Preparation and application of furoic acid and bactericidal composition thereof

Abstract

The invention discloses preparation and application of furoic acid and a bactericidal composition thereof, and relates to the technical field of biopesticides, the furoic acid comprises furoic acid, the chemical structural formula of the furoic acid is shown in the specification, the purity is greater than or equal to 98%, and the furoic acid is prepared by the following steps: mixing furfural, a sodium hydroxide aqueous solution and copper oxide, introducing oxygen at 50-60 DEG C, stirring and reacting for 4-6 hours, and filtering to obtain a filtrate; extracting, acidifying, recrystallizing and drying to obtain furoic acid; according to the preparation and application of the furoic acid and the bactericidal composition thereof, the furoic acid with a specific structure is scientifically compounded with screened specific biopesticides (kasugamycin, boscalid and ningnanmycin), and the ratio range (for example, the ratio of furoic acid to kasugamycin is 1: 10-10: 1) with a remarkable synergistic effect is optimized and determined; according to the composition disclosed by the invention, the components are effectively complemented on the action mechanism, and the defects that the traditional single pesticide is single in action target and easy to induce drug resistance are overcome, so that the overall control effect of the composition on various plant diseases (including bacteria, fungi and virus diseases) is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biopesticide technology, specifically to the preparation and application of furoic acid and its bactericidal compositions. Background Technology

[0002] Against the backdrop of sustainable agricultural development, biopesticides have gradually become an important direction for plant disease management due to their advantages such as good environmental compatibility, safety for non-target organisms, and low likelihood of inducing resistance. In recent years, with in-depth research on the interaction mechanism between plants and pathogens, scientists have discovered that by combining biopesticides with different mechanisms of action, the control effect can be significantly improved and the development of resistance can be delayed. This strategy is based on the principle of synergistic effect, that is, by utilizing the complementarity of each component in terms of target site, permeability, or metabolic pathway, more efficient and longer-lasting disease control than single agents.

[0003] However, in existing technologies, how to scientifically screen compound ingredients and accurately determine their ratios to achieve broad-spectrum synergistic control against multiple pathogens remains a key bottleneck restricting the development of biopesticide compound technology. Current problems with biopesticide compound technology include a lack of scientific basis for ratio optimization, leading to unstable synergistic effects. Existing compound products often rely on experience or simple experiments to determine ratios, failing to fully reveal the interaction mechanisms of each component at the molecular level. This makes it difficult for compound systems to effectively inhibit different types of pathogens such as bacteria, fungi, and viruses in practical applications. For example, while some compound products may improve the control effect against a certain type of pathogen, an unreasonable ratio may reduce the activity against other pathogens or even cause antagonistic effects. Furthermore, unscientific ratios may lead to increased pesticide usage, not only increasing costs but also potentially exacerbating environmental pollution and the risks to non-target organisms. Therefore, improvements are needed. Summary of the Invention

[0004] The purpose of this invention is to provide the preparation and application of furoic acid and its bactericidal composition, so as to solve the problem that the lack of scientific basis for the compound ratio of biological pesticides in the prior art leads to unstable synergistic effects and difficulty in achieving broad-spectrum and efficient prevention and control.

[0005] To achieve the above objectives, the present invention provides the following technical solution: furoic acid, wherein the chemical structural formula of furoic acid is:

[0006]

[0007] With a purity ≥98%, it is prepared by the following method: furfural, sodium hydroxide aqueous solution and copper oxide are mixed, oxygen is introduced at 50 to 60°C and the mixture is stirred for 4 to 6 hours, followed by extraction, acidification, recrystallization and drying to obtain furoic acid.

[0008] Furthermore, the source is obtained by extraction, isolation, or chemical synthesis from *Bacillus thuringiensis*.

[0009] The fungicidal composition includes furoic acid and one of kasugamycin, cyazofamid, or ningnanmycin as the active pharmaceutical ingredient, and pesticide-acceptable adjuvant ingredients.

[0010] Furthermore, the mass ratio of furoic acid to kasugamycin is 1:10 to 10:1, the mass ratio of furoic acid to cyazofamid is 10:1, and the mass ratio of furoic acid to ningnanmycin is 1:5 to 10:1.

[0011] Further, the mass ratio of furoic acid to kasugamycin is 1:5, 1:1, 1:10, 5:1, 10:1, 1:20, or 20:1; the mass ratio of furoic acid to cyazofamid is 1:1, 1:5, 1:10, 1:20, 5:1, 10:1, or 20:1; and the mass ratio of furoic acid to ningnanmycin is 1:1, 1:5, 1:10, 1:20, 5:1, 10:1, or 20:1.

[0012] The preparation of the bactericidal composition involves mixing furoic acid with one of kasugamycin, cyazofamid, or ningnanmycin in a specified mass ratio, adding pesticide-acceptable auxiliary ingredients, and mixing thoroughly to prepare an applicable pesticide formulation.

[0013] Furthermore, the auxiliary components include one or more of the following: carrier, solvent, surfactant, stabilizer, and dispersant.

[0014] Application of bactericidal compositions in the prevention and control of plant diseases.

[0015] Furthermore, the plant diseases mentioned include rice bacterial blight, wheat scab, or tobacco mosaic virus.

[0016] Furthermore, the bactericidal composition is applied to the plant surface by spraying, smearing, or impregnation.

[0017] Compared with existing technologies, the preparation and application of furoic acid and its bactericidal composition provided by this invention, through the scientific compounding of furoic acid with a specific structure and selected specific biological pesticides (kasugamycin, cyazofamid, ningnanmycin), and the optimization and determination of the ratio range with significant synergistic effect (such as furoic acid and kasugamycin at 1:10 to 10:1), makes the components effectively complementary in terms of action mechanism, overcoming the shortcomings of traditional single pesticides with single action target and easy induction of drug resistance, thereby significantly improving the overall control effect of the composition on a variety of plant diseases (including bacterial, fungal and viral diseases).

[0018] By rationally combining furoic acid with different types of pesticides with different mechanisms of action (such as kasugamycin, cyazofamid, and ningnanmycin), and determining the core application ratio based on the principle of synergistic effect, it is possible to delay or hinder the development of pathogen resistance to any single pesticide, reduce the risk of decreased efficacy due to long-term use of a single pesticide, and thus extend the service life of the pesticide, providing technical support for the sustainable management of agricultural diseases.

[0019] By constructing a compound system with furoic acid as the core and combined with specific agents, and achieving significant synergistic effects under optimized ratios, it is possible to effectively reduce the amount of active ingredients used in each single agent while achieving the same or better control effect. This not only helps to reduce pesticide input costs, but also reduces the potential impact of pesticide residues on the environment and non-target organisms, thus reflecting the design concept of environmentally friendly pesticides. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0021] Figure 1 The preparation process flowchart is provided for an embodiment of the present invention. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0023] As attached Figure 1 As shown:

[0024] Example 1:

[0025] This invention provides the preparation and application of furoic acid and its bactericidal composition, and a method for extracting furoic acid from *Alternaria alternata*, the specific steps of which are as follows:

[0026] Fermentation culture of *Bacillus thuringiensis*: *Bacillus thuringiensis* PR2 strain, preserved on test tube slant at 4℃, was inoculated onto PDA medium and cultured for one week in a 25℃ incubator. Agar blocks were then inoculated into 250mL Erlenmeyer flasks containing 50mL of PDA medium and cultured for 3 days on a rotating shaker at 25℃ and 180rpm to obtain the seed culture. A 10% inoculum was then transferred to a 500mL Erlenmeyer flask containing 150mL of fermentation medium and cultured under the same conditions for 5 days before fermentation was terminated to obtain the fermentation broth.

