limited.

3. Plant disease control method One aspect of the present invention is a plant disease control method. The plant disease control method of the present invention includes a step of contacting a target plant with a plant disease control composition containing sodium chloride of more than 0 mass% and less than 0.9 mass%, and a bacteriophage that exhibits lytic activity against bacteria. The plant disease control method can control plant disease in a target plant by contacting the plant disease control composition with the target plant. As the plant disease control composition used in the plant disease control method, the above-mentioned plant disease control composition of the present invention can be used.

In other words, one aspect of the present invention is a method for controlling plant diseases, which includes a step of contacting a target plant with the plant disease control composition of the present invention.

Another aspect of the present invention includes a step of mixing sodium chloride with a bacteriophage that exhibits lytic activity against bacteria to prepare a plant disease control composition. That is, this aspect is a plant disease control method including a step of mixing sodium chloride with a bacteriophage that exhibits lytic activity against bacteria to prepare a plant disease control composition, and a step of contacting a target plant with the plant disease control composition that contains more than 0 mass% and less than 0.9 mass% sodium chloride and a bacteriophage that exhibits lytic activity against bacteria.

In this specification, "contact" refers to contact between the plant disease control composition and the target plant. More specifically, it refers to contact of the plant disease control composition with at least a part of the target plant. This contact step is intended to infect the target bacterium with the phage, which is the active ingredient, thereby lysing the target bacterium. As a result, a control effect against plant diseases caused by the target bacterium can be exerted.

The contact is preferably carried out by direct contact, and direct contact refers to applying, spraying, scattering or immersing the target plant in a liquid or gel plant disease control composition. In this case, the parts of the plant that are the subject of contact are mainly the leaves, flowers, fruits, stems, branches and/or trunks. In one embodiment of the present invention, the step of contacting the target plant with the plant disease control composition is a step of foliar spraying the plant disease control composition on the target plant. Foliar spraying can efficiently exert the effect of the phage, and since leaves are generally exposed to ultraviolet light, a decrease in bacteriophage activity can be a problem with conventional plant disease control compositions and lysing agents, but by using the plant disease control composition of the present invention, it is possible to suppress the decrease in bacteriophage activity, and it is extremely useful.

4. Uses of Sodium Chloride One aspect of the present invention is the use of sodium chloride.

In other words, one aspect of the present invention is the use of sodium chloride in a plant disease control composition containing a bacteriophage that exhibits lytic activity against bacteria, in order to improve the residual activity of the bacteriophage that exhibits lytic activity against bacteria in a target plant.

In the use of sodium chloride in the present invention, the residual activity may be the residual activity under direct sunlight and/or at a temperature of 1 to 60°C (e.g., 1 to 50°C, 1 to 40°C, 1 to 30°C, 10 to 30°C, 10 to 25°C, or 15 to 20°C). Whether sodium chloride improves the residual activity of bacteriophage in a target plant can be determined, for example, by the following method. A predetermined amount of aqueous sodium chloride solution or water (e.g., 0.2 mL) is added to a predetermined amount of bacteriophage-containing liquid (e.g., 1.8 mL). The resulting bacteriophage-containing sodium chloride-containing composition and bacteriophage-containing non-sodium chloride-containing composition are allowed to stand for a predetermined period of time (e.g., 1 day) under conditions for growing plants. The phage titer based on the number of plaques (Plaque Forming Units, PFU) is measured for each composition before and after the standing by a plaque assay method. The potency of the composition after standing is calculated as the residual activity relative to the potency of the composition before standing. If the residual activity of the bacteriophage-containing sodium chloride-containing composition is 10% or more higher than the residual activity of the bacteriophage-containing non-sodium chloride-containing composition, it can be determined that sodium chloride has residual activity.

Another aspect of the present invention is the use of sodium chloride in the production of a plant disease control composition containing a bacteriophage that exhibits lytic activity against bacteria.

