Use of phytotoxic substances for the control of plant diseases

Abstract

The present application relates to the field of biopharmaceuticals, and particularly relates to the application of crotonic acid in the prevention and treatment of plant diseases. The present application provides a secondary metabolite of methylotrophic Bacillus, i.e. crotonic acid, which has antibacterial activity, and an application method thereof in the broad-spectrum inhibition of plant pathogenic fungi such as pepper fusarium, pepper blight fungus, banana anthracnose fungus and tomato botrytis blight fungus. The inhibition rate of the substance on the mycelial growth of plant pathogenic fungi such as pepper fusarium, pepper blight fungus, banana anthracnose fungus and tomato botrytis blight fungus can reach 85.24% to 100% at a concentration of 2 ppm, and the control effect on pepper fusarium, pepper blight, banana anthracnose, tomato botrytis blight on detached leaves and fruits is 71.09% to 86.10% at a concentration of 20 ppm, which lays a foundation for further development of the crotonic acid for use in fumigation and antibacterial agents for melon and vegetable transportation and storage.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and more particularly to the application of crotonic acid in the prevention and control of plant diseases. Background Technology

[0002] Crotonic acid, also known as trans-2-butenoic acid, has the following chemical formula: Figure 1 As shown, its chemical formula is C4H6O2, its relative molecular weight is 86.09, and its CAS number is 107-93-7. It is mainly used to prepare various resins, surface coatings, and plasticizers. It can also be used as an important pharmaceutical intermediate, pesticide intermediate, and other organic chemical intermediates. Its most important use is as a raw material for polyvinyl acetate coatings. There are no reports or applications of its ability to inhibit the activity of plant pathogenic fungi and prevent and control plant diseases. Summary of the Invention

[0003] In view of this, the present invention provides the application of crotonic acid in the prevention and control of plant diseases. The present invention provides crotonic acid, a secondary metabolite of methyltrophic Bacillus, with antibacterial activity, and its application method in broadly inhibiting plant pathogens such as *Fusarium wilt*, *Pyrophyllaria rubrifolia*, *Anthracnose*, and *Botrytis cinerea*. At a concentration of 2 ppm, this substance can inhibit the mycelial growth of these plant pathogens by 85.24%–100%. At a concentration of 20 ppm, it shows a control efficacy of 71.09%–86.10% against *Fusarium wilt*, *Pyrophyllaria rubrifolia*, *Anthracnose*, and *Botrytis cinerea* on detached leaves and fruits. This lays the foundation for further development of crotonic acid as a fumigation antibacterial agent for the transportation and storage of melons and vegetables.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the use of crotonic acid in any of the following: (a) Prevention and control of plant diseases; and / or (b) Preparing products for the prevention and control of plant diseases; and / or (c) Inhibition of plant pathogens; and / or (d) Prepare products that inhibit plant pathogens.

[0005] In some embodiments of the present invention, in the above applications, the structural formula of the crotonic acid is as follows: Figure 1 As shown.

[0006] In some embodiments of the present invention, in the above applications, the plant pathogens include one or more of the following: pepper wilt, pepper blight, banana anthracnose, and tomato gray mold.

[0007] In some embodiments of the present invention, in the above applications, the crotonic acid is derived from a metabolite of methyltrophic Bacillus.

[0008] In some embodiments of the present invention, the above-mentioned application uses the methyltrophic Bacillus accession number CGMCC NO. 14478.

[0009] In some embodiments of the present invention, the application concentration of the crotonic acid in the above-described applications is 0.5 ppm to 2 ppm.

[0010] In some embodiments of the present invention, the above-described applications include: pesticides and / or antibacterial agents.

[0011] In some embodiments of the present invention, in the above applications, the antibacterial agent in the product includes: a fumigation antibacterial agent.

[0012] In some embodiments of the present invention, in the above applications, the crotonic acid acts on the plant pathogen in a volatile form.

[0013] The present invention also provides pesticides and / or antibacterial agents, including crotonic acid and acceptable excipients.

[0014] In some embodiments of the present invention, the crotonic acid in the above-mentioned pesticides and / or antibacterial agents is derived from the metabolites of Bacillus methyltrophicus with accession number CGMCC NO. 14478.