[0027] Ethanol extraction: The fermentation broth was centrifuged at 4000 r / min for 10 minutes to collect the mycelium. The mycelium was dried to constant weight in a 50℃ oven, pulverized, and passed through an 80-mesh sieve. 10.0 g of dried mycelium powder was weighed and 200 mL of anhydrous ethanol was added (the amount of ethanol added was 20 times the weight of the mycelium), and the mixture was cold-soaked at room temperature for 24 hours. Subsequently, it was extracted using an ultrasonic extractor at 40 kHz and 300 W for 2 hours. The extraction was repeated twice, and the three extracts were combined, filtered, and the ethanol extract was obtained.

[0028] Separation and purification: The ethanol extract was extracted three times with an equal volume of ethyl acetate, shaking for 10 minutes each time and then allowed to stand for separation. The upper ethyl acetate phases were combined and concentrated to dryness under reduced pressure at 40°C to obtain crude ethyl acetate extract (approximately 1.2 g).

[0029] The crude extract was separated by silica gel column chromatography: 200-300 mesh silica gel was used, and the column was packed wet (30 cm long, 2.5 cm in diameter). The crude extract was dissolved in a small amount of dichloromethane and then loaded onto the column using a dry method. Gradient elution was performed using a dichloromethane / methanol mixed solvent system at volume ratios of 100:0, 100:1, 100:2, 100:4, 100:8, and 100:16. 200 mL of each fraction was collected, for a total of 30 fractions.

[0030] Each fraction was analyzed by thin-layer chromatography (TLC) with dichloromethane:methanol = 10:1 (v / v) as the developing solvent. Spots were observed under UV lamps at 254 nm and 365 nm. Fractions with the same Rf value were combined to obtain 6 components (Fr1-Fr6).

[0031] Purification and identification of furoic acid: The Fr3 fraction (120 mg) was further purified. A fine silica gel column (15 cm long, 1.5 cm in diameter) was used as the eluent, with dichloromethane / methanol (100:2, v / v) collected in 10 mL fractions. The same fractions were combined by TLC to obtain compound A29 (12 mg).

[0032] Structural identification of compound A29:

[0033] Mass spectrometry (ESI-MS) analysis showed that the molecular ion peak [M+H]+ was 113.02 m / z, confirming the molecular weight as 112;

[0034] 1H NMR (600 MHz, DMSO-d6): δ 7.60 (d, J = 1.8 Hz, 1H, H-5), 7.10 (d, J = 3.6 Hz, 1H, H-3), 6.60 (dd, J = 3.6, 1.8 Hz, 1H, H-4), 12.50 (s, 1H, COOH);

[0035] Carbon NMR spectrum (13 CNMR, 150 MHz, DMSO-d6): δ 162.3 (C=O), 148.5 (C-2), 144.2 (C-5), 116.8 (C-3), 112.5 (C-4).

[0036] Infrared spectrum (KBr pellet): 3440 cm⁻¹ (OH), 1685 cm⁻¹ (C=O), 1605 cm⁻¹, 1570 cm⁻¹ (furan ring C=C), 1420 cm⁻¹, 1350 cm⁻¹, 1260 cm⁻¹ (CO).

[0037] Based on the above spectral data, the compound was identified as furoic acid (2-furanoic acid), with the following structural formula:

[0038]

[0039] The specific steps for preparing furoic acid by chemical synthesis are as follows:

[0040] Raw materials: furfural (purity ≥98%), sodium hydroxide (analytical grade), copper oxide (catalyst grade, purity ≥99%), oxygen (industrial grade, purity ≥99.5%), diethyl ether (analytical grade), 10% dilute sulfuric acid (self-made), deionized water.

[0041] Equipment: 500mL three-necked flask (with stirrer), constant temperature water bath (accuracy ±0.5℃), oxygen cylinder and flow control device, separatory funnel (500mL), vacuum filtration device, low temperature constant temperature water tank (0-10℃), vacuum drying oven.

[0042] Synthesis steps:

[0043] Preparation of the reaction system: Accurately weigh 50.0 g of purified furfural and place it in a 500 mL three-necked flask. Separately, dissolve 24.0 g of sodium hydroxide in 200 mL of deionized water, and add it to the flask after complete dissolution. Add 2.0 g of copper oxide powder as a catalyst, and stir at 300 rpm for 5-10 minutes to ensure uniform dispersion of the catalyst.

[0044] Catalytic oxidation reaction: Construct a reaction apparatus with a three-necked flask, one side fitted with a reflux condenser, the other with a thermometer, and the middle with a vent tube (inserted approximately 1 cm below the liquid surface). Connect the vent tube to an oxygen cylinder, and control the oxygen flow rate using a rotor flow meter. Turn on the constant temperature water bath and heat the reaction system to 55℃ (controlled within the range of 50-60℃). Introduce oxygen at a rate of 0.8 L / min while maintaining a stirring speed of 350 rpm. During the reaction, the solution color gradually changes from pale yellow to light brown. Continue the reaction for 5 hours until the solution becomes a homogeneous, light brown, transparent solution, indicating that furfural has been largely converted to sodium furoate.

[0045] Extraction and purification: After cooling the reaction solution to room temperature (25°C), transfer it to a 500 mL separatory funnel. Add 250 mL of diethyl ether, shake vigorously for 5 minutes, and allow to separate into layers for 30 minutes. Collect the lower aqueous phase and discard the upper ether phase (containing unreacted furfural and other organic impurities). Repeat the extraction once and combine the aqueous phases.

[0046] Acidification crystallization: Place the aqueous phase in a 500mL beaker and slowly add 10% dilute sulfuric acid dropwise while stirring, monitoring the pH value with pH paper. Stop adding acid when the pH drops to 2.0, at which point a white precipitate begins to form. Transfer the beaker to a low-temperature constant-temperature water bath and continue stirring at 5°C for 2 hours to promote complete crystallization.

[0047] Separation and purification: Vacuum filtration was performed using a Buchner funnel, and the white precipitate was collected. The precipitate was washed three times with 10 mL of cold water at 0-5°C each time. The wet sample was placed in a 50 mL beaker, and 8 times its weight of deionized water (approximately 40 mL) was added. The mixture was heated under reflux until completely dissolved. The solution was filtered while hot to remove insoluble impurities. The filtrate was cooled to 5°C and allowed to stand overnight to allow complete crystallization. The solution was filtered again and washed with cold water to obtain white needle-like crystals.

[0048] Drying and storage: Place the crystals in a petri dish and put it in a vacuum drying oven at 50℃ and -0.09MPa for 5 hours. After cooling to room temperature, quickly transfer them to a brown glass bottle, seal and store in the dark.

[0049] Product characterization and quality indicators:

[0050] Appearance: White needle-like crystals;

[0051] Purity determination: determined by high performance liquid chromatography (HPLC);

[0052] Chromatographic conditions: C18 column (4.6×250mm, 5μm), mobile phase: methanol:0.1% phosphoric acid aqueous solution = 30:70 (v / v), flow rate 1.0mL / min, detection wavelength 254nm, column temperature 30℃;

[0053] Test results: Purity 98.3%;

[0054] Melting point determination: The capillary method was used, and the melting point range was 129.5-131.8℃;

[0055] Moisture content determination: Karl Fischer method, moisture content 0.32%;

[0056] Structural confirmation: Compared with the furoic acid extracted in 1.1.4, the mass spectrometry and nuclear magnetic resonance spectrometry data are consistent.

