Agent for controlling gray mold disease, method for controlling gray mold disease, method for obtaining bacterial strain for controlling gray mold disease, and bacterial strain

Abstract

To provide a new agent for controlling gray mold disease that employs a microorganism as an active component.SOLUTION: An agent for controlling gray mold disease of plants, the agent containing a plant-derived C1 microorganism.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a control agent for Botrytis cinerea, a control method for Botrytis cinerea, a method for obtaining a bacterial strain for controlling Botrytis cinerea, and a bacterial strain. [Background technology]

[0002] Gray mold damages many plants and causes significant damage to agricultural production. Currently, chemical pesticides and microbial pesticides are used as methods for controlling gray mold. Chemical pesticides have antifungal effects, but there is a limit to the number of times they can be used, so they cannot be used frequently in agricultural fields (Non-Patent Document 1). On the other hand, microbial pesticides have issues such as no therapeutic effect after the onset of disease due to nutrient competition, and their effectiveness does not last because they do not easily establish after spraying. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Ogawara et al., "Current status of tomato gray mold control and reduction of the number of chemical synthetic fungicide applications using microbial fungicides," Research Report of the Horticultural Research Institute, Ibaraki Prefectural Agricultural Center (2006) 14, 35-42 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a novel control agent which contains a microorganism as an active ingredient and is capable of controlling Botrytis cinerea.

[0005] Another object of the present invention is to provide a method for obtaining a microorganism capable of controlling Botrytis cinerea, and a novel bacterial strain obtained by the method. [Means for solving the problem]

[0006] The present inventors focused on plant symbiotic C1 microorganisms and completed the present invention by selecting plant symbiotic C1 microorganisms that have a growth inhibitory effect on Botrytis cinerea.

[0007] That is, the present invention relates to, for example, the following inventions. [1] A plant gray mold control agent containing a plant-derived C1 microorganism. [2] The agent described in [1], wherein the C1 microorganism is selected from the group consisting of bacteria of the species Serratia rubidae, bacteria of the genus Pantoea, bacteria of the genus Cartobacterium, and combinations thereof. [3] The agent according to [1] or [2], characterized in that it controls gray mold by inhibiting the germination and / or growth of Botrytis cinerea by the C1 microorganism. [4] The C1 microorganism is selected from the group consisting of Serratia rubidaea bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, Serratia rubidaea bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 2, Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 3, Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4, Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5, Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 6, and Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 7. The agent described in any one of [1] to [3] is selected from the group consisting of Serratia rubidae bacteria having an rRNA gene, Pantoea bacteria having a 16S rRNA gene consisting of a base sequence having 99% or more sequence identity to the base sequence shown in SEQ ID NO: 8, Curtobacterium bacteria having a 16S rRNA gene consisting of a base sequence having 99% or more sequence identity to the base sequence shown in SEQ ID NO: 9, and combinations thereof. [5] the C1 microorganism is a bacterium of the species Serratia rubidaea selected from the group consisting of SY163 strain (NITE-AP-04103), SY183 strain (NITE-AP-04104), SY135 strain, SY89 strain, SY131 strain, SY339 strain, SY50 strain, and mutant strains thereof; a Pantoea bacterium selected from the SY685 strain (NITE-AP-04105) or a mutant thereof; The agent according to any one of [1] to [4], wherein the agent is selected from the group consisting of a bacterium belonging to the genus Cartobacterium selected from the group consisting of the SY851 strain (NITE-AP-04106) or a mutant strain thereof, and a combination thereof. [6] A method for controlling Botrytis cinerea in plants, comprising a step of treating a plant with the Botrytis cinerea control agent according to any one of [1] to [5]. [7] The C1 microorganism is selected from the group consisting of Serratia rubidaea bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, Serratia rubidaea bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 2, Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 3, Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4, Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5, Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 6, and Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 7. The method described in [6], wherein the bacterium is selected from the group consisting of Serratia rubidae bacteria having an rRNA gene, Pantoea bacteria having a 16S rRNA gene consisting of a base sequence having 99% or more sequence identity to the base sequence shown in SEQ ID NO: 8, Curtobacterium bacteria having a 16S rRNA gene consisting of a base sequence having 99% or more sequence identity to the base sequence shown in SEQ ID NO: 9, and combinations thereof. [8] the C1 microorganism is a bacterium belonging to the species Serratia rubidaea selected from the group consisting of SY163 strain (NITE-AP-04103), SY183 strain (NITE-AP04104), SY135 strain, SY89 strain, SY131 strain, SY339 strain, SY50 strain, and mutant strains thereof; a Pantoea bacterium selected from the SY685 strain (NITE-AP-04105) or a mutant thereof; The method according to [6] or [7], wherein the bacterium is selected from the group consisting of a bacterium belonging to the genus Cartobacterium selected from the group consisting of the SY851 strain (NITE-AP-04106) or a mutant thereof, and a combination thereof. [9] The method according to any one of [6] to [8], wherein the treatment of the plant includes treatment of the above-ground parts of the plant.

[10] In the step of applying to plants, the concentration of the C1 microorganism is 2 x 10 based on the total amount of the gray mold control agent. 5 ~1×10 12 The method according to any one of [6] to [9], characterized in that the treatment is carried out with a gray mold control agent at a concentration of 1 CFU / ml.

[11] The method according to any one of [6] to

[10] , wherein the treatment of the plant is carried out before, after, or both before and after the onset of Botrytis cinerea on the plant.

[12] Cultivating a single strain of C1 microorganism; contacting the single strain with cultured Botrytis cinerea; and a step of determining the C1 microorganism as a strain inhibiting the germination and / or growth of Botrytis cinerea when the germination and / or growth of Botrytis cinerea is inhibited; Including, A method for obtaining a plant-derived C1 microbial strain that inhibits the germination and / or growth of Botrytis cinerea.

[13] A method for producing a gray mold control agent using a plant-derived C1 microbial strain that inhibits the germination and / or growth of Botrytis cinerea, obtained by the method described in

[12] .

[14] SY163 strain (NITE-AP-04103), SY183 strain (NITE-AP04104), SY135 strain, SY89 strain, SY131 strain, SY339 strain, SY50 strain, SY685 strain (NITE-AP-04105), or SY851 strain (NITE-AP-04106). [Effects of the Invention]

[0008] According to the present invention, there are provided a novel control agent that uses a microorganism as an active ingredient and is capable of controlling Botrytis cinerea, a method for obtaining a microorganism capable of controlling Botrytis cinerea, and a novel bacterial strain obtained by the method. [Brief explanation of the drawings]