[0015] The present invention also provides a method for preventing and controlling plant diseases, which involves applying crotonic acid and / or the above-mentioned pesticides and / or antibacterial agents to plants or plant pathogens infected with plant pathogens.

[0016] In some embodiments of the present invention, the crotonic acid in the above method is derived from the metabolite of Bacillus methyltrophicus with accession number CGMCCNO. 14478.

[0017] In some embodiments of the present invention, the plant pathogens in the above method include one or more of the following: pepper wilt, pepper blight, banana anthracnose, and tomato gray mold.

[0018] In some embodiments of the present invention, the application concentration of crotonic acid in the above method is 0.5 ppm to 2 ppm.

[0019] The beneficial effects of this invention include: (1) This invention proposes the application of crotonic acid, a methyl-nutritive Bacillus metabolite, in inhibiting plant pathogens and controlling diseases on plant leaves and fruits. Experimental studies have shown that crotonic acid has significant inhibitory effects on various plant pathogens, including *Fusarium solani*, *Phytophthora capsici*, *Colletotrichummusae*, and *Botrytis cinerea*, providing a natural source of bactericidal active substance for plant disease control. Crotonic acid, isolated and identified from methyl-nutritive Bacillus VS15, exhibits volatile antibacterial activity when dissolved in ethanol. Simply placing a small amount of the solution near the plant or pathogens, without the need for spraying or soaking, can inhibit the growth of plant pathogens and control diseases on plant leaves and fruits. Crotonic acid is characterized by high efficiency, low toxicity, and safety, meeting the current requirements for the creation of new pesticides and possessing broad application prospects.

[0020] (2) To evaluate the agricultural fungicidal activity of crotonic acid, a volatile product of methyl-nutritive Bacillus, this invention uses the growth rate method to determine the inhibitory effect of crotonic acid on the mycelial growth of four plant pathogens: *Fusarium wilt*, *Pyrophyllaria dichotoma*, *Anthracnose*, and *Botrytis cinerea*. The in vitro plant tissue method is used to evaluate the control effect of this compound on these four plant diseases. Experiments have shown that at a test concentration of 2 ppm, crotonic acid exhibits an inhibition rate of 85.24%–100% against the four plant pathogens, which is comparable to or better than the inhibitory effects of carbendazim and pyraclostrobin at a 10-fold concentration (20 ppm), indicating that this substance has good inhibitory activity against plant pathogens. Furthermore, through in vitro plant tissue experiments, at a test concentration of 20 ppm, crotonic acid showed a control efficacy of 71.09%–86.10% against four plant diseases: pepper wilt, pepper blight, banana anthracnose, and tomato gray mold. Compared with the reference agents carbendazim (12.65%–86.89%) and azoxystrobin (14.50%–68.48%) at a 10-fold concentration (200 ppm), the efficacy was comparable to or significantly better.

[0021] Biological Preservation Instructions

[0022] Biological material: VS15; Classification and nomenclature: Bacillus methylotrophicus; Deposited on July 31, 2017 at the China General Microbiological Culture Collection Center (CGMCC); Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; Accession number: CGMCC No. 14478. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0024] Figure 1 The chemical structure of shecrotonic acid; Figure 2 GC-MS results of metabolites from methyltrophic Bacillus VS15 (ethanol extract); Figure 3 The effect of crotonic acid on inhibiting the mycelial growth of plant pathogenic fungi (test concentration 1 ppm); where: A1 is the control of *Fusarium wilt* (pepper wilt pathogen); A2 is the crotonic acid treatment of *Fusarium wilt* (pepper wilt pathogen); B1 is the control of *Pyrophyllaria lobata* (pepper rot pathogen); B2 is the crotonic acid treatment of *Pyrophyllaria lobata* (pepper rot pathogen); C1 is the control of *Anthracnose* (banana anthelmintic pathogen); C2 is the crotonic acid treatment of *Anthracnose* (banana anthelmintic pathogen); D1 is the control of *Botrytis cinerea* (tomato rot pathogen); D2 is the crotonic acid treatment of *Botrytis cinerea* (tomato rot pathogen). Figure 4 The control effects of crotonic acid on pepper wilt on pepper leaves and pepper blight on pepper fruits; where: A is 20 ppm crotonic acid; B is 200 ppm carbendazim; C is 200 ppm azoxystrobin; D is ethanol control; Figure 5 The control effect of crotonic acid on banana anthracnose on banana fruit; where: A is 20 ppm crotonic acid; B is 200 ppm carbendazim; C is 200 ppm azoxystrobin; D is ethanol control; Figure 6 The control effect of crotonic acid on gray mold on tomato fruit; where: A is 20 ppm crotonic acid; B is 200 ppm carbendazim; C is 200 ppm azoxystrobin; D is ethanol control. Detailed Implementation