The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

<Preparation of phage>
A plate lysate (PL) method, which is an amplification method using a plaque assay, was carried out using a phage (Myoviridae) having the genomic DNA sequence shown in SEQ ID NO: 3, which was isolated from the environment in Japan, and a bacterial strain (MAFF No. 311351) obtained from the National Agriculture and Food Research Organization, in which this phage exhibits bacteriolytic activity.

The bacteria/phage mixture was adjusted so that many plaques would form on the YPG agar, and then mixed with Top agar, spread on the YPG agar, and cultured. Then, 3 mL of SM Buffer was added to the Top agar on which the plaques had formed, and the mixture was shaken at 25°C for about 30 minutes, and the supernatant was passed through a 0.2 μm filter to recover a recovery liquid containing the phage. The phage was amplified to about 10 ⁸ PFU/mL by the above operation, and the amplified liquid was diluted with sterilized ultrapure water to about 10 ³ PFU/mL to be used as a phage-containing liquid.

(Preparation of medium)
The medium used in the PL method was prepared as follows.
1 g of peptone, 1 g of yeast extract, and 2 g of glucose were dissolved in 1 L of H ₂ O and autoclaved to prepare a liquid medium (YPG Broth).

Agar medium (YPG Agar) was prepared by adding 15 g of agar per liter to YPG Broth and autoclaving. In addition, soft agar medium (Top Agar) to be layered on top of the agar medium was prepared by adding 5 g of agarose per liter to YPG Broth and autoclaving. Top Agar was stored at approximately 50°C and used as needed.

<Stabilization rating 1>
To 1.8 mL of the phage-containing solution, 0.2 mL of a mixture of 10% sodium chloride and sterilized ultrapure water was added so that the final concentration of sodium chloride was 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0% (mass%) to prepare a total of 2 mL of phage-containing aqueous sodium chloride solution. For example, to make the final concentration of sodium chloride 0.1%, a mixture of 10% sodium chloride and sterilized ultrapure water in a ratio of 1:9 was added.

0.02 mL was taken from the prepared sodium chloride aqueous solution containing various phages and used as the pre-standing sample (day 0). The sodium chloride aqueous solution containing various phages was then poured into a UV-transparent cell, which was then covered to suppress evaporation and left in a greenhouse environment (20°C during the day/approximately 15°C at night, in direct sunlight during the day). After leaving it alone, 0.02 mL was taken at appropriate intervals, such as one day later (day 1) or two days later (day 2), and used as the post-standing sample.

The titer [PFU/mL] based on the number of plaques (Plaque Forming Units, PFU) was calculated for the collected samples before and after standing using a plaque assay, and the ratio of the titer after standing (day 1) to the titer before standing (day 0) was quantified as the residual activity (Residual Activity Ratio vs. Day 0). In this assay, the number of plaques was counted using 0.02 mL of phage-containing aqueous solution, so the titer (number of plaques per mL) was calculated by multiplying the counted number of plaques by 50. Note that, from the perspective of making it easier to evaluate the residual activity, compositions with a lower titer [PFU/mL] before standing were used in Stabilization Evaluation 1 and Stabilization Evaluation 2 described below, compared to the compositions actually applied to plants.

Specifically, the titer was determined by preparing a plate with YPG agar medium as the bottom layer and 0.6% soft agar medium containing a specified amount of host bacteria and phage as the top layer, co-culturing the host bacteria and phage, and counting the number of plaques formed.

The results of stability evaluation 1 are shown in Table 1 and Figure 1. The stability evaluation was performed twice, and in Table 1 and Figure 1, the first test is shown as Exp. 1 and the second test is shown as Exp. 2. Table 1 and Figure 1 summarize the results before standing (day 0) and after standing (day 1).

<Stabilization rating 2>
To 1.4 mL of the phage-containing solution, 0.2 mL of each of the aqueous solutions with 10 times the concentration of each additive was added so that the final concentration of each additive was the desired one. For example, when 0.2 mL each of 1.0% skim milk (SM), 5.0% sodium chloride (NaCl), and 0.3% Silwet (registered trademark) L-77 (SL) was added, a 0.1% SM/0.5% NaCl/0.03% SL solution was obtained in which the phage was present at a titer of 10 ² PFU/mL or more. All percentages are by mass. Various phage-containing aqueous solutions (samples) were prepared by combining the presence or absence of each additive. When a certain additive was not added, 0.2 mL of sterilized ultrapure water was added instead of the aqueous solution of the additive. That is, all phage-containing aqueous solutions were prepared so that the total volume was 2.0 mL.