[0009] [Figure 1] In Example 2, the antifungal activity of 1,000 isolated methanol-utilizing bacterial strains was evaluated using the agar well diffusion method on PDA medium coated with a suspension of Botrytis cinerea. The method and results are shown in Figure 1(A). SY135, SY163, and SY183 exhibited zones of inhibition against Botrytis cinerea. The inhibitory effect of methanol-utilizing bacterial strains on growing Botrytis cinerea mycelia was also investigated using a dual culture assay. The method and results are shown in Figure 1(B). Zones of inhibition were observed for SY135, SY163, and SY183. [Figure 2] As in Figure 1(A), the antifungal activity of 1,000 isolated methanol-utilizing bacteria was evaluated using the agar well diffusion method on PDA plates coated with a suspension of Botrytis cinerea. SY50, SY89, SY131, SY135, SY339, SY685, SY851, SY685, and SY851 showed zones of inhibition against Botrytis cinerea. [Figure 3] Similar to Figure 1(B), the inhibitory effect of methanol-utilizing bacterial strains on growing Botrytis cinerea mycelia was evaluated using a dual culture assay. Zones of inhibition were observed for SY50, SY89, SY131, and SY339. [Figure 4]This is a phylogenetic tree showing the results of a homology search of the 16S rRNA gene sequences for seven strains of the genus Serratia that have been found to have antifungal activity against Botrytis cinerea. [Figure 5] This is a phylogenetic tree showing the results of a homology search using the gyrB gene for seven strains of the genus Serratia that were found to have antibacterial activity against Botrytis cinerea. [Figure 6] 1 is a graph showing the results of examining the inhibitory effects of the inhibitory strains SY163 and SY183 on Botrytis cinerea on tomato foliage in Example 3. The graph shows the change in disease severity up to day 22 when treated under conditions A to D for (A) the SY163 strain and (B) the SY183 strain, respectively. [Figure 7] The severity of the disease was evaluated using a four-point scale (0-3) based on the percentage of diseased leaf area (0 = no symptoms, 1 = 0.1-24.9%, 2 = 25-49.9%, 3 = 50-100%). Examples of disease indices of 0, 1, and 3 are shown in (A). The percentage of leaves with spores after 22 days is shown in (B). Condition A: Botrytis cinerea only was applied on day 1; Condition B: Botrytis cinerea and each inhibitory strain were applied on day 1; Condition C: Each inhibitory strain was applied on day 0, Botrytis cinerea and each inhibitory strain were applied on day 1; Condition D: Botrytis cinerea and each inhibitory strain were applied on day 1, and each inhibitory strain was applied on day 2. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0011] [Control agent or composition for controlling Botrytis cinerea in plants] In one embodiment of the present invention, a plant Botrytis control agent or composition for controlling Botrytis cinerea comprises a C1 microorganism. The C1 microorganism is derived from a plant and is selected from the group consisting of bacteria of the species Serratia rubidaea, bacteria of the genus Pantoea, bacteria of the genus Curtobacterium, and combinations thereof. Any description of a control agent in this specification can be interpreted as a description of a control composition.

[0012] (C1 microorganism) As used herein, microorganisms that can utilize reduced C1 compounds such as methane and methanol as carbon and energy sources are referred to as C1 microorganisms. C1 microorganisms are also referred to as C1-utilizing bacteria. Among C1 microorganisms, microorganisms that can utilize methanol as a carbon and energy source are referred to as methanol-utilizing bacteria, and bacteria that can utilize methane as a carbon and energy source are referred to as methanotrophs and methanotrophs. Most methanol-utilizing bacteria are facultative methanol-utilizing bacteria, which utilize carbon sources other than C1 compounds. Microorganisms that use only C1 compounds as a carbon source are also referred to as obligate methylotrophs. C1 microorganisms may be methanol-utilizing bacteria.

[0013] C1 microorganisms normally present on plant leaves are said to be in a symbiotic relationship with plants, utilizing methane and methanol emitted by the plant to produce plant hormones that promote plant growth. The present inventors hypothesized that bacteria in a symbiotic relationship with plants could be expected to establish themselves on plants, and came up with the idea of ​​selecting bacteria from C1 microorganisms that can be used as microbial pesticides against gray mold. However, there have been no reports to date of the use of C1 microorganisms as microbial pesticides against gray mold. Furthermore, even if C1 microorganisms exist, such as bacteria of the species Serratia rubidae, Pantoea, or Cartobacterium, bacteria of the same genus and species are not necessarily C1 microorganisms; in fact, it is expected that they are rare.

[0014] C1 microorganisms can be obtained by methods known to those skilled in the art. C1 microorganisms can be selected, for example, by culturing microorganisms in a solid medium containing a C1 compound as the sole carbon source and collecting colonies (isolated strains). For example, methanol-assimilating bacteria can be obtained by culturing and selecting microorganisms in a solid medium containing methanol as the sole carbon source, such as a low-nutrient methanol inorganic salts medium plate. If microorganisms present on the surface of plants (e.g., leaves and stems) are used as the microorganisms to be cultured, C1 microorganisms derived from plants (e.g., derived from aboveground parts such as leaves and / or stems) can be obtained.

[0015] A solid medium containing a C1 compound as the sole carbon source can be prepared by adding ingredients for solidifying the liquid, such as agar and polysaccharides, to a liquid medium containing a C1 compound such as methane, methanol, or formaldehyde, inorganic salts such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, potassium nitrate, or magnesium sulfate, and distilled water. The solid medium may further contain an antibiotic such as tunicamycin.

[0016] The C1 microorganism contained in the control agent of this embodiment is selected from the group consisting of bacteria of the species Serratia rubidae, bacteria of the genus Pantoea, bacteria of the genus Cartobacterium, and combinations thereof. This means bacteria that are C1 microorganisms among bacteria of the species Serratia rubidae, bacteria of the genus Pantoea, and bacteria of the genus Cartobacterium.

[0017] Serratia rubidae bacteria, which are C1 microorganisms, include Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 2, Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 3, Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4, Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5, Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 6, and Serratia rubidae bacteria having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 7. The bacteria may be Serratia rubidae species that have rRNA genes.

[0018] Furthermore, the C1 microorganism, Serratia rubidae bacterium, may be a Serratia rubidae bacterium selected from the SY163 strain (NITE-AP-04103), the SY183 strain (NITE-AP-04104), the SY135 strain, the SY89 strain, the SY131 strain, the SY339 strain, the SY50 strain, or a mutant strain thereof, or may be a Serratia rubidae bacterium selected from the SY163 strain (NITE-AP-04103), the SY183 strain (NITE-AP-04104), or a mutant strain thereof.

[0019] The Pantoea bacterium, which is a C1 microorganism, may be a Pantoea bacterium having a 16S rRNA gene consisting of a nucleotide sequence that has 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO:8.

[0020] Furthermore, the Pantoea bacterium, which is a C1 microorganism, may be a Pantoea bacterium selected from the SY685 strain (NITE-AP-04105) or a mutant strain thereof.

[0021] The C1 microorganism, the genus Cartobacterium, may be a bacterium of the genus Cartobacterium having a 16S rRNA gene consisting of a nucleotide sequence that has 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO:9.

[0022] Furthermore, the C1 microorganism, the genus Cartobacterium, may be a Cartobacterium selected from the SY851 strain (NITE-AP-04106) or a mutant strain thereof.

[0023] The SY163 strain (NITE-AP-04103), SY183 strain (NITE-AP-04104), SY135 strain, SY89 strain, SY131 strain, SY339 strain, SY50 strain, SY685 strain (NITE-AP-04105), and SY851 strain (NITE-AP-04106) are bacterial strains newly discovered by the present inventors. Those assigned numbers beginning with NITE-AP have been deposited at the National Patent Microorganisms Depositary (NPMD), Biotechnology Center, National Institute of Technology and Evaluation (NITE) (2-5-8-122 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) (received on April 11, 2024).

[0024] The sequence of the 16S rRNA gene of the SY163 strain is shown in SEQ ID NO: 1, the sequence of the 16S rRNA gene of the SY183 strain is shown in SEQ ID NO: 2, the sequence of the 16S rRNA gene of the SY135 strain is shown in SEQ ID NO: 3, the sequence of the 16S rRNA gene of the SY89 strain is shown in SEQ ID NO: 4, the sequence of the 16S rRNA gene of the SY131 strain is shown in SEQ ID NO: 5, the sequence of the 16S rRNA gene of the SY339 strain is shown in SEQ ID NO: 6, the sequence of the 16S rRNA gene of the SY50 strain is shown in SEQ ID NO: 7, the sequence of the 16S rRNA gene of the SY685 strain is shown in SEQ ID NO: 8, and the sequence of the 16S rRNA gene of the SY851 strain is shown in SEQ ID NO: 9.

[0025] The sequence of the gyrB gene of the SY163 strain is shown in SEQ ID NO: 14, the sequence of the gyrB gene of the SY183 strain is shown in SEQ ID NO: 15, the sequence of the gyrB gene of the SY135 strain is shown in SEQ ID NO: 16, the sequence of the gyrB gene of the SY89 strain is shown in SEQ ID NO: 17, the sequence of the gyrB gene of the SY131 strain is shown in SEQ ID NO: 18, the sequence of the gyrB gene of the SY339 strain is shown in SEQ ID NO: 19, and the sequence of the gyrB gene of the SY50 strain is shown in SEQ ID NO: 20.

