Plant-derived fungicide combination for preventing and treating brassica nigra black rot

Abstract

This invention relates to the field of biological pesticides, specifically disclosing a plant-derived fungicide combination for controlling black rot in broccoli. Its active ingredients are a compound of 0.2% benzoyl mycotoxin and 0.5% matrine. Under the technical solution of this invention, indoor fungicide toxicity tests show that both 0.2% benzoyl mycotoxin and 0.5% matrine have strong inhibitory effects on black rot in broccoli. When the mass ratio of 0.2% benzoyl mycotoxin to 0.5% matrine is 3:7 to 6:4, the co-toxicity coefficient against the broccoli black rot pathogen Xcc is greater than 120, exhibiting a synergistic effect. Indoor pot experiments show that when the mass ratio of 0.2% benzoyl mycotoxin to 0.5% matrine is 3:7 to 6:4, the control effect is significantly higher than that of 200 mg / L 15% agricultural streptomycin.

Description

Technical Field

[0001] This invention relates to the field of biological pesticides, specifically to a combination of plant-derived fungicides for the prevention and control of black rot in broccoli. Background Technology

[0002] Broccoli (Brassica oleracea), also known as green cauliflower, is a variety of cabbage belonging to the Brassicaceae family. Its edible head consists of multiple fleshy pedicels and underdeveloped flower buds. In recent years, with increased intensive cultivation and longer planting periods, black rot disease in broccoli has become increasingly severe, posing a significant challenge to the development of the broccoli industry.

[0003] Broccoli black rot is a bacterial disease caused by Xanthomonas campestris Pv. Campestris (Xcc). Its main transmission routes include seeds, soil, and diseased plant debris. The pathogen invades leaves through stomata, water pores, or wounds, causing leaf infection, which then spreads to the stems and bulbs, leading to plant wilting and bulb rot. Furthermore, black rot can induce other diseases such as soft rot and sclerotinia rot, causing severe decay of plant tissues and significantly impacting broccoli yield and quality, resulting in substantial economic losses for the crop.

[0004] Rainy and humid environments are particularly prone to disease outbreaks. Continuous cropping, excessive nitrogen fertilization, excessive vegetative growth, and pests all exacerbate black rot in broccoli. Current prevention and control measures primarily focus on strictly implementing sterilization and disinfection of seeds and seedling growing media, strengthening cultivation management, and implementing crop rotation. Furthermore, effective control of black rot mainly relies on chemical methods, such as the use of copper hydroxide, thiamethoxam, metalaxyl, and streptomycin. However, long-term pesticide use leads to increased pathogen resistance. To enhance control, the frequency and dosage of pesticides are often increased, resulting in higher pesticide residues in broccoli, posing a risk to environmental pollution and food safety. Plant-derived fungicides are active ingredients extracted from plants that inhibit plant pathogens and are