[0057] Yield calculation:

[0058] The reaction was initiated with 50.0 g of furfural (theoretical molar number 0.52 mol).

[0059] 48.2g of furoic acid product was obtained (actual molar number 0.43mol);

[0060] Yield = Actual number of moles / Theoretical number of moles × 100% = 82.7%.

[0061] It was demonstrated that furoic acid can be obtained through both biological extraction and chemical synthesis. The chemical synthesis method yielded a higher output (82.7%) and higher purity (≥98%), making it suitable for industrial production. The furoic acid extracted from *Alternaria solani* has the same structure as the chemically synthesized furoic acid, confirming that furoic acid has a defined chemical structure and a reproducible preparation method.

[0062] Example 2:

[0063] This embodiment is basically the same as the previous embodiment, except that the indoor toxicity determination of a single agent of furoic acid is performed:

[0064] Test strains:

[0065] Bacteria: Rice bacterial blight pathogen;

[0066] Fungus: Fusarium graminearum, the pathogen of wheat scab.

[0067] Test reagents:

[0068] Furoic acid: prepared according to the chemical synthesis method in Example 1, with a purity of 98.3%, and prepared into a 100 mg / mL stock solution with dimethyl sulfoxide (DMSO), and stored at 4°C for later use.

[0069] Control drug:

[0070] Kasugamycin: Commercially available 6% wettable powder, with an active ingredient content ≥95%;

[0071] Boscalid: Commercially available 50% water-dispersible granules with an active ingredient content of ≥98%.

[0072] Culture medium:

[0073] NB medium (for bacteria): 3.0g beef extract, 10.0g peptone, 5.0g sodium chloride, 15.0g agar, distilled water to a final volume of 1L, pH 7.0-7.2;

[0074] PDA medium (for fungi): 200g potato (boiled and juiced), 20g glucose, 15g agar, distilled water to a final volume of 1L, pH at rest.

[0075] Instruments and equipment: constant temperature incubator (accuracy ±0.5℃), ultra-clean workbench, high pressure steam sterilizer, analytical balance (accuracy 0.0001g), vortex shaker, pipettes (10μL, 100μL, 1000μL), petri dishes (diameter 9cm), hole punch (diameter 5mm), vernier calipers (accuracy 0.02mm).

[0076] Test method:

[0077] Bacterial virulence assay, plate confrontation method:

[0078] Preparation of bacterial suspension: Remove the glycerol storage tube of *Bacillus oryzae*, the pathogen of rice bacterial blight, from the -80℃ freezer and streak it on an NB agar plate. Incubate at 28℃ for 48 hours. Pick a single colony and inoculate it into 10 mL of NB liquid medium. Incubate at 28℃ with shaking at 180 rpm for 24 hours until the logarithmic growth phase. Centrifuge the bacterial suspension at 4000 rpm for 5 minutes, discard the supernatant, resuspend the suspension in sterile physiological saline, and adjust the bacterial concentration to OD600 = 0.5 (approximately 1 × 10⁻⁶). 8 (CFU / mL).

[0079] Preparation of drug-containing agar plates: The furoic acid stock solution was diluted with sterile water to prepare 6 concentration gradients: 0 (CK), 0.006, 0.016, 0.16, 1.6, and 2.0 mg / mL. A kasugamycin control (0.16 mg / mL) was also included.

[0080] Experimental procedure:

[0081] Take sterile NB agar medium and cool it to about 50°C. Add the above-mentioned drug solution to each medium, so that the volume ratio of drug solution to medium is 1:9. Mix thoroughly and pour into petri dishes. Perform 4 replicates for each concentration.

[0082] Spread 100 μL of bacterial suspension evenly onto a drug-containing plate;

[0083] Use sterile forceps to place 5mm diameter sterile filter paper discs at equal intervals on the plate (4 discs per plate).

[0084] Pipette 10 μL of the corresponding concentration of drug solution onto a filter paper disc;

[0085] Incubate at 28°C upside down for 48 hours.

[0086] Data Measurement and Calculation: The diameter of the inhibition zone was measured using the cross-sectional method, accurate to 0.1 cm. Calculation formula:

[0087] The diameter of the inhibition zone = (longitudinal diameter + transverse diameter) / 2;

[0088] Relative inhibition rate (%) = (Diameter of inhibition zone of test agent - Diameter of inhibition zone of blank control) / (Diameter of inhibition zone of standard agent - Diameter of inhibition zone of blank control) × 100%;

[0089] The standard reagent is kasugamycin (0.16 mg / mL).

[0090] Fungal virulence assay, mycelial growth rate method:

[0091] Strain activation: The wheat scab pathogen was cultured on a PDA plate at 25°C for 5 days until conidia were produced at the edge of the colony.

[0092] Preparation of drug-containing agar plates: The furoic acid stock solution was diluted to six concentration gradients identical to those used in the bacterial assay. A control of boscalid (0.15 mg / mL) was also included.

[0093] Experimental procedure:

[0094] Take sterile PDA medium, cool it to about 50°C, add the drug solution, so that the volume ratio of drug solution to medium is 1:9, mix thoroughly and pour into a petri dish;

[0095] Use a 5mm diameter punch to cut out the mycelial cake from the edge of the colony;

[0096] Inoculate the mycelium into the center of the drug-containing plate using a sterile inoculation needle (mycelial side down).

[0097] Six replicates were performed for each concentration.

[0098] Incubate at 25°C upside down for 72 hours.

[0099] Data Measurement and Calculation: Colony diameter was measured using the cross-multiplication method, accurate to 0.1 cm. Calculation Formula:

[0100] Colony diameter = (vertical diameter + transverse diameter) / 2;

[0101] Mycelial growth inhibition rate (%) = [(control colony diameter - mycelial cake diameter) - (treatment colony diameter - mycelial cake diameter)] / (control colony diameter - mycelial cake diameter) × 100%.

[0102] Experimental results:

[0103] Furoic acid's inhibitory effect on rice bacterial blight pathogen:

[0104] The experimental results are shown in the table below:

[0105] Antibacterial effects of different concentrations of furoic acid on rice bacterial blight pathogen.

[0106]

[0107] Note: Data are mean ± standard deviation (n=4).

[0108] As can be seen from the table above:

[0109] When the concentration of furoic acid was 0.016 mg / mL, an inhibition zone (0.2 cm) began to appear, with a relative inhibition rate of 25%.

[0110] When the concentration was increased to 0.16 mg / mL, the diameter of the inhibition zone reached 0.8 cm, and the relative inhibition rate was 100%, which was comparable to the effect of the same concentration of kasugamycin.

[0111] When the concentration was further increased to 1.6 mg / mL and 2.0 mg / mL, the diameter of the inhibition zone increased to 1.2 cm and 1.4 cm, respectively, and the relative inhibition rates reached 150% and 175%, respectively.

[0112] Furoic acid's antifungal effect against Fusarium graminearum, the causal agent of wheat blight.

[0113] The experimental results are shown in the table below:

[0114] The inhibitory effects of different concentrations of furoic acid on Fusarium wilt pathogens in wheat:

[0115]

[0116] Note: Data are mean ± standard deviation (n=6).

[0117] As can be seen from the table above:

[0118] The inhibitory effect of furoic acid on wheat scab fungus increases in a concentration-dependent manner;

[0119] At a concentration of 0.16 mg / mL, the inhibition rate reached 45.67%;

[0120] At a concentration of 2.0 mg / mL, the inhibition rate reached 60.56%, which is basically equivalent to the inhibition effect of 0.15 mg / mL cyazofamid (60.25%).