[0026] The bacterial mutant strains mentioned above include those that have naturally occurring mutations or morphological variations in each bacterial strain, those obtained by screening for mutations induced in each bacterial strain using radiation such as gamma rays or X-rays or chemical agents such as ethyl methanesulfonate (EMS), or those obtained by genetic engineering techniques such as transposon introduction or CRISPR-Cas9, and have altered characteristics while maintaining the taxonomic properties of each bacterial strain as a new species. For example, they may have germination and / or growth inhibitory activity against Botrytis cinerea and maintain 99% or more sequence identity based on 16S rRNA gene analysis.

[0027] Strains SY163, SY183, SY135, SY89, SY131, SY339, and SY50 are Serratia rubidae bacteria, Gram-stain-negative, grow at 25-37°C, and are facultative anaerobic. Strain SY685 is a Pantoea bacterium, Gram-stain-negative, grow at 25-37°C, and is either aerobic or facultative anaerobic. Strain SY851 is a Cartobacterium bacterium, Gram-stain-positive, grow at 25-37°C, and is an obligate aerobic bacterium.

[0028] As used herein, "sequence identity" refers to the percentage (%) of identical DNA bases relative to the total overlapping DNA sequence in the optimal alignment of two DNA sequences, as determined by a mathematical algorithm known in the art. Alignment can be performed using, for example, CLUSTAL X or MAFFT, and sequence identity can be calculated using, for example, CLUSTAL X or MAFFT.

[0029] In this specification, the sequence identity to the base sequences shown in SEQ ID NOs: 1 to 20 may be, for example, 98.7% or more, 99% or more, 99.5% or more, 99.7% or more, 99.8% or more, 99.9% or more, 99.96% or more, 99.97% or more, 99.96% or more, or 100%.

[0030] In the present specification, the 16S rRNA gene or the gyrB gene may contain 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 2 or 3 nucleotide sequence differences with respect to the nucleotide sequences shown in SEQ ID NOs: 1 to 9 and 10 to 20. The nucleotide sequence differences may be insertions, substitutions, deletions, or combinations thereof, when the nucleotide sequences shown in SEQ ID NOs: 1 to 9 and 10 to 20 are used as a reference.

[0031] The bacteria contained in the control agent of this embodiment may be live bacteria or dead bacteria. For example, a bacterial culture may be used as is, or the bacteria may be isolated after culture and used as is, or may be dried and used, for example, after further pulverization.

[0032] (Botrytis) Gray mold is a disease caused by Botrytis cinerea. Botrytis cinerea is a filamentous fungus belonging to the imperfect fungi, and is prolific, causing gray mold on many vegetables and fruit trees, including ornamental plants. It occurs mainly on stems, leaves, and flowers, and on leaves, the tips and margins turn brown, and the lesions eventually expand. On flowers, small brown to amber-colored spots appear on the petals and expand. In humid conditions, conidia are produced in the affected areas.

[0033] (plant) As used herein, unless otherwise specified, the term "plant" includes whole plants, plant cells, plant protoplasts, plant callus, or plant parts such as embryos, pollen, ovules, gametes, seeds, leaves, flowers, branches, fruits, stems, roots, anthers, etc.

[0034] The plants to be controlled from gray mold are not particularly limited, but are preferably agricultural products, including, but not limited to, vegetables, grains, fruits, flowers, and legumes. Specific examples thereof include Solanaceae plants (Solanum plants such as eggplant, tomato, potato, etc., Capsicum plants such as bell peppers and capsicum), Rosaceae plants (Fragaria plants, Prunus plants such as cherry, peach, plum, etc., Loquat plants), Cucurbitaceae plants (Cucumber plants such as cucumber, melon, etc., Melon plants such as watermelon, Cucurbita plants such as pumpkin, zucchini, Bottle gourd plants such as bottle gourd, Luffa plants such as loofah, Wax gourd plants such as wax gourd, Ecclesiastes plants such as Easter melon, Momordica charantia plants such as bitter melon), Rutaceae plants (citrus), Leguminosae plants (soybean, Glycine soja, etc., Purple gourd, etc.), Examples include Medicago plants such as soybean (alfalfa), Vitis vinifera plants such as cowpea and adzuki bean, Phaseolus vulgaris plants such as kidney beans, Pisum sativum plants such as peas, Winged Beans such as winged beans, Vicia faba plants such as broad beans, Jack Beans such as jack beans, Arachis hypogaea plants such as peanuts, and Lentils such as lentils, Asteraceae plants (Lactuca sativa plants such as lettuce, Larch plants such as burdock, Garland chrysanthemum plants such as crown daisy), Liliaceae plants (Allium spp. such as scallions, chives, leeks, and onions), Vitaceae plants (Vitis vinifera plants, Euonymus plants, Triticum aestivum plants, and Ivy plants), and Brassicaceae plants (cabbage, broccoli). In this embodiment, the plant to be controlled from gray mold may be selected from Solanaceae plants, Rosaceae plants, Cucurbitaceae plants, Leguminosae plants, Alliaceae plants, and Vitaceae plants, or may be a Solanaceae plant or a tomato.

[0035] The plant from which the C1 microorganism is derived is also not particularly limited and may be selected from the agricultural products described above, for example, from Solanaceae plants, Rosaceae plants, Cucurbitaceae plants, Legumes, Alliaceae plants, and Vitaceae plants, or may be a Solanaceae plant or a tomato. The plant from which the C1 microorganism is derived may be selected from, for example, tomato, corn, cotton, sunflower, soybean, red clover, wheat, and rice.

[0036] (form) The form of the control agent of this embodiment may be a liquid, solid, gel, paste, etc., and can be appropriately selected depending on the usage situation, etc. When the form is a liquid, the bacteria can be applied to the plant or soil by an operation such as application, spraying (spraying), dripping, or immersion. When the form is a solid, the bacteria may be applied by placing the solid on or in the soil, or by contacting the solid with the surface of the plant. When the form is a gel or paste, the bacteria may be applied by placing the gel or paste on or in the soil, or by applying the gel or paste to the surface of the plant, or by applying the gel or paste to the surface of the seed (for example, coating).

[0037] The amount of C1 microorganisms contained in the control agent of this embodiment can be appropriately set depending on the type of plant to which it is applied, the type of formulation, the application method, etc. The bacterial concentration of the control agent is not particularly limited, but can be, for example, 2 × 10 5 ~1×10 12 CFU / ml, 5 × 10 5 ~5×10 11 CFU / ml, or 2 x 10 6 ~1×10 11 The control agent of this embodiment is effective in smaller amounts than existing microbial formulations. When two or more types of C1 microorganisms are used, the bacterial concentration refers to the total concentration of all C1 microorganisms.

[0038] The control agent of this embodiment may be in the form of a concentrate. In this case, the concentration ratio is not particularly limited and can be, for example, 2 to 1000 times, 5 to 500 times, 1 to 100 times, or 10 to 50 times. When the control agent of this embodiment is a concentrate, it can be appropriately diluted with a solvent such as water, and the diluted solution can be applied to plants. The content of bacteria in the control agent of this embodiment can be set according to the concentration ratio.

[0039] The control agent of this embodiment can be used as a microbial pesticide effective in controlling Botrytis cinerea. In addition to using the C1 microorganism of this embodiment as the microorganism capable of controlling Botrytis cinerea, known materials, components, etc. used in the formulation of known microbial pesticides can also be used. For example, diluents (carriers, bases) and adjuvants commonly used in the formulation of microbial pesticides can be used. Examples of diluents include carriers such as Akadama soil, calcined Akadama soil, Kanuma soil, Ando soil, lime, ceramics, alumina powder, silica gel, zeolite, hydroxyapatite, zirconium phosphate, titanium phosphate, titanium oxide, zinc oxide, hydrotalcite, kaolinite, montmorillonite, talc, clay, diatomaceous earth, bentonite, white carbon, kaolin, vermiculite, perlite, and peat moss. Examples of adjuvants include surfactants, dispersants, anti-decomposition agents, stabilizers, and emulsifiers.