[0025] This invention discloses the application of crotonic acid in the prevention and control of plant diseases.

[0026] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0027] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0028] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0029] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0030] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0031] The technical solution adopted in this invention includes: 1. Plate test to inhibit the growth of plant pathogens Pour PDA medium into one compartment of a two-part plate, allow it to solidify, and then inoculate with plant pathogens (fusarium wilt, wilt, anthracnose, and gray mold). Add 5 μL, 10 μL, or 20 μL of a 10-fold ethanol dilution to the other compartment, respectively. The control group was similar, except the drug dilution was replaced with an equal volume of ethanol. After discarding the top cap, the plates were sealed in a sterile container with 1000 mL of solvent. Each treatment was replicated six times. After incubation at 26°C for 5 days, the diameter of the pathogen colonies was measured, and the inhibition rate was calculated using the formula: "(average diameter of control group - average diameter of treatment group) ÷ (average diameter of control group - diameter of inoculated mycelial block)".

[0032] 2. Disease control experiment on detached plant leaves and fruits

[0033] After cleaning and drying the detached leaves or fruits, place them in a sterile, sealed box. For crotonic acid treatment, add a few drops of diluted crotonic acid solution to a corner of the box; for carbendazim and pyraclostrobin treatment, soak the fruit in the solution and then dry it; for the control, only add or soak the fruit in a solvent-free solution. Apply a few drops of diluted spores (10⁻⁶ spores) of plant pathogens (fusarium wilt, wilt, anthracnose, and gray mold) to the surface of the fruit. 5 (cfu / g). Seal the container and store at a constant temperature of 26°C. Investigate the severity of the disease after 15 days.

[0034] 3. Disease grading criteria: Level 0: Basically the same as when it was freshly picked.

[0035] Grade 1: The color is lighter or the gloss is reduced, with no discoloration, no blemishes, and no obvious change in hardness.

[0036] Grade 3: Discoloration area ≤ 5%, no lesions, no obvious change in hardness.

[0037] Level 5: 5% < discolored area ≤ 20% or slightly softened or rotten area ≤ 5%.

[0038] Level 7: 20% < discoloration area ≤ 50% or obvious softening and wilting or 5% < rotten area ≤ 20%.

[0039] Level 9: Discoloration area > 50% or decay area > 20%.

[0040] Disease index = ∑ (number of diseased plants at each level × relative level value) / (total number of plants surveyed) × (highest disease level value) × 100.

[0041] Prevention and control effect = (control disease index - treatment disease index) / control disease index × 100%.

[0042] The significance of differences was analyzed by performing one-way multiple comparisons using the Fisher's LSD method with SPSS software.

[0043] In Examples 1 to 5 of this invention, all raw materials and reagents used can be purchased from the market.

[0044] The present invention will be further illustrated below with reference to the embodiments: Example 1: Inhibition test of crotonic acid on four plant pathogens Pour PDA medium into one compartment of a two-part plate, let it solidify, and then inoculate it with plant pathogens (fusarium wilt of pepper, wilt of pepper, anthracnose of banana, and gray mold of tomato). Add 5 μL, 10 μL, or 20 μL of 10-fold crotonic acid ethanol solution to the other compartment, respectively. For the control group, replace the drug dilution with an equal amount of ethanol. After discarding the top of the plate, place it in a sterile sealed box with 1000 mL of solvent and seal it. Each treatment has 6 replicates.