The prepared phage-containing aqueous solutions were evaluated in the same manner as in Stability Evaluation 1 described above, and the ratio of the titer after standing to the titer before standing was quantified as the residual activity. However, the amount of phage-containing aqueous solution taken before and after standing was 0.1 mL, and the titer was calculated by multiplying the number of plaques counted by 10.

The results of stability evaluation 2 are shown in Table 2. The relative values of the average residual activity in Table 2 are relative values when the average residual activity of sample 1 is set to 1.0.

<Tests on plant disease control effects>
The following bacterial strains were used to prepare the phage spray used on each plant:
Tomato: Xanthomonas campestris pv. vesicatoria (MAFF No. 301256)
Broccoli: Xanthomonas campestris pv. campestris (MAFF No. 106765)

Phages used (cocktail of four phages)
Tomato: phage (Siphoviridae) having the genomic DNA sequence shown in SEQ ID NO:2, phage (Myoviridae) having the genomic DNA sequence shown in SEQ ID NO:1, phage (Siphoviridae) having the genomic DNA sequence shown in SEQ ID NO:6, phage (Siphoviridae) having the genomic DNA sequence shown in SEQ ID NO:5
Broccoli: phage (Siphoviridae) having the genomic DNA sequence shown in SEQ ID NO:2, phage (Myoviridae) having the genomic DNA sequence shown in SEQ ID NO:1, phage (Autographiviridae) having the genomic DNA sequence shown in SEQ ID NO:4, phage (Siphoviridae) having the genomic DNA sequence shown in SEQ ID NO:5

(1) Preparation of samples The phage spray solution used in this test was prepared as follows. First, the bacterial strain was inoculated into YPG culture medium and cultured overnight in a shaker set at 25°C to prepare a bacterial culture medium with an OD ₆₀₀ (turbidity at 600 nm) of about 1.0. Then, this bacterial culture medium and a separately prepared purified phage solution (with a titer of about 10 ⁸ PFU/mL) were mixed in equal amounts and inoculated into 100 times the amount of YPG culture medium, co-cultured for about 8 to 12 hours in a shaker set at 25°C, and the culture medium was recovered. If the titer after culture was insufficient, the mixture ratio of the culture medium and the purified phage solution in the co-culture was finely adjusted and co-cultured again. 1/10 volume of chloroform was added to the recovered liquid, which was vigorously stirred and then centrifuged, and the supernatant was recovered without touching the chloroform at the bottom. The supernatant was then filtered through a 0.2 μm filter to obtain a phage-containing liquid free of bacterial cell contamination. A phage-containing liquid was prepared for each phage, and equal amounts of the phage-containing liquids were mixed together to obtain a stock solution before dilution, with the titers adjusted to the same order.

This stock solution was diluted with an aqueous solution containing each component and sterile tap water so that the titer was about ¹⁰ PFU/mL and the concentrations of sodium chloride, skim milk (SM), and Silwet (registered trademark) L-77 (SL) were included in the concentrations shown in Table 3. The final phage dispersal solution (sample) was used. Note that all percentages in the table are by mass.

The bacterial spray solution used to infect plants in this test was prepared by applying a solution prepared by diluting the bacterial cell culture solution, without (before) mixing the above-mentioned phage purification solution, approximately 10,000 times with sterile tap water to a YPG plate and culturing the plates on a solid state for approximately 1-3 days in an incubator set at 25°C. The colonies on the solid culture plate were collected while suspending them in sterile tap water, and the bacteria-containing solution was diluted with sterile tap water to a final _OD600 (turbidity at 600 nm) of approximately 0.5 to use as the bacterial spray solution.