[0040] As described above, the control agent of this embodiment can be used as a microbial pesticide effective in controlling Botrytis cinerea. However, it may also be incorporated into, for example, fertilizers, biostimulants, potting soil, and other pesticides for the purpose of imparting a Botrytis cinerea control effect to plants. Fertilizer components include, for example, nitrogen (N), phosphoric acid (PO), potassium (KO), lime (CaO), magnesia (MgO), manganese (MnO), silicic acid (SiO), boron (BO), and sulfur (S or SO). Potting soil includes the diluents mentioned above, such as Akadama soil, calcined Akadama soil, Kanuma soil, and Andosol soil. When combined with other pesticides, it may be combined with other Botrytis cinerea control agents or other pesticides for disease control.

[0041] (Pest control) The control agent of this embodiment is effective in smaller amounts than existing microbial preparations. Furthermore, since the C1 microorganisms contained in the control agent of this embodiment are C1 microorganisms that live symbiotically with plants, they can be expected to establish on plants. Treatment methods for control will be explained in the section on control methods below.

[0042] Control by the control agent of this embodiment may be characterized by inhibiting the germination and / or growth of Botrytis cinerea by C1 microorganisms, rather than by nutritional competition. Furthermore, control by the control agent of this embodiment may also include suppressing spore formation. Inhibiting the growth of Botrytis cinerea may include stopping mycelial growth of Botrytis cinerea by swelling and rupturing the tips of its hyphal growth. Because of these characteristics, the agent exerts an antifungal effect directly on Botrytis cinerea, the causative agent of gray mold, so that the plants targeted for gray mold control are not particularly limited. Furthermore, because control is not by nutritional competition, the agent is effective even after the onset of disease.

[0043] The severity of disease can be determined by a disease index, and this disease index can be used as an indicator to determine the effectiveness of control. For example, the disease index can be evaluated from 0 to 3, representing the percentage of diseased leaf area (0 = no disease symptoms, 1 = 0.1-24.9%, 2 = 25-49.9%, 3 = 50-100%) (Konishi et al., Bull. Hort. Inst. Ibaraki Agr. Center (2010) 17: 43-46.), and the severity of disease can be calculated by {Σ(disease index × number of leaves per disease index) / (number of leaves examined)}. The percentage of spore-forming leaves can also be used as an indicator to determine the effectiveness of control. For example, the percentage of spore-forming leaves can be calculated by (number of spore-forming leaves) / (total number of leaves) × 100 (%).

[0044] The disease-suppressing effect of the control agent of this embodiment on Botrytis cinerea disease may be determined based on the disease severity and / or the proportion of spore-forming leaves. For example, a preventive effect may be determined when the disease severity is suppressed to 1 or less, or an effect of suppressing the spread of Botrytis cinerea infection and / or Botrytis cinerea disease may be determined when the disease severity is suppressed to 1 or less after Botrytis cinerea infection or Botrytis cinerea disease onset. Alternatively, a disease-suppressing effect may be determined when the disease severity and / or the proportion of spore-forming leaves is significantly reduced compared to identical plants that are not treated with the control agent of this embodiment.

[0045] The disease suppression effect of the control agent of this embodiment on gray mold disease may be obtained when the amount of C1 microorganisms contained in the control agent is 0.5 to 100, i.e., the ratio of Botrytis cinerea:C1 microorganisms is 1:0.5 to 100, assuming that the amount of Botrytis cinerea cultured in a solid medium is 1. The ratio of Botrytis cinerea:C1 microorganisms may be 1:0.5 to 50, 1:0.5 to 20, 1:0.5 to 10, 1:0.5 to 2, or 1:0.5 to 1.

[0046] [Method for controlling gray mold in plants] As one embodiment, the present invention also provides a method for controlling Botrytis cinerea in plants, which comprises a step of treating a plant with the above-described Botrytis cinerea control agent. The Botrytis cinerea control agent is as described above.

[0047] In the step of applying the control agent of this embodiment to a plant, the control agent may be applied to above-ground plant parts such as flowers, stems, leaves, and fruits, and / or underground plant parts such as roots, and preferably includes application to above-ground plant parts. When the control agent is a liquid, application methods include coating, sprinkling (spraying), dripping, or immersion of the plant or soil. When the control agent is a solid, the solid may be applied by mixing it into the soil, or by contacting the solid with the surface of the plant. When the control agent is a gel or paste, the gel or paste may be applied by placing it on or in the soil, or by applying the gel or paste to the surface of the plant, or by applying the gel or paste to the surface of the seed (e.g., coating).

[0048] The amount of C1 microorganisms used in the treatment step can be appropriately determined depending on the type of plant to be treated, the type of formulation, the application method, etc. The concentration of C1 microorganisms during treatment is not particularly limited, but may be, for example, 2 × 10 5 ~1×10 12 CFU / ml, 5 × 10 5 ~5×10 11 CFU / ml, or 2 x 10 6 ~1×10 11 The control agent of this embodiment is effective in smaller amounts than existing microbial formulations. When two or more types of C1 microorganisms are used, the bacterial concentration refers to the total concentration of all bacteria.

[0049] When the control agent to be applied is in a concentrated form, it is diluted appropriately with a solvent such as water to an appropriate bacterial concentration, and the diluted solution is applied to the plant.

[0050] The timing of treatment of plants is not particularly limited, and may be before, after, or both before and after the onset of Botrytis cinerea in plants, and from the viewpoint of high effectiveness, treatment is preferably performed both before and after the onset. The number and frequency of treatments are also not particularly limited, and various conditions may be applied, such as the number of treatments being 1 to 10 times, 1 to 3 times, 4 to 6 times, or 7 to 10 times, and the treatment frequency being every 10 days, every 15 days, every 20 days, every 30 days, or every 45 days.

[0051] [Method for obtaining a plant-derived C1 microbial strain that inhibits the germination and / or growth of Botrytis cinerea] In one embodiment, the present invention also provides a method for obtaining a plant-derived C1 microbial strain that inhibits the germination and / or growth of Botrytis cinerea. The method includes the steps of culturing a single strain of a plant-derived C1 microorganism, contacting the single strain with the cultured Botrytis cinerea, and, if the germination and / or growth of Botrytis cinerea is inhibited, determining that the C1 microorganism is a strain that inhibits the germination and / or growth of Botrytis cinerea. The plant-derived C1 microbial strain that inhibits the germination and / or growth of Botrytis cinerea obtained by this method can be used to control gray mold, as described above.

[0052] The method of this embodiment may include selecting a single strain of plant-derived C1 microorganism before the step of culturing the single strain of plant-derived C1 microorganism. Selection of a single strain of plant-derived C1 microorganism can be carried out, for example, by culturing a microorganism (group) present on the surface of a plant (e.g., leaves and stems) in a solid medium containing a reduced C1 compound (e.g., methanol) as the sole carbon source and collecting an isolated strain. Examples of solid media containing methanol as the sole carbon source include low-nutrient methanol inorganic salts medium plates.

[0053] The step of culturing a single strain of plant-derived C1 microorganism in the method of this embodiment can be carried out by culturing the collected single strain (colony) in a liquid medium. The liquid medium containing the cultured bacteria is called a culture solution. Examples of liquid media that can be used include methanol-inorganic salts liquid medium and NB liquid medium. The culture may be either static culture or shaking culture. The culture temperature is preferably 15°C to 35°C, more preferably 20°C to 30°C. Whether the bacteria have been cultured can be confirmed by whether the culture solution is suspended or not.