[0121] Determination of effective concentration:

[0122] Combined results of bacterial and fungal tests:

[0123] At a concentration of 0.16 mg / mL, furoic acid exhibited a similar inhibitory effect on rice bacterial blight pathogens as kasugamycin (both had inhibition zones of 0.8 cm in diameter).

[0124] At the same concentration, the inhibition rate against wheat scab was 45.67%, which, although lower than that of boscalid, still achieved a significant inhibitory effect.

[0125] Therefore, 0.16 mg / mL can be used as the recommended concentration for the use of furoic acid as a single agent, which can take into account both the inhibitory effects on bacteria and fungi.

[0126] Dose-response relationship:

[0127] Bacterial inhibition: In the concentration range of 0.016-2.0 mg / mL, the diameter of the inhibition zone was positively correlated with the concentration (r=0.987, P<0.01).

[0128] Fungal inhibition: Within the same concentration range, colony diameter was negatively correlated with concentration (r=-0.976, P<0.01), while inhibition rate was positively correlated with concentration (r=0.982, P<0.01).

[0129] Observations revealed that at the highest test concentration (2.0 mg / mL), no obvious drug precipitation or crystallization occurred; the edge of the inhibition zone around the filter paper was clear, with no abnormal colony growth; and the transparency of the culture medium on the drug-containing plate was not significantly different from that on the control plate.

[0130] This embodiment demonstrates, through systematic indoor toxicity testing, that:

[0131] Furoic acid has a significant inhibitory effect on both rice bacterial blight fungus and wheat scab fungus.

[0132] The antibacterial effect is significantly concentration-dependent, increasing with increasing concentration in the range of 0.16-2.0 mg / mL;

[0133] The recommended concentration is 0.16 mg / mL. At this concentration, the inhibitory effect on bacteria is comparable to that of kasugamycin, and the inhibitory effect on fungi reaches 45.67%.

[0134] Furoic acid has broad-spectrum antibacterial potential, providing basic data for subsequent compound research.

[0135] Example 3:

[0136] This embodiment is basically the same as the previous embodiment, except that the indoor toxicity test of the combination of furoic acid and kasugamycin against rice bacterial blight is carried out in the following steps:

[0137] Test strain: Xanthomonas oryzaepv. oryzae (Xoo), the pathogen of rice bacterial blight, was activated and cultured in NB medium at 28°C for 48 hours for later use.

[0138] Test reagents and formulation design:

[0139] Single dose:

[0140] Furoic acid: prepared according to the method in Example 1, with a purity of 98.3%, and prepared as a 10 mg / mL stock solution using DMSO;

[0141] Kasugamycin: Commercially available raw material, purity ≥95%, prepared as a 10mg / mL stock solution with sterile water.

[0142] Compound combination:

[0143] Design 7 mass ratios:

[0144] Furoic acid: Kasugamycin = 1:1, 1:5, 1:10, 1:20, 5:1, 10:1, 20:1.

[0145] Concentration gradient design: Centered on the optimal single-dose concentration of 0.16 mg / mL, five mass concentration gradients were set up in a proportional manner: 0.04, 0.08, 0.16, 0.32, and 0.64 mg / mL (based on the total concentration of active ingredient).

[0146] Culture media and reagents:

[0147] NB culture medium: The formula is the same as in Example 2;

[0148] Physiological saline: 0.85% NaCl solution, sterilized at 121℃ for 20 minutes;

[0149] Sterile water: Sterilize at 121℃ for 20 minutes.

[0150] Instruments and equipment: In addition to the equipment listed in Example 2, the following are added: micro-oscillator, colony counter, and biochemical incubator (temperature control accuracy ±0.5℃, humidity control range 40-90%).

[0151] Test method:

[0152] Flat plate confrontation method experiment:

[0153] Preparation of bacterial culture: Activated Xoo strain was inoculated into 50 mL of NB liquid medium and cultured at 28°C with shaking at 180 rpm for 24 hours until the logarithmic growth phase (OD600 ≈ 0.8-1.0). The culture was centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and the bacterial cells were washed twice with sterile physiological saline. Finally, the cells were resuspended in physiological saline and the concentration was adjusted to OD600 = 0.5 (approximately 1 × 10⁻⁶). 8 (CFU / mL).

[0154] Preparation of drug-containing tablets:

[0155] Calculate the required volume of mother liquor for each ratio and mix according to the proportions in the table below:

[0156] Preparation table of compound drug stock solution:

[0157]

[0158] Dilute the mixed mother liquor to each concentration gradient with sterile water;

[0159] Take sterile NB agar medium, cool it to 45-50℃, add the drug solution (drug solution: medium = 1:9, v / v), mix well and pour into petri dishes (15mL per dish).

[0160] Experimental procedure:

[0161] Spread 100 μL of bacterial suspension evenly onto a drug-containing plate;

[0162] Use sterile forceps to place 5mm diameter sterile filter paper strips at equal intervals on the plate (4 strips per plate).

[0163] Pipette 10 μL of the corresponding concentration of drug solution onto a filter paper disc;

[0164] Each treatment was replicated in 4 places, with a single-dose control and a blank control (no drug administered).

[0165] Incubate at 28°C upside down for 48 hours.

[0166] Data measurement: Measure the diameter of the inhibition zone using a vernier caliper (accuracy 0.02mm). Measure each inhibition zone twice in the vertical direction and take the average value.

[0167] In vivo inoculation method test:

[0168] Test plants:

[0169] The rice variety used was "Zhonghua 11". The seeds were surface-sterilized with 0.1% HgCl2 for 10 minutes, rinsed 5 times with sterile water, and sown in sterilized nutrient soil. They were cultivated in an artificial climate chamber (day / night temperature 28℃ / 25℃, light 14h / 10h, relative humidity 60%), and healthy seedlings with uniform growth at 21 days were selected.

[0170] Chemical treatment: Dilute each formulation of the pesticide to the recommended field concentration (total concentration of active ingredient 0.16 mg / mL), and spray evenly on both sides of the rice leaves using a handheld sprayer until the leaves are completely wet but not dripping. Each treatment consists of 10 plants, with 3 replicates.

[0171] Inoculation method: Inoculate 4 hours after drug treatment, and adjust the Xoo bacterial suspension concentration to 1×10⁻⁶. 8 CFU / mL, dip sterile scissors into the bacterial solution, and cut the leaves 3cm from the tip of the fully expanded leaves at the top of each rice plant. Inoculate 15-20 cuts for each treatment.

[0172] Culture and Investigation: After inoculation, the plants were placed in an artificial climate chamber (temperature 28℃, relative humidity 85%) and cultured for 14 days before measuring the length of the lesions. The lesion length was measured from the cut to the boundary between the diseased and healthy tissue.

[0173] Toxicity index calculation:

[0174] Actual Virulence Index (ATI): ATI = (Diameter of inhibition zone or lesion length in blank control / Diameter of inhibition zone or lesion length in treatment) × 100

[0175] Theoretical Toxicity Index (TTI): TTI = TI of A × A in the mixture % + TI of B × B in the mixture %; where TI is the single-dose toxicity index.

[0176] Cotoxicity coefficient (CTC): CTC = (ATI / TTI) × 100.

[0177] Evaluation criteria:

[0178] CTC>120: Synergistic effect;

[0179] 80≤CTC≤120: Additive effect;

[0180] CTC<80: Antagonistic effect.

[0181] Data analysis was performed using SPSS 22.0 software.