(2) Plants Commercially available tomato and broccoli seeds were sown and grown in a greenhouse. Tomato seedlings with 50 or more leaves and broccoli seedlings with 5 or more leaves were used as evaluation leaf specimens. Phage spray solution was sprayed twice before and after bacterial infection treatment, with an interval of 2 to 3 days. Eight specimens were prepared for each spray solution. For the untreated group, sterile tap water was used instead of the phage purified solution described above in (1), and the culture solution obtained by mass-culturing only bacteria and then sterilizing it was diluted with sterile tap water and sprayed on the leaves. As a result, the untreated group was almost the same except that the spray solution did not contain phages. In some cases, multiple branches of the same seedling were divided into two groups and sprayed with phages. For bacterial infection treatment, the bacterial spray solution was sprayed on the leaves, and then the specimens were placed in a simple vinyl house with the inside sufficiently moistened with sterile tap water, sealed, and left for about two days under conditions with a higher humidity than the surrounding area. Then, one week or more after the infection treatment, when a certain degree of disease was confirmed in the untreated group, the disease incidence rate was investigated when each phage spray solution was applied. In the case of peach bacterial hole disease, characteristic brown spots appear on the leaf surface, so the disease incidence rate was calculated as the ratio of the number of leaves that were confirmed to be infected to the total number of leaves at the time of investigation.

(3) Testing the control effect of various phages By checking the disease incidence according to the above method, both tomato and broccoli were evaluated 20 days after infection. Note that broccoli has a smaller total number of leaves (denominator) and a larger leaf area than tomato, so there is a high risk that the spray will not reach all areas. Therefore, the disease incidence rate of broccoli may be worse than that of tomato in terms of the values of the untreated group.

(4) Results The results are shown in Table 4 below.

As shown in Table 4, samples B, C, E, and F, which correspond to the embodiment, were confirmed to have a lower disease incidence rate than the other examples. This is thought to be because sodium chloride inhibits the decline in phage activity.

The upper and/or lower limit values of the numerical ranges described in this specification can be combined in any manner to define a preferred range. Although the present embodiment has been described in detail above, the specific configuration is not limited to this embodiment, and even if there are design changes within the scope of the present disclosure, they are included in the present disclosure.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

A method for controlling plant diseases, comprising the step of contacting a target plant with a plant disease control composition containing sodium chloride greater than 0% by mass and less than 0.9% by mass, and a bacteriophage that exhibits lytic activity against bacteria.
The plant disease control method according to claim 1, wherein the step is a step of foliar spraying of the plant disease control composition on a target plant.
The method for controlling plant diseases according to claim 1, comprising the step of mixing sodium chloride with a bacteriophage that exhibits lytic activity against bacteria to prepare a plant disease control composition.
The plant disease control method according to claim 1, wherein the plant disease control composition further comprises a protective agent.
The plant disease control method according to claim 4, wherein the protective agent includes skim milk.
The plant disease control method according to claim 1, wherein the plant disease control composition further comprises a spreading agent.
The plant disease control method according to claim 6, wherein the wetting agent includes an organic silicone wetting agent.
The plant disease control method according to claim 1, wherein the plant disease control composition further comprises at least one component selected from an agriculturally acceptable carrier and an agriculturally acceptable solvent.
A plant disease control composition containing more than 0% by mass and less than 0.9% by mass of sodium chloride, and a bacteriophage that exhibits lytic activity against bacteria.
The plant disease control composition according to claim 9, further comprising a protective agent.
The plant disease control composition according to claim 10, wherein the protective agent comprises skim milk.
The plant disease control composition according to claim 9, further comprising a spreading agent.
The plant disease control composition according to claim 12, wherein the spreading agent comprises an organic silicone-based spreading agent.
The plant disease control composition according to claim 9, further comprising at least one component selected from an agriculturally acceptable carrier and an agriculturally acceptable solvent.
The use of sodium chloride in a plant disease control composition containing a bacteriophage exhibiting lytic activity against bacteria, in order to improve the residual activity of the bacteriophage exhibiting lytic activity against bacteria in a target plant.
Use of sodium chloride in the manufacture of a plant disease control composition containing a bacteriophage exhibiting lytic activity against bacteria.