[0054] In the method of this embodiment, the step of contacting the cultured Botrytis cinerea with the single strain can be carried out by dripping the culture solution onto a solid medium on which Botrytis cinerea has been cultured. Alternatively, an agar piece on which Botrytis cinerea has been cultured and the culture solution can be dripped onto the solid medium, and as each is cultured, Botrytis cinerea and the single strain can eventually be brought into contact. Conversely, this can also be carried out by applying or dripping Botrytis cinerea or a culture solution in which Botrytis cinerea has been cultured onto a solid medium on the surface of which the culture solution has been applied.

[0055] In the method of this embodiment, when the germination and / or growth of Botrytis cinerea is inhibited, the step of determining the C1 microorganism as a strain inhibiting the germination and / or growth of Botrytis cinerea (selection step) includes culturing Botrytis cinerea on the solid medium and observing the germination and / or growth of Botrytis cinerea in contact with a single strain of plant-derived C1 microorganism. When the germination and / or growth of Botrytis cinerea is inhibited, a clear zone called an inhibition zone is formed around the single strain of plant-derived C1 microorganism. When the inhibition zone and / or inhibition circle are circular or approximately circular, they are also referred to as an inhibition circle. When this inhibition zone and / or inhibition circle are observed, the C1 microorganism can be determined (selected) as a strain inhibiting the germination and / or growth of Botrytis cinerea.

[0056] The method of this embodiment may include culturing the single strain of the C1 microorganism that has been determined (selected) after the above-mentioned determination (selection) step. The method for culturing the single strain of the C1 microorganism is as described above.

[0057] The method of this embodiment may also include recovering a single strain (selected strain) of the cultured C1 microorganism, which can be recovered, for example, by separating the supernatant (medium) from the precipitate (bacteria) by centrifugation and removing the supernatant.

[0058] As one embodiment, the present invention also provides a method for producing a gray mold control agent using a plant-derived C1 microbial strain that inhibits the germination and / or growth of Botrytis cinerea, obtained by the method described above.

[0059] The production method of this embodiment may include formulating a culture medium obtained by culturing the C1 microorganism strain by the method described above, or may include formulating a C1 microorganism recovered from the culture medium.

[0060] For formulation, known materials, components, etc. used in the formulation of microorganisms can be used. For example, diluents (carriers, bases) and adjuvants commonly used in the formulation of microbial pesticides can be used. Examples of diluents include carriers such as Akadama soil, calcined Akadama soil, Kanuma soil, Ando soil, lime ash, lime, ceramics, alumina powder, silica gel, zeolite, hydroxyapatite, zirconium phosphate, titanium phosphate, titanium oxide, zinc oxide, hydrotalcite, kaolinite, montmorillonite, talc, clay, diatomaceous earth, bentonite, white carbon, kaolin, vermiculite, perlite, and peat moss. Examples of adjuvants include surfactants, dispersants, antidecomposition agents, stabilizers, emulsifiers, etc.

[0061] As described above, the control agent of this embodiment can be used as a microbial pesticide effective in controlling Botrytis cinerea. However, it may also be incorporated into, for example, fertilizers, biostimulants, potting soil, and other pesticides to impart Botrytis cinerea control effects to plants. Therefore, as one embodiment, the present invention also provides a method for producing a fertilizer, biostimulant, potting soil, or pesticide effective in controlling Botrytis cinerea by adding the control agent described above to a fertilizer, biostimulant, potting soil, or other pesticide. Fertilizer components include, for example, nitrogen (N), phosphoric acid (PO), potassium (KO), lime (CaO), magnesia (MgO), manganese (MnO), silicic acid (SiO), boron (BO), sulfur (S or SO), and the like. Potting soil examples include, as diluents, Akadama soil, calcined Akadama soil, Kanuma soil, and Andosol soil, as described above. When used in combination with other pesticides, it may be combined with other pesticides for controlling gray mold, or may be combined with other pesticides for controlling other diseases. [Example]

[0062] Materials and Methods (biological material) The test used Botrytis cinerea (No. 241680, National Institute of Agrobiological Sciences Genebank, Ministry of Agriculture, Forestry and Fisheries), known as the causative agent of gray mold disease in tomatoes. Tomato leaves and stems (Reiki; Sakata Seed, Kanagawa Prefecture) used for microbial collection were grown in a greenhouse on a farm in Kumamoto Prefecture. The grafted tomato seedlings used were the "Momotaro Haruka" variety.

[0063] (Buffer and medium composition) Phosphate-buffered saline (PBS) was prepared by dissolving 8.0 g of NaCl, 0.2 g of KCl, 1.15 g of Na2HPO4, and 0.2 g of KH2PO4 in 1.0 L of distilled water, and was used to recover microorganisms from tomato leaf surfaces.

[0064] The composition of the methanolic inorganic salts liquid medium is as follows: 2.0 g KH2PO4, 2.4 g K2HPO4, 1.0 g (NH4)2SO4, 1.0 g KNO3, 1.0 g MgSO4, 1.0 g trace element mixture, 5 mL methanol, and 1.0 L distilled water. KH2PO4, K2HPO4, KNO3, (NH4)2SO4, MgSO4, and the trace element mixture were dissolved in 1.0 L distilled water to give a concentration of 1.05 kg / cm 2 The medium was autoclaved at 4°C for 20 minutes. Methanol was added to the medium and dispensed into test tubes. Methanol inorganic salts solid medium was prepared by adding 10.0 g of gellan gum and 0.4 μg of tunicamycin to methanol inorganic salts liquid medium. All ingredients except methanol and tunicamycin were dissolved in distilled water and sterilized by autoclaving. The medium to which methanol and tunicamycin had been added was dispensed into Petri dishes (90 mm diameter) to prepare methanol inorganic salts liquid medium plates.

[0065] The NB liquid medium consisted of Nutrient Broth (3.0 g of beef extract, 5.0 g of peptone) and 1.0 L of distilled water. NB was dissolved in 1.0 L of distilled water to give a concentration of 1.05 kg / cm. 2 The plates were sterilized in an autoclave at RT for 20 minutes. The NB liquid medium was dispensed into test tubes.

[0066] Potato dextrose agar (PDA) was prepared as follows: 4.0 g of potato infusion powder (Sigma, 52424), 20.0 g of glucose, 20.0 g of agar, and 1.0 L of distilled water. Glucose, agar, and potato dextrose were dissolved in 1.0 L of distilled water to give a concentration of 1.05 kg / cm. 2 The mixture was sterilized in an autoclave at RT for 20 minutes. The PDA medium was dispensed into petri dishes (diameter 90 mm).

[0067] (sampling) Approximately 3 kg of tomato leaves and stems were randomly collected from a tomato greenhouse on July 1, 2020. All samples were placed in a polyethylene bag. Within 3 hours of collection, approximately 1 kg of tomato leaves was divided into three polyethylene bags. 50 mL of PBS was added to each polyethylene bag using a thermos syringe (10 mL) and a sterile syringe filter CA (0.22 μm). The polyethylene bags containing the leaves, stems, and washing buffer were shaken vigorously by hand and left at room temperature for 1 hour. The suspension was then collected in 50 mL tubes.

[0068] (Cultivation and isolation of methanol-utilizing bacteria) Using the suspension sample, 1 / 5 dilutions (5x dilutions), 1 / 25, 1 / 125, 1 / 625, 1 / 3125, and 1 / 15625 dilutions were prepared. Seven different dilutions of the suspension sample were coated onto methanol-based mineral salts medium plates using a sterile Drigalski Spatel stick, with 50 μl of each. These plates were incubated at 30°C and visually inspected daily for colony formation. After 6–14 days, colonies of various sizes and colors (e.g., red, yellow, and white) were obtained. Once colony formation was confirmed, isolates were selected based on morphological characteristics, and each colony was picked by prodding with a platinum loop. Selection of similar colonies that appeared simultaneously on the plate was avoided. Colonies picked with the platinum loop were then cultured in methanol-based mineral salts liquid medium and NB liquid medium. Culture was performed in two ways: static culture (30°C) and shaking culture (150 RPM, 30°C). Once the culture medium was confirmed to be suspended, it was stored at -60°C together with glycerol.