[0045] Because the control agent is non-volatile, the efficacy test was conducted by adding the agent to PDA medium. After melting the medium and cooling it to 50°C, 50 μL, 100 μL, or 200 μL of 100-fold diluted dimethyl sulfoxide (DMSO) solution were added to each 100 mL of medium, respectively. After mixing, the mixture was poured into plates, solidified, and then inoculated with plant pathogens (fusarium wilt, wilt, anthracnose, and gray mold). In the control group, the amount of agent added was replaced with an equal amount of DMSO. Each treatment was replicated 6 times.

[0046] After culturing at 26℃ for 5 days, the diameter of the pathogen colony was measured, and the inhibition rate was calculated using the formula "(average diameter of control group - average diameter of treatment group) ÷ (average diameter of control group - diameter of inoculated mycelial block)".

[0047] Table 1. Antibacterial effects of crotonic acid on four plant pathogens.

[0048] Note: There were no significant differences between data containing the same letter in the same column (LSD, P < 0.05).

[0049] As shown in Table 1, at the three tested concentrations, crotonic acid exhibited an inhibition rate of 63.67%–100% against the four plant pathogens; carbendazim at a 10-fold concentration showed an inhibition rate of 3.54%–100% against the four plant pathogens; and azoxystrobin at a 10-fold concentration showed an inhibition rate of 38.26%–95.23% against the four plant pathogens. This indicates that the antibacterial effect of crotonic acid against the four plant pathogens was comparable to or significantly better than that of carbendazim and azoxystrobin at 10-fold concentrations.

[0050] Example 2: In vitro test of crotonic acid in the control of pepper wilt disease

[0051] After cleaning and drying the detached pepper leaves, place them in a sterile, sealed box. For crotonic acid treatment, add 10 μL or 20 μL of a 10-fold ethanol dilution of crotonic acid to a corner of the box (1000 mL solvent). For carbendazim and pyraclostrobin treatment, soak the leaves in a 10 mg / L or 20 mg / L solution for 30 seconds and then air dry. For the control, simply soak the leaves in an equal volume of ethanol or sterile water and then air dry. Apply a suspension of plant pathogen spores (containing 10 mg / L of spores) to the surface of the fruit. (Note: The original text contains some formatting issues and may require further context for accurate translation.) 5 (CFU / g), add 10 μL to 3 spots for each sample. Seal the container and store at 26°C. Investigate disease severity after 15 days.

[0052] Disease severity grading criteria: Level 0: Basically the same as when it was freshly picked.

[0053] Grade 1: The color is lighter or the gloss is reduced, with no discoloration, no blemishes, and no obvious change in hardness.

[0054] Grade 3: Discoloration area ≤ 5%, no lesions, no obvious change in hardness.

[0055] Level 5: 5% < discolored area ≤ 20% or slightly softened or rotten area ≤ 5%.

[0056] Level 7: 20% < discoloration area ≤ 50% or obvious softening and wilting or 5% < rotten area ≤ 20%.

[0057] Level 9: Discoloration area > 50% or decay area > 20%.

[0058] Disease index = ∑ (number of diseased plants at each level × relative level value) / (total number of plants surveyed) × (highest disease level value) × 100; Prevention and control effect = (Control disease index - Treatment disease index) / Control disease index × 100%; The significance of differences was analyzed by performing one-way multiple comparisons using the Fisher's LSD method with SPSS software.

[0059] Table 2. Control effects of crotonic acid on pepper wilt disease.

[0060] Note: There were no significant differences between data containing the same letter in the same column (LSD, P < 0.05).

[0061] As shown in Table 2, at the two tested concentrations, crotonic acid achieved a control efficacy of 82.66%–85.26% against wilt of pepper on detached leaves; carbendazim at a 10-fold concentration achieved a control efficacy of 82.02%–86.89% against the disease; and azoxystrobin at a 10-fold concentration achieved a control efficacy of 53.19%–64.48% against the disease. This indicates that the control effect of crotonic acid against wilt of pepper is comparable to that of carbendazim at a 10-fold concentration, and significantly better than that of azoxystrobin at a 10-fold concentration.

[0062] Example 3: In vitro test of crotonic acid in the prevention and control of pepper blight

[0063] The experimental method was the same as in Example 2, except that the detached pepper leaves to be tested were replaced with detached pepper fruit clusters. Fruit clusters with plump but not yet discolored berries were selected, with a maturity of approximately 70-80%.