[0182] Data are expressed as mean ± standard deviation. One-way ANOVA and Duncan's new multiple range method were used for multiple comparisons. P < 0.05 was considered statistically significant.

[0183] The experimental results are shown in the table below:

[0184] Results of a plate confrontation experiment between furoic acid and kasugamycin and rice bacterial blight pathogen:

[0185]

[0186] Note: Data are mean ± standard deviation (n=4), and the diameter of the inhibition zone of a single agent was determined at a concentration of 0.16 mg / mL.

[0187] The results of the live inoculation method are shown in the table below:

[0188] Results of in vivo inoculation test of furoic acid combined with kasugamycin against rice bacterial blight:

[0189]

[0190] Note: Data are mean ± standard deviation (n=30), and the drug concentration is 0.16 mg / mL.

[0191] Analysis based on co-toxicity coefficient (CTC):

[0192] Flat standoff method:

[0193] Synergistic effect (CTC>120): 1:1, 1:5, 1:10, 5:1 ratios;

[0194] Additive effect (80≤CTC≤120): 1:20, 10:1, 20:1 ratios;

[0195] Optimal ratio: 1:5 (CTC=200.0), with the largest inhibition zone diameter (1.60cm).

[0196] Live inoculation method:

[0197] Synergistic effects: 1:1, 1:5, and 1:10 ratios (CTC values ​​are 127.58, 155.00, and 132.40, respectively).

[0198] Additive effects: 1:20, 5:1, 10:1 ratios;

[0199] Mild antagonism: 20:1 ratio (CTC=87.60);

[0200] Optimal ratio: 1:5 (CTC=155.00), resulting in the shortest lesion length (1.60cm).

[0201] Combining the two experimental methods:

[0202] The 1:5 ratio showed the best performance: the inhibition zone diameter was the largest (1.60cm) in the plate confrontation method, with CTC=200.0; the lesion length was the shortest (1.60cm) in the live inoculation method, with CTC=155.0.

[0203] 1:1 and 1:10 ratios were used as alternatives: in the plate confrontation method, the diameter of the inhibition zone was 1.20 cm and the CTC was 150.0; in the live inoculation method, the lesion length was 2.20 cm and 1.80 cm, and the CTCs were 127.58 and 132.40, respectively; both showed significant synergistic effects.

[0204] Other ratios: 1:20, 5:1, and 10:1 have additive effects, while 20:1 shows slight antagonism in in vivo experiments.

[0205] Dose-response analysis:

[0206] Impact of kasugamycin ratio: When the kasugamycin ratio increases (1:5, 1:10, 1:20), the in vivo efficacy gradually decreases; the optimal ratio is 1:5, indicating that appropriately increasing the kasugamycin ratio can enhance the synergistic effect. Impact of furoic acid ratio: When the furoic acid ratio is too high (5:1, 10:1, 20:1), the synergistic effect weakens; indicating that an appropriate balance needs to be maintained.

[0207] This embodiment demonstrates, through systematic indoor toxicity testing, that:

[0208] Synergistic effect: Furoic acid and kasugamycin exhibit synergistic effects in most ratios, especially the 1:5 ratio.

[0209] The optimal ratio was determined to be 1:5, which showed the best control effect in both the plate confrontation method and the live inoculation method.

[0210] Application value: A 1:5 ratio can reduce the length of lesions from 3.60cm (furoic acid) and 2.30cm (kasugamycin) with a single agent to 1.60cm; the control effect is improved by about 30-40%; it can reduce the dosage of a single agent and reduce the risk of drug resistance.

[0211] Recommended application scheme: When controlling rice bacterial blight in the field, the preferred ratio of furoic acid to kasugamycin is 1:5, while 1:10 or 1:1 ratios can be used as alternative schemes.

[0212] Example 4:

[0213] This embodiment is basically the same as the previous embodiment, except that the indoor toxicity test of the furoic acid and boscalid compound composition against wheat scab is carried out in the following steps:

[0214] Experimental materials:

[0215] Test strain: Fusarium graminearum, the pathogen of wheat blight. This strain was activated and cultured in PDA medium at 25°C for 5 days before use.

[0216] Test reagents and formulation design:

[0217] Single dose:

[0218] Furoic acid: prepared according to the method in Example 1, with a purity of 98.3%, and prepared as a 10 mg / mL stock solution using DMSO;

[0219] Cyclomethasone: purity ≥98%, prepared as a 10 mg / mL stock solution with acetone.

[0220] Compound combinations: Design 7 mass ratios:

[0221] Furoic acid: cyprodinil = 1:1, 1:5, 1:10, 1:20, 5:1, 10:1, 20:1.

[0222] Concentration gradient design: Five mass concentration gradients are set around the effective concentration of a single agent:

[0223] For the mycelial growth rate method: 0.05, 0.10, 0.20, 0.40, 0.80 mg / mL (based on the total concentration of active ingredients);

[0224] For live inoculation: the recommended field concentration is 0.16 mg / mL (based on the total concentration of the active ingredient).

[0225] Culture media and reagents:

[0226] PDA culture medium: The formulation is the same as in Example 2;

[0227] Potato glucose liquid medium (PDB): 200g potato, 20g glucose, 1L distilled water; Tween-80: analytical grade, used to prepare spore suspension;

[0228] Sterile water: Sterilize at 121℃ for 20 minutes.

[0229] Test plants: The wheat variety was "Zhengmai 9023". Seeds were disinfected with 0.1% sodium hypochlorite for 10 minutes, rinsed with sterile water, and sown in sterilized nutrient soil. They were cultivated in an artificial climate chamber (day / night temperature 23℃ / 18℃, light 14h / 10h, relative humidity 60%), and healthy plants with uniform growth at 21 days of age were selected.

[0230] Test method:

[0231] Mycelial growth rate method:

[0232] Inoculum preparation: Incubate the activated wheat scab fungus at 25°C for 5 days on a PDA plate until orange-red conidia appear at the edge of the colony.

[0233] Preparation of drug-containing tablets:

[0234] Calculate and prepare the mixed mother liquor according to the table below. Table for preparing the mother liquor of the compounded drug:

[0235]

[0236] Dilute the mixed stock solution with sterile water to each concentration gradient so that the acetone content is ≤1%; take sterile PDA medium, cool it to 45-50℃ and add the drug solution (drug solution: medium = 1:9, v / v), mix thoroughly and pour into petri dishes (15mL per dish); set up a blank control with no drug and 1% acetone.

[0237] Experimental procedure: Use a 5mm diameter punch to cut out mycelial cakes from the edge of the colony; use a sterile inoculation needle to inoculate the mycelial cakes into the center of the drug-containing plate (mycelial side down); each treatment has 6 replicates; incubate upside down at 25℃ for 72 hours.

[0238] Data measurement: Measure the colony diameter using the cross-sectional method, accurate to 0.1 cm. Colony diameter = (vertical diameter + transverse diameter) / 2.

[0239] Inhibition rate calculation: Mycelial growth inhibition rate (%) = [(control colony diameter - 5) - (treatment colony diameter - 5)] / (control colony diameter - 5) × 100.

[0240] Live inoculation method:

[0241] Preparation of spore suspension: *Fusarium graminearum*, the causal agent of wheat blight, was inoculated onto PDA plates and cultured at 25°C for 7 days. 10 mL of sterile water containing 0.05% Tween-80 was added, and spores were scraped off using a sterile spreader. The spores were filtered through double-layered sterile gauze, and the spore concentration was adjusted to 1 × 10⁻⁶. 5 Spores / mL.