[0069] The composition of the methanol inorganic salt liquid medium was 2.0 g of KH2PO4, 2.4 g of K2HPO4, 1.0 g of (NH4)2SO4, 1.0 g of KNO3, 0.2 g of MgSO4, 1.0 g of a trace element mixture, 5 mL of methanol, and 1.0 L of distilled water. KH2PO4, K2HPO4, (NH4)2SO4, KNO3, MgSO4, and the trace element mixture were dissolved in 1.0 L of distilled water to obtain a solution of 1.05 kg / cm 2The medium was sterilized in an autoclave at 47°C for 20 minutes, and methanol was added to the medium, which was then dispensed into test tubes. Methanol-based inorganic salt solid medium was prepared by adding 10.0 g of gellan gum and 0.4 μg of tunicamycin to methanol-based inorganic salt liquid medium.

[0070] (Screening of inhibitory strains) Botrytis cinerea suspension (4.2 × 10 5 spores / ml) was applied to a PDA plate (diameter 90 mm) and cultured in an incubator at 25°C. The time it took for the mycelia of Botrytis cinerea to cover the plate was measured.

[0071] Two to eight holes (9 mm in diameter) were drilled around the center of the plate on which the Botrytis cinerea suspension had been applied (Figure 1(A)). The holes were equidistant from each other and positioned 14 mm from the edge of the plate. The strain suspension (2 × 10 6 50 μl of 1000 μL ...

[0072] Growth inhibitors of Botrytis cinerea were examined using a dual culture assay. Grown Botrytis cinerea were excised from a PDA plate and inoculated onto another PDA plate. At the same time, 1.0 μl of the inhibitory strain suspension was dropped onto the edge of the plate and allowed to stand at 25°C. The inhibitory effect of the inhibitory strain suspension on Botrytis cinerea in the culture supernatant was also examined. The culture supernatant for the inhibitory strain suspension was prepared by filtering the inhibitory strain cells after cultivation. Botrytis cinerea was spread onto a PDA plate, four wells were drilled, and a control (water), an inhibitory strain, the inhibitory strain suspension supernatant, or a 10x concentrated solution of the inhibitory strain suspension supernatant were placed into each well of the PDA plate and incubated at 25°C.

[0073] (Identification of inhibitory strains) The V3-V4 region of the 16S rRNA gene from each inhibitor strain was approximately 400-450 bp and was amplified using primer pair 341F (5'-CCTACGGGNGGCWGCAG-3' (SEQ ID NO: 10)) and 805R (5'-GACTACHVGGTATCTAATCC-3' (SEQ ID NO: 11)) [Klindworth et al., Nucleic Acids Res. (2013) 7:41, e1. doi:10.1093 / na r / gks808.]. The gyrB gene region from each inhibitor strain was approximately 1100 bp and was amplified with the primer pair gyrF (5'-GAAGTCATCATGACCGTTCTGCATCGCTCAGGGTCAGGGTCAGAAAGTTTCGA-3' (SEQ ID NO: 12)) and gyrR (5'-AGCAGGTACGATGTGCGA GCCAGTCTCAGACAGTCTCAGGCAGTCTCAGGTAT-3' (SEQ ID NO: 13)). For each inhibitory strain, the PCR mixture (50 μL) used for amplification consisted of 1.0 μL metagenomic DNA, 5.0 μL 10x PCR Buffer for KOD-Plus-Neo, 5.0 μL 2 mM dNTPs, 3.0 μL 25 mM MgSO, 1.0 μL KOD-Plus-Neo, 1.5 μL 10 μM forward primer, 1.5 μL 10 μM reverse primer, and 33 μL PCR-grade water (Promega) in a microcentrifuge tube. PCR of 16S rRNA was performed with the following thermocycling parameters: initial denaturation at 94°C for 2 min, followed by 25 cycles of 98°C for 10 s, 50°C for 30 s, and 68°C for 20 s. PCR of the gyrB gene was performed: initial denaturation at 94°C for 2 minutes, followed by 25 cycles of 98°C for 10 seconds, 50°C for 30 seconds, and 68°C for 35 seconds. PCR products were visualized under UV light after standard ethidium bromide gel electrophoresis. Amplification products were purified using the PCR Clean-Up System (Promega, USA). Seven PCR products (SY50, SY89, SY131, SY135, SY163, SY183, and SY339) were purified using the PCR Clean-Up System (Promega).50 μL of Membrane Binding Solution was added to each tube containing the PCR reaction mixture and mixed. Each sample was transferred to a column tube and centrifuged (25°C, 12,000 rpm, 1 minute). The waste liquid was discarded, and the tube was centrifuged with 700 μL of Membrane Wash Solution (Promega, USA) (25°C, 12,000 rpm, 1 minute). The waste liquid was discarded, and 500 μL of Membrane Wash Solution (Promega, USA) was added, followed by centrifugation (25°C, 12,000 rpm, 1 minute). The column was placed in the tube, and the product was eluted from the column by centrifugation (25°C, 12,000 rpm, 1 minute) with 50 μL of Nuclease-Free Water (Promega, USA). The purified PCR product was stored at -20°C. PCR products of the V3-V4 region of the 16S rRNA gene were similarly obtained for the inhibitory strain of the genus Pantoea (SY685) and the inhibitory strain of the genus Cartobacterium (SY851).

[0074] The PCR products were subjected to agarose gel electrophoresis and cloned into TOPO-Blunt (Invitrogen, Carlsbad, CA). The PCR products were cloned into pCR Blunt II plasmid using the Zero Blunt TOPO PCR Cloning Kit according to the manufacturer's protocol. The amplified PCR fragments were subjected to DNA sequencing. Homology searches were performed using the determined DNA sequences as queries in NCBI blast [Johnson et al., Nucleic Acids Research, (2008) 36, 2, 1, W5-W9; https: / / blast.ncbi.nlm.nih.gov / Blast.cgi], and molecular phylogenetic analysis was performed using the 16S rRNA gene and gyrB gene. Alignment and phylogenetic tree inference were performed using MAFFT 6.861 and RAxML 8.2.11 in ETE v3.1.2 (Huerta-Cepas et al., Mol. Biol. Evol. (2016) 33, 1635-1638), and the results were plotted using iTOL (Letunic and Bork et al., Nucleic Acids Res (2021) 49:W293-W296). A similar homology search was performed on the 16S rRNA gene of a Pantoea spp. inhibitor strain (SY685) and a Cartobacterium spp. inhibitor strain (SY851). The 16S rRNA gene sequences of each inhibitor strain are shown in Table 1.

[0075] [Table 1] TIFF2025167723000002.tif204149

[0076] (Botrytis blight control assay in plants) The strains that showed the highest inhibitory effect on the pathogen in the petri dish test were then tested in a greenhouse to determine their inhibitory effect on Botrytis cinerea disease. Pots of tomatoes were watered with 20 ml of water per pot in the morning and evening, and the plants were grown in a greenhouse placed near a window in the laboratory.

[0077] Forty-five grafted tomato seedlings (2 months old) were prepared (control group + 4 treatment conditions (A-D) x 2 plants = 9 groups, 9 groups x 5 plants). In the morning, 20 ml of water per pot was sprayed onto the soil surface using a Falcon tube. SY163 and SY183 were used as inhibitor strains. The experimental groups were as follows: Control group: normal growth (no application) Condition A: Only Botrytis cinerea was applied on the first day Condition B: Botrytis cinerea and each inhibitor were applied on the first day. Condition C: Each inhibitor was applied on day 0, and Botrytis cinerea and each inhibitor were applied on day 1. Condition D: Botrytis cinerea and each inhibitor were applied on the first day, and each inhibitor was applied on the second day. A spore suspension of Botrytis cinerea (4.2 × 10 5 spores / ml) was sprayed onto each leaf using a pipette at a rate of 10 μl per leaf. 6 CFU / ml) was sprayed at 10 μl per leaf using a pipette.