[0064] Table 3. Control effects of crotonic acid on pepper blight

[0065] Note: There were no significant differences between data containing the same letter in the same column (LSD, P < 0.05).

[0066] As shown in Table 3, at the two tested concentrations, crotonic acid showed a control efficacy of 79.03%–86.10% against pepper wilt on detached leaves; carbendazim at a 10-fold concentration showed a control efficacy of 6.59%–12.65% against the disease; and azoxystrobin at a 10-fold concentration showed a control efficacy of 46.77%–64.10% against the disease. This indicates that crotonic acid is significantly more effective than carbendazim and azoxystrobin at 10-fold concentrations in controlling pepper wilt.

[0067] Example 4: In vitro test of crotonic acid in the prevention and control of banana anthracnose

[0068] The experimental method was the same as in Example 2, except that the detached pepper leaves to be tested were replaced with freshly harvested small bananas with a maturity of about 70-80%.

[0069] Table 4. Control effects of crotonic acid on banana anthracnose.

[0070] Note: There were no significant differences between data containing the same letter in the same column (LSD, P < 0.05).

[0071] As shown in Table 4, at the two tested concentrations, crotonic acid achieved a control efficacy of 72.18%-80.81% against anthracnose in banana plants; 10 times the concentration of carbendazim achieved a control efficacy of 39.85%-59.23% against the disease; and 10 times the concentration of azoxystrobin achieved a control efficacy of 6.89%-14.50% against the disease. This indicates that crotonic acid is significantly more effective than 10 times the concentrations of carbendazim and azoxystrobin in controlling anthracnose in banana plants.

[0072] Example 5: In vitro test of crotonic acid in controlling gray mold in tomatoes

[0073] The experimental method was the same as in Example 2, except that the detached pepper leaves to be tested were replaced with freshly harvested cherry tomatoes with a maturity of about 80-90%.

[0074] Table 5. Control effect of crotonic acid on gray mold in tomatoes

[0075] Note: There were no significant differences between data containing the same letter in the same column (LSD, P < 0.05).

[0076] As shown in Table 5, at the two tested concentrations, crotonic acid showed a control efficacy of 58.09%–71.09% against gray mold on cherry tomatoes; 10 times the concentration of carbendazim showed a control efficacy of 24.74%–37.46% against the disease; and 10 times the concentration of azoxystrobin showed a control efficacy of 35.46%–50.19% against the disease. This indicates that crotonic acid is significantly more effective than 10 times the concentrations of carbendazim and azoxystrobin in controlling gray mold on cherry tomatoes.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of crotonic acid in any of the following: (a) Control of plant diseases; and / or (b) Preparing products for the prevention and control of plant diseases; and / or (c) Inhibition of plant pathogens; and / or (d) Prepare products that inhibit plant pathogens.

2. The application as described in claim 1, characterized in that, The plant pathogens include one or more of the following: pepper wilt, pepper blight, banana anthracnose, and tomato gray mold.

3. The application as described in claim 1 or 2, characterized in that, The crotonic acid is derived from a metabolite of methyltrophic Bacillus.

4. The application as described in claim 3, characterized in that, The preservation number of the methyltrophic Bacillus is CGMCC NO. 14478.

5. The application as described in any one of claims 1 to 4, characterized in that, The application concentration of crotonic acid is 0.5 ppm to 20 ppm.

6. The application as described in any one of claims 1 to 5, characterized in that, The products include pesticides and / or antibacterial agents.

7. A pesticide and / or bacteriostatic agent, characterized in that, include: Crotonic acid and acceptable excipients.

8. A method for preventing and controlling plant diseases, characterized in that, Crotonic acid and / or the pesticide and / or antibacterial agent as described in claim 7 are applied to plants or plant pathogens infected with plant pathogens.

9. The method as described in claim 8, characterized in that, The plant pathogens include one or more of the following: pepper wilt, pepper blight, banana anthracnose, and tomato gray mold.

10. The method as described in claim 8 or 9, characterized in that, The application concentration of crotonic acid is 0.5 ppm to 20 ppm.

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