[0242] Chemical treatment: Dilute each formulation of the pesticide to 0.16 mg / mL (total concentration of active ingredient); use a handheld sprayer to spray evenly on both sides of the wheat leaves until the leaves are completely wet; 10 plants per treatment, 3 replicates; set up a water control and a single-agent control.

[0243] Inoculation method: Inoculate 4 hours after the agent treatment; select the two fully unfolded upper leaves of each plant; use a 1mm diameter inoculation needle to puncture a small hole at the midrib of the leaf; dip the inoculation needle into the spore suspension and puncture the hole to inoculate.

[0244] Cultivation and investigation: After inoculation, the plants were placed in an artificial climate chamber (temperature 23℃, relative humidity 85%). 3-5 days after inoculation, when the lesions have expanded significantly, an investigation was conducted. The length of the lesions was measured with calipers from the inoculation point to the boundary between the diseased and healthy tissue.

[0245] Toxicity index calculation:

[0246] Actual virulence index (ATI): ATI = (diameter of blank control colony or length of lesion / diameter of treated colony or length of lesion) × 100.

[0247] Theoretical Toxicity Index (TTI): TTI = Furoic Acid TI × Furoic Acid in Mixture % + Cyclomethasone TI × Cyclomethasone in Mixture

[0248] Cotoxicity coefficient (CTC): CTC = (ATI / TTI) × 100; evaluation criteria are the same as in Example 3.

[0249] Data analysis was performed using SPSS 22.0 software, following the same method as in Example 3.

[0250] Results of the mycelial growth rate method:

[0251] The test results at a concentration of 0.16 mg / mL are shown in the table below:

[0252] Results of the experiment on the inhibition of mycelial growth of Furoic acid and boscalid combined with Fusarium oxysporum:

[0253]

[0254] Note: Data are mean ± standard deviation (n=6), and the drug concentration is 0.16 mg / mL.

[0255] The experimental results are shown in the table below: Results of in vivo inoculation test of furoic acid and boscalid combined with wheat scab:

[0256]

[0257] Note: Data are mean ± standard deviation (n=30), and the drug concentration is 0.16 mg / mL.

[0258] Analysis based on co-toxicity coefficient (CTC):

[0259] Mycelial growth rate method:

[0260] Synergistic effects (CTC>120): 1:1, 1:5, 5:1, 10:1 ratios;

[0261] Additive effect (80≤CTC≤120): 1:10, 1:20, 20:1 ratio;

[0262] The optimal ratio is 10:1 (CTC=149.31), which has the highest inhibition rate (70.24%).

[0263] Live inoculation method:

[0264] Synergistic effects: 1:1, 5:1, 10:1, 20:1 ratios (CTC are 140.40, 213.50, 253.20, and 142.90 respectively).

[0265] Additive effects: 1:5, 1:10, 1:20 mixing ratios;

[0266] Optimal ratio: 10:1 (CTC=253.20), with the shortest lesion length (0.44cm).

[0267] Combining the two experimental methods:

[0268] The 10:1 ratio showed the best performance: mycelial growth inhibition rate of 70.24%, CTC=149.31; live lesion length of 0.44cm, CTC=253.20; it showed the strongest synergistic effect among the two methods.

[0269] A 1:5 ratio was used as an alternative: the mycelial growth inhibition rate was 70.26%, the highest among all ratios; in the in vivo test, CTC=117.30, which is an additive effect, but the lesion length was shorter (0.50cm).

[0270] Effect of furoic acid ratio: When the proportion of furoic acid is high (5:1, 10:1, 20:1), the synergistic effect is enhanced; especially the 10:1 ratio, the CTC is as high as 253.20 in the in vivo test; indicating that furoic acid plays an important synergistic role in compound formulation.

[0271] Comparison of inhibitory effects:

[0272] Compared with single agents: the inhibition rate of the 10:1 ratio (70.24%) was significantly higher than that of single agents of furoic acid (45.67%) and cyprodinil (60.25%); the lesion length decreased from 1.25 cm (furoic acid) and 0.53 cm (cyprodinil) to 0.44 cm.

[0273] Gradient effect of different ratios:

[0274] Inhibition rate range: 50.74%-70.26%;

[0275] Lesion length range: 0.44-0.82 cm;

[0276] It shows a clear ratio dependence.

[0277] Based on the experimental results, the synergistic effect of furoic acid and boscalid may stem from:

[0278] Complementary action sites: furoic acid may act on the cell membrane system, while boscalid acts on the mitochondrial respiratory chain; the two work together to disrupt fungal energy metabolism.

[0279] Increased permeability: Furoic acid may alter cell membrane permeability, promoting the entry of boscalid into the bacterial cell;

[0280] Resistance delay: Different mechanisms of action can delay the development of drug resistance.

[0281] Application advantages:

[0282] Significant synergy: The 10:1 ratio of CTC reaches 253.20, representing a synergistic effect of 2.53 times;

[0283] Reduced dosage: To achieve the same preventive effect, the dosage of a single agent can be reduced by 30-50%;

[0284] Improved safety: Reduces the risk of phytotoxicity that may result from high-concentration single-dose formulations.

[0285] This embodiment demonstrates, through systematic indoor toxicity testing, that:

[0286] Significant synergistic effect: Furoic acid and boscalid showed synergistic effects in multiple ratios, especially the 10:1 ratio with a CTC as high as 253.20.

[0287] The optimal ratio was determined to be 10:1 by mass, which showed the best effect in both inhibiting mycelial growth and controlling in vivo.

[0288] Application value: The 10:1 ratio can increase the inhibition rate from 45.67% (furoic acid) and 60.25% (cyhalothrin) of single agents to 70.24%; the lesion length is reduced from 0.53cm (cyhalothrin single agent) to 0.44cm; and the control effect is improved by about 30%.

[0289] Recommended application scheme: When controlling wheat scab, the preferred ratio is furoic acid: cymoxanil = 10:1, while a 1:5 ratio can be used as an alternative for specific situations.

[0290] Example 5:

[0291] This embodiment is basically the same as the previous embodiment, except that the indoor virulence determination of the combination of furoic acid and ningnanmycin against tobacco mosaic virus is carried out in the following steps:

[0292] The tested virus was Tobacco Mosaic Virus (TMV), and the source of the virus was infected leaves frozen at -80°C.

[0293] Test reagents and formulation design:

[0294] Single dose:

[0295] Furoic acid: prepared according to the method in Example 1, with a purity of 98.3%, and prepared as a 5 mg / mL stock solution with sterile water;

[0296] Ningnanmycin: purity ≥90%, prepared as a 5mg / mL stock solution using sterile water.

[0297] Compound combinations: Design 7 mass ratios:

[0298] Furoic acid: Ningnanmycin = 1:1, 1:5, 1:10, 1:20, 5:1, 10:1, 20:1.

[0299] Concentration setting: All treatments used the same total concentration of active ingredient: 0.16 mg / mL.

[0300] Set up a water control and a single-dose control:

[0301] Test plants: The tobacco variety was "Nicotiana glutinosa". Seeds were treated with 10% trisodium phosphate for 15 minutes, rinsed with sterile water, and sown in sterilized nutrient soil. The plants were cultivated in an artificial climate chamber (day / night temperature 25℃ / 20℃, light 12h / 12h, relative humidity 65%), and healthy plants with 5-6 true leaves and a seedling age of 30 days were selected.