[0078] The control and treated plants were grown at room temperature (18–22°C) for 22 days after treatment. The Botrytis cinerea disease index on each tomato leaf was recorded daily, and the disease severity for each plant was calculated. The disease index was calculated based on the percentage of diseased leaf area (0 = no disease symptoms, 1 = 0.1–24.9%, 2 = 25–49.9%, 3 = 50–100%), and scored on a scale of 0–3 (Konishi et al., Bull. Hort. Inst. Ibaraki Agr. Center. (2010) 17: 43–46). The disease severity was calculated as {Σ(disease index × number of leaves per disease index) / (number of leaves surveyed)}. Spore formation on all leaves after 22 days was also evaluated. The percentage of spore-bearing leaves was calculated as (number of spore-bearing leaves) / (total number of leaves) × 100 (%).

[0079] Example 1: Isolation of a Methanol-Assimilating Bacterial Strain Tomato leaf samples were collected from three locations in a greenhouse. The leaf surfaces were washed with PBS to obtain a gray suspension. Methanol-utilizing bacteria were collected from the gray suspension washed from the leaf surfaces using the method described above. The gray suspension was then spread onto low-nutrient methanol-mineral salts medium plates and cultured. After 6 to 14 days, colonies of various colors (e.g., red, yellow, white) and sizes were obtained. Colonies were collected from the methanol-mineral salts medium plates and cultured in methanol-mineral salts liquid medium and NB liquid medium. Isolate culture samples were obtained after 1 to 14 days of static culture and 1 to 13 days of shaking culture. 1,000 isolates were obtained and frozen in glycerol.

[0080] Example 2 Screening and identification of inhibitory strains Spores of Botrytis cinerea were cultured on PDA plates at 25°C, and on the 10th day, the mycelium was visually confirmed to have covered the plate. Previously, spores of Botrytis cinerea were spread on PDA plates and cultured at 22°C for 7 days (Vidal et al., Botrytis cinerea J. Fungi (2020) 6, 149), and the mycelium was similar to that reported. The mycelium of Botrytis cinerea is characterized by gray, branched, and arborescent hyphae.

[0081] Growth inhibitory strains were selected based on their ability to suppress the spread of Botrytis cinerea mycelia on PDA plates. The antifungal activity of 405 isolated strains was evaluated using the agar well diffusion method on PDA plates coated with a Botrytis cinerea suspension (Figure 1(A)). A well was drilled in a PDA plate coated with Botrytis cinerea spores, and a suspension of each strain was placed in the well and incubated at 25°C for 10 days. If a strain exhibited growth inhibitory effects against Botrytis cinerea, a distinct zone called an inhibition zone was formed around the well. Many plates were completely covered with Botrytis cinerea mycelia. Of all 405 strains tested, nine strains (SY50, SY89, SY131, SY135, SY163, SY183, SY339, SY685, and SY851) exhibited a zone of inhibition against Botrytis cinerea (Figures 1 and 2). Among the strains for which an inhibition zone was confirmed, the SY163 strain had the largest zone of 50 mm, followed by SY183 at 40 mm, SY135 at 37 mm, SY131 and SY339 at 34 mm, SY89 at 32 mm, SY851 at 27 mm, and SY50 and SY685 at 25 mm.

[0082] The inhibitory effects of nine strains on growing Botrytis cinerea mycelia were examined using a dual culture assay, following the procedure in Figure 1(B). Although the size of the zone of inhibition varied for each strain, zones of inhibition were observed for all strains (Figures 1 and 3). Zones of inhibition were observed for seven strains, with inhibition rates ranging from 11.9 to 35.7%. A similar trend was observed with Metarhizium anisopliae, which inhibited the radial growth of Botrytis cinerea by approximately 43.9% compared to the control (Sarven et al., Pathogens (2020) 9, 213). Note that Metarhizium anisopliae is a filamentous fungus, not a methanol-utilizing bacterium. The inhibition zones of each strain were formed without contact between Botrytis cinerea and the inhibitory strains, suggesting that the inhibitory strains secrete some substance (e.g., antibiotics, enzymes, or volatile substances) that inhibits the growth of Botrytis cinerea.

[0083] The results of a homology search of the 16S rRNA gene sequences of the seven strains that demonstrated antibacterial activity against Botrytis cinerea are shown in a phylogenetic tree (Figure 4). Six of the seven strains, except for SY163, were closely related to Serratia rubidaea JCM1240 and Serratia rubidaea NBRC103169, but were not identical. They were significantly different from strains such as Serratia marcescens and Serratia ureilytica. Although SY163 shared a 16S rRNA gene sequence with Serratia rubidaea JCM1240, it is considered a novel strain for reasons described below. Based on these results, all seven strains were closely related to Serratia rubidaea. A homology search of the 16S rRNA gene sequences was also performed on the other two strains (Pantoea (SY685 strain) and Curtobacterium (SY851 strain)). The results indicated that Patoea belongs to the genus Pantoea, and that the closest 16S rRNA gene sequence is that of Pantoea ananatis strain PA13, but differs by two bases. The 16S rRNA gene sequence of Curtobacterium matches that of Curtobacterium luteum strain JCM 1480, indicating that it belongs to the genus Curtobacterium.

[0084] A homology search using the gyrB gene was performed on seven strains that showed antifungal activity against Botrytis cinerea: strains SY50, SY89, SY131, SY135, SY163, SY183, and SY339. The phylogenetic tree is shown (Fig. 5). The differences between strains SY50, SY131, SY135, SY183, and SY339 were within one base pair. These five strains differed from SY89 by 7 bp in the 1171 bp region, and SY163 had a 4-bp substitution in the 1171 bp region. Furthermore, there was a 5-bp difference between SY89 and SY163 in the 1171 bp region. Based on these results, the inhibitory strains obtained were classified into three groups: (1) SY163 strain, (2) SY89 strain, and (3) SY50 strain, SY131 strain, SY135 strain, SY183 strain, and SY339 strain. The sequences of the gyrB gene of each inhibitory strain are shown in Table 2.

[0085] [Table 2] TIFF2025167723000004.tif200149 TIFF2025167723000005.tif200149 TIFF2025167723000006.tif101149

[0086] According to the "Explanation of Guidelines for the Use of Microbial Bioremediation" (July 2005, partially revised in March 2012, Bioscience Industry Division, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, and Environmental Management Technology Office, General Affairs Division, Water and Air Environment Bureau, Ministry of the Environment), when it comes to identifying microorganisms to be used, in the case of microorganisms newly isolated from nature and without an officially recognized scientific name, "bacteria are identified by analysis of the 16S ribosomal RNA gene (determination of the base sequence). In this case, a species is considered to be of the same species if the 16S ribosomal RNA gene has 98.5% or more homology. If there are multiple independent species among those with 98.5% or more homology, they can be identified by whole genome gene analysis (Appendix 4), analysis of the gyrase B gene (gyrB), or methods centered on phenotype." While the sequences of the 16S rRNA and gyrB genes of known strains have been determined, there are also cases for which only one of these has been identified. The 16S rRNA gene is used to classify prokaryotes, allowing for provisional identification. If the identification is 99% or higher, there is a high probability that the species is the same, but discussion at the species level is considered difficult. On the other hand, the gyrase gene encodes a functional protein that is more widely present in organisms than the 16S rRNA. Using both 16S rRNA and gyrB will likely clarify phylogenetic relationships and allow for more accurate discussion of differences with known strains.

[0087] A strain for which there is no known strain with matching 16S rRNA gene and gyrB gene can be said to be a novel strain solely on that basis.

[0088] Furthermore, we have also conducted whole genome analysis (ANI analysis) of the SY163 and SY183 strains, and have confirmed that no species are 100% identical.