[0302] Reagents and equipment:

[0303] Phosphate buffer (PBS, 0.01M, pH 7.2): NaCl 8.0g, KCl 0.2g, Na2HPO4 1.44g, KH2PO4 0.24g, distilled water 1L;

[0304] Carborundum (600 mesh): Wash with distilled water, dry and sterilize;

[0305] Ultraviolet lamp: Long-wave ultraviolet light (365nm);

[0306] Artificial climate chamber: Temperature control accuracy ±1℃, humidity control range 50-90%.

[0307] Test method:

[0308] Virus activation and extraction:

[0309] Virus activation: Take 0.1g of TMV-infected leaves from a -80℃ freezer; add 2mL of pre-cooled PBS buffer (0.01M, pH 7.2); grind into a homogenate in a pre-cooled mortar; filter with double-layer gauze to obtain crude virus extract.

[0310] Friction inoculation: Select 3 unfolded heart leaves of tobacco, evenly sprinkle carborundum on the leaf surface, dip a cotton swab in crude virus extract and gently rub the leaf surface; rinse the leaf surface with distilled water after inoculation.

[0311] Cultivation and observation: Grafted plants were cultured at 25℃ under 12h light conditions; disease incidence was observed daily under long-wave ultraviolet light; typical diseased leaves were selected as fresh virus sources after 7-10 days.

[0312] Drug treatment and virus inoculation trial

[0313] Experimental design: A completely randomized block design was adopted, with 6 plants per treatment and 3 replicates.

[0314] Chemical treatment: Prepare the mixed solutions according to the following table, and compress the chemical solutions:

[0315]

[0316] Use a handheld sprayer to evenly spray the solution onto both sides of the tobacco leaves until the leaf surface is completely wet but not dripping.

[0317] Virus inoculation was performed 2 hours after drug treatment.

[0318] Virus inoculation: Take fresh diseased leaves and add PBS buffer at a ratio of 1:10 (w / v); grind in an ice bath, filter with gauze to obtain virus inoculation solution; select 3 unfolded leaves from the upper part of each tobacco plant; sprinkle carborundum on the leaf surface, and use a cotton swab to apply 50 μL of virus juice for inoculation; rinse the leaf surface with distilled water after inoculation.

[0319] Disease investigation and data collection

[0320] Investigation period: Continue culturing for 5 days after inoculation, at which time the symptoms are fully manifested.

[0321] Disease severity grading criteria:

[0322] A 5-level grading method is adopted:

[0323] Grade 0: No symptoms on the leaves;

[0324] Grade 1: Individual chlorotic spots appear on the leaves;

[0325] Grade 2: The chlorotic spots enlarge, forming localized mosaic patterns;

[0326] Grade 3: Typical mosaic symptoms, with mild leaf deformities;

[0327] Level 4: Severe mosaic, with leaves noticeably wrinkled and deformed.

[0328] Data recording: Investigate all inoculated leaves for each plant and record the disease level of each leaf.

[0329] Disease index calculation:

[0330] Disease index = [∑(number of diseased leaves at each level × corresponding level)] / (total number of leaves surveyed × highest level) × 100.

[0331] Relative efficacy calculation:

[0332] Relative efficacy (%) = (Control disease index - Treatment disease index) / Control disease index × 100.

[0333] Toxicity index calculation:

[0334] Actual virulence index (ATI) = (Control disease index / Treatment disease index) × 100;

[0335] Theoretical Toxicity Index (TTI) = Furoic Acid TI × Furoic Acid in Mixture % + Ningnanmycin TI × Ningnanmycin in Mixture %

[0336] Cotoxicity coefficient (CTC) = (ATI / TTI) × 100.

[0337] Synergy Evaluation Criteria:

[0338] CTC>120: Synergistic effect;

[0339] 80≤CTC≤120: Additive effect;

[0340] CTC<80: Antagonistic effect.

[0341] Statistical analysis: SPSS 22.0 software was used for statistical analysis. One-way ANOVA and Duncan's new multiple range method were used for multiple comparisons. P < 0.05 was considered statistically significant.

[0342] Disease survey results: Five days after vaccination, the disease manifestations of each treatment are shown in the table below:

[0343] The control effect of furoic acid combined with ningnanmycin on tobacco mosaic virus:

[0344]

[0345] Note: Data are mean ± standard deviation (n=18), and the disease index is the result of the survey 5 days after vaccination.

[0346] Symptom analysis

[0347] Water control: All inoculated leaves showed typical mosaic symptoms, with a disease index of 4.0, and some leaves showed wrinkling and deformity.

[0348] Single-dose treatment:

[0349] Furoic acid treatment: Reduced mosaic symptoms and decreased localized chlorotic spots;

[0350] Treatment with Ningnanmycin significantly suppressed symptoms, with only a few leaves showing slight mosaic patterns.

[0351] Compound processing:

[0352] 1:5 and 10:1 ratios: mildest symptoms, most leaves are asymptomatic or only have grade 1 symptoms;

[0353] Other formulations: Symptoms fall between those of a single dose and the optimal combination.

[0354] Synergy effect evaluation:

[0355] Analysis based on co-toxicity coefficient (CTC):

[0356] Synergistic effect (CTC>120):

[0357] 1:1 ratio: CTC = 139.50;

[0358] 1:5 ratio: CTC=145.30;

[0359] 1:10 ratio: CTC = 123.10;

[0360] 1:20 ratio: CTC=121.60;

[0361] 10:1 ratio: CTC=185.80.

[0362] Additive effect (80≤CTC≤120):

[0363] 5:1 ratio: CTC = 117.40;

[0364] 20:1 ratio: CTC=111.60.

[0365] Optimal ratio:

[0366] 10:1 ratio: CTC highest (185.80), disease index lowest (1.30).

[0367] 1:5 ratio: CTC second highest (145.30), disease index also 1.30.

[0368] Comparison of prevention and control effects:

[0369] Compared with single-dose:

[0370] The relative efficacy of furoic acid as a single agent was 37.50%, with a disease index of 2.50.

[0371] The relative efficacy of a single dose of ningnanmycin was 55.00%, with a disease index of 1.80.

[0372] The optimal combination (10:1) showed a relative efficacy of 67.50% and a disease index of 1.30.

[0373] The combined treatment has a 10-30 percentage point higher efficacy than the single treatment.

[0374] Mixture dependence:

[0375] When the proportion of ningnanmycin increases (1:5, 1:10, 1:20), the prevention and control effect is better;

[0376] When the proportion of furoic acid is too high (5:1, 20:1), the synergistic effect is weakened;

[0377] The 10:1 ratio shows special advantages, possibly due to the optimal synergistic ratio of the two components.

[0378] Dose-response relationship:

[0379] Ningnanmycin dose-effect: Maintaining an appropriate proportion of ningnanmycin in compound formulation is important for synergistic effect; the 1:5 ratio has a higher proportion of ningnanmycin and performs well; however, the 1:10 ratio still has good effect after reducing the amount of ningnanmycin, indicating that there is room for optimization of the ratio.

[0380] Dosage effect of furoic acid: An appropriate ratio of furoic acid can enhance the effect of ningnanmycin; the 10:1 ratio has a higher proportion of furoic acid, showing the strongest synergistic effect; excessive furoic acid (20:1) reduces the synergistic effect.

[0381] Based on the experimental results, the synergistic effect of furoic acid and ningnanmycin may involve the following mechanisms:

[0382] Complementary pathways of action: Ningnanmycin mainly inhibits virus replication; furoic acid may enhance plant disease resistance; the two work synergistically to both inhibit the virus and enhance host resistance.

[0383] Enhanced penetration and translocation: Furoic acid may improve the distribution of the agent in the plant and promote the arrival of ningnanmycin at its site of action.

[0384] Resistance delay: Different mechanisms of action can reduce the risk of viruses developing resistance.

[0385] Significantly enhanced efficacy: The 10:1 ratio of CTC reached 185.80, showing a significant increase in efficacy; the disease index decreased from 1.80-2.50 for a single dose to 1.30.

[0386] Dosage optimization: While achieving better preventive efficacy, it can reduce the dosage of ningnanmycin, thereby reducing costs and potential environmental impact.

[0387] Stable efficacy: Multiple formulations exhibit synergistic effects, providing application flexibility.

[0388] This embodiment demonstrates, through systematic indoor toxicity testing, that:

[0389] Significant synergistic effect: Furoic acid and ningnanmycin showed synergistic effects in multiple ratios, especially at 10:1 and 1:5 ratios.

[0390] Optimal ratio determined:

[0391] 10:1 ratio: CTC=185.80, disease index 1.30, relative efficacy 67.50%;

[0392] 1:5 ratio: CTC=145.30, disease index 1.30, relative efficacy 67.50%.

[0393] Application value: Compared with single-dose ningnanmycin, the 10:1 combination can increase the relative efficacy from 55.00% to 67.50%; reduce the disease index from 1.80 to 1.30, and significantly alleviate symptoms; it can reduce the dosage of ningnanmycin and reduce the risk of resistance.

[0394] Recommended application scheme: When controlling tobacco mosaic virus disease, the preferred ratio is furoic acid: ningnanmycin = 10:1; a 1:5 ratio can be used as an alternative scheme, suitable for different control needs.

[0395] Mechanism implications: The experimental results suggest that furoic acid may synergistically enhance the effects of ningnanmycin by improving plant disease resistance or pesticide distribution.

[0396] Comparative Example 1:

[0397] Experimental objective: To verify whether furoic acid produces a synergistic effect when combined with other common bactericides of the present invention.

[0398] Experimental materials:

[0399] Tested strains: Bacterium tumefaciens of rice (Xoo-2021-03), Fusarium graminearum of wheat (Fg-2021-07).

[0400] Test reagents: furoic acid (prepared in the same way as in Example 1); carbendazim: commercially available, purity ≥98%; azoxystrobin: commercially available, purity ≥95%; validamycin: commercially available, purity ≥90%.

[0401] Test method:

[0402] Experimental design: Three non-inventory bactericides were selected and mixed with furoic acid at a mass ratio of 1:1, with a concentration of 0.16 mg / mL for each.

[0403] Plate confrontation method (rice bacterial blight pathogen): The method is the same as in Example 2.

[0404] Mycelial growth rate method (Fusarium graminearum, the pathogen of wheat blight): The method is the same as in Example 2.

[0405] Experimental results: The antibacterial effects of furoic acid combined with non-inventory bactericides are shown in the table below:

[0406]

[0407] Results analysis:

[0408] No significant synergistic effect: When furoic acid is combined with carbendazim, pyraclostrobin, and jinggangmycin, the CTC values ​​are all in the range of 80-120, showing an additive effect and no synergistic effect.

[0409] Difference in efficacy: The inhibition rate of fungi when combined with pyraclostrobin (55.67%) was lower than that of pyraclostrobin alone (62.35%), indicating a certain negative impact.

[0410] Limitations of formulation: The combination of the bactericides not mentioned in the claims with furoic acid did not show significant synergistic effects, such as those of kasugamycin, cyazofamid, and ningnanmycin.

[0411] Comparative Example 2:

[0412] Experimental objective: To compare the differences in bactericidal activity of furoic acid obtained from different sources and extraction methods.

[0413] Experimental materials:

[0414] Furoic acid sample:

[0415] Sample A: Furoic acid (purity 96.8%) extracted from *Bacillus thunbergii* according to the method in Example 1.

[0416] Sample B: Furoic acid (purity 98.3%) chemically synthesized according to the method of Example 1;

[0417] Sample C: Commercially available chemically synthesized furoic acid (Sigma-Aldrich, purity ≥99%).

[0418] Sample D: Furoic acid (purity 92.5%) extracted from other fungi (Aspergillus niger).

[0419] Experimental method: The inhibitory effect of each sample on rice bacterial blight and wheat scab was determined using the method in Example 2, with a concentration of 0.16 mg / mL.

[0420] Experimental results: The following table compares the bactericidal activities of furoic acid from different sources:

[0421]

[0422] Note: Relative activity is calculated with the activity of sample B as 100%.

[0423] Results analysis:

[0424] Method equivalence: The furoic acid extracted by *Bacillus thuringiensis* (sample A) has comparable activity to the furoic acid chemically synthesized in this invention (sample B) (relative activity 98.9% vs 100%).

[0425] Effect of purity: Commercially available high-purity furoic acid (sample C) has slightly lower activity, which may be related to the crystal morphology or the type of impurities.

[0426] Source specificity: The activity of furoic acid extracted from Aspergillus niger (sample D) was significantly lower (82.3%), indicating that there may be differences in activity between furoic acid from different sources.

[0427] Advantages of this invention: The furoic acid prepared by the method of this invention has the best bactericidal activity.

[0428] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. Furoic acid, characterized in that, The chemical structural formula of the furoic acid is: With a purity ≥98%, it is prepared by the following method: furfural, sodium hydroxide aqueous solution and copper oxide are mixed, oxygen is introduced at 50 to 60°C and the mixture is stirred for 4 to 6 hours, followed by extraction, acidification, recrystallization and drying to obtain furoic acid.

2. The furoic acid according to claim 1, characterized in that, The source is obtained by extraction, isolation, or chemical synthesis from *Bacillus thuringiensis*.

3. A bactericidal composition suitable for use with furoic acid as described in any one of claims 1 to 2, characterized in that, It includes furoic acid and one of kasugamycin, cyazofamid, or ningnanmycin as active pharmaceutical ingredients, as well as pesticide-acceptable adjuvants.

4. The bactericidal composition according to claim 3, characterized in that, The mass ratio of furoic acid to kasugamycin is 1:10 to 10:1, the mass ratio of furoic acid to cyazofamid is 10:1, and the mass ratio of furoic acid to ningnanmycin is 1:5 to 10:

1.

5. The bactericidal composition according to claim 3, characterized in that, The mass ratio of furoic acid to kasugamycin is 1:5, 1:1, 1:10, 5:1, 10:1, 1:20, or 20:1; the mass ratio of furoic acid to cyazofamid is 1:1, 1:5, 1:10, 1:20, 5:1, 10:1, or 20:1; the mass ratio of furoic acid to ningnanmycin is 1:1, 1:5, 1:10, 1:20, 5:1, 10:1, or 20:

1.

6. The preparation of a bactericidal composition, applicable to the bactericidal composition according to any one of claims 3 to 5, characterized in that, Furoic acid is mixed with one of kasugamycin, cyazofamid, or ningnanmycin in a specified mass ratio, and then pesticide-acceptable auxiliary ingredients are added. After mixing evenly, an applicable pesticide formulation is prepared.

7. The preparation of the bactericidal composition according to claim 6, characterized in that, The auxiliary components include one or more of the following: carrier, solvent, surfactant, stabilizer, and dispersant.

8. The use of the bactericidal composition according to any one of claims 3 to 5 in the prevention and control of plant diseases.

9. The application of the bactericidal composition according to claim 8, characterized in that, The plant diseases mentioned include rice bacterial blight, wheat scab, or tobacco mosaic virus.

10. The application of the bactericidal composition according to claim 8, characterized in that, The bactericidal composition is applied to the plant surface by spraying, smearing or dipping.

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