[0089] Example 3: Biological control activity of bacterial isolates in plants Grafted tomato seedlings were grown in a laboratory greenhouse with regular watering. Under conditions of room temperature between 18 and 22°C, and only water was provided for 22 days, the tomato plants were able to grow without developing diseases such as gray mold on their leaves. This growth method was confirmed to be suitable for evaluating biological control activity.

[0090] The inhibitory effect of inhibitors on gray mold disease on tomato foliage was examined. The inhibitors used were strains SY163 and SY183, which share significant differences in the gyrB gene sequence. The disease index was scored on a 4-point scale (0–3) (Figure 7(A)). Under condition A, tomato leaves were sprayed with Botrytis cinerea on day 1, and disease development was observed from day 5. Figure 6 shows the disease severity up to day 22. Disease severity gradually increased, reaching a score of 2 or higher by day 22. Under condition B, Botrytis cinerea and either strain SY163 (B-SY163) or strain SY183 (B-SY183) were sprayed on day 1. Compared to condition A, B-SY163 showed less disease suppression, while B-SY183 showed some disease suppression. In condition D, Botrytis cinerea and strain SY163 (D-SY163) or strain SY183 (D-SY183) were sprayed on day 1, and strain SY163 or strain SY183 was sprayed on day 2. D-SY163 and D-183 showed a tendency to suppress disease compared to conditions A and B, respectively. In condition C, two suppressive strains, strain SY163 or strain SY183, were applied the day before, and Botrytis cinerea and two suppressive strains, strain SY163 (C-SY163) or strain SY183 (C-SY183), were applied on day 1 and grown. In condition C, disease suppression was observed for all strains compared to conditions A, B, and D. Spraying the disease inhibitor strain suppressed the onset of gray mold under all conditions, but C-SY183 was particularly effective, with the disease severity reaching 1 or less by the 22nd day, the final day of observation. This suggests that spraying the S183 strain before gray mold onset may be effective in preventing the onset of gray mold. A significant difference was found in the state of sporulation on the leaves. Sporulation causes the disease to become more severe, but Serratia fungi are thought to prevent this sporulation.

[0091] In addition to the difference in disease severity scores, the percentage of leaves on which spores were observed was also evaluated. As the severity of Botrytis cinerea disease increased, spores formed, as indicated by a score of 3 in Figure 7(A). The percentage of leaves on which spores formed is shown in Figure 7(B). Compared to condition A, in which only Botrytis cinerea was sprayed, spore formation was significantly suppressed under all conditions sprayed with SY163 or SY183. These two strains of Selezia bacteria were found to be effective in suppressing Botrytis cinerea spore formation. Because spore formation increases the risk of infection spreading due to spore dispersal (Barnes et al., Phytopathology. (2003) May;93(5):573-8), these strains are expected to significantly contribute to suppressing the spread of gray mold infection.

[0092] This is the first time that a novel strain of Serratia rubidae has been evaluated for the biological control of gray mold caused by the genus Serratia on tomato plants at various stages of development, and it is expected that this new strain will be useful as an agricultural material in the future. Similar to strain SY163 or SY183, the five novel strains of Serratia rubidae (strains SY50, SY89, SY131, SY135, and SY339), Pantoea bacteria, and Cartobacterium bacteria identified in Example 2, which showed growth inhibitory effects against Botrytis cinerea, are also expected to have similar effects.

Claims

1. A plant gray mold control agent comprising a plant-derived C1 microorganism.

2. The agent according to claim 1, wherein the C1 microorganism is selected from the group consisting of bacteria of the species Serratia rubidae, bacteria of the genus Pantoea, bacteria of the genus Cartobacterium, and combinations thereof.

3. The agent according to claim 1, which controls gray mold by inhibiting the germination and / or growth of Botrytis cinerea by the C1 microorganism.

4. The C1 microorganism is a bacterium of the species Serratia rubidaea having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, a bacterium of the species Serratia rubidaea having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 2, a bacterium of the species Serratia rubidaea having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 3, a bacterium of the species Serratia rubidaea having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4, a bacterium of the species Serratia rubidaea having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5, a bacterium of the species Serratia rubidaea having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 6, a bacterium of the species Serratia rubidaea having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 7, The agent according to any one of claims 1 to 3, wherein the bacterium is selected from the group consisting of Serratia rubidae bacteria having an rRNA gene, Pantoea bacteria having a 16S rRNA gene consisting of a base sequence having 99% or more sequence identity to the base sequence shown in SEQ ID NO: 8, Curtobacterium bacteria having a 16S rRNA gene consisting of a base sequence having 99% or more sequence identity to the base sequence shown in SEQ ID NO: 9, and combinations thereof.

5. the C1 microorganism is a bacterium belonging to the species Serratia rubidaea selected from the group consisting of SY163 strain (NITE-AP-04103), SY183 strain (NITE-AP-04104), and mutant strains thereof; SY685 strain (NITE-AP-04105), or a mutant strain thereof; The agent according to claim 4, wherein the agent is selected from the group consisting of Curtobacterium bacteria selected from the group consisting of SY831 strain (NITE-AP-04106) or mutant strains thereof, and combinations thereof.

6. A method for controlling Botrytis cinerea in plants, comprising a step of treating plants with the Botrytis cinerea control agent according to claim 1.

7. The C1 microorganism is a bacterium of the species Serratia rubidaea having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, a bacterium of the species Serratia rubidaea having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 2, a bacterium of the species Serratia rubidaea having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 3, a bacterium of the species Serratia rubidaea having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4, a bacterium of the species Serratia rubidaea having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5, a bacterium of the species Serratia rubidaea having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 6, a bacterium of the species Serratia rubidaea having a 16S rRNA gene consisting of a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 7, The method of claim 6, wherein the bacterium is selected from the group consisting of Serratia rubidae bacteria having an rRNA gene, Pantoea bacteria having a 16S rRNA gene consisting of a base sequence having 99% or more sequence identity to the base sequence shown in SEQ ID NO: 8, Curtobacterium bacteria having a 16S rRNA gene consisting of a base sequence having 99% or more sequence identity to the base sequence shown in SEQ ID NO: 9, and combinations thereof.

8. the C1 microorganism is a bacterium belonging to the species Serratia rubidaea selected from the group consisting of SY163 strain (NITE-AP-04103), SY183 strain (NITE-AP-04104), and mutant strains thereof; a Pantoea bacterium selected from the SY685 strain (NITE-AP-04105) or a mutant thereof; The method according to claim 6, wherein the bacterium is selected from the group consisting of Curtobacterium genus bacteria selected from the group consisting of SY831 strain (NITE-AP-04106) or a mutant strain thereof, and combinations thereof.

9. The method according to any one of claims 6 to 8, wherein the treatment of the plant includes treatment of the above-ground parts of the plant.

10. In the step of applying the agent to a plant, the concentration of the C1 microorganism is 2 x 10 based on the total amount of the agent for controlling gray mold. ５ ~1 x 10 １２ The method according to any one of claims 6 to 8, characterized in that the treatment is carried out with a gray mold control agent at a concentration of CFU / ml.

11. The method according to any one of claims 6 to 8, wherein the treatment of the plant is carried out before, after, or both before and after the onset of Botrytis cinerea on the plant.

12. Cultivating a single strain of plant-derived C1 microorganism; contacting the single strain with cultured Botrytis cinerea; and a step of determining the C1 microorganism as a strain inhibiting the germination and / or growth of Botrytis cinerea when the germination and / or growth of Botrytis cinerea is inhibited. Including, A method for obtaining a plant-derived C1 microbial strain that inhibits the germination and / or growth of Botrytis cinerea.

13. A method for producing a control agent for Botrytis cinerea, using a plant-derived C1 microbial strain that inhibits the germination and / or growth of Botrytis cinerea, obtained by the method according to claim 12.

14. SY163 strain (NITE-AP-04103), SY183 strain (NITE-AP-04104), SY685 strain (NITE-AP-04105), or SY831 strain (NITE-AP-04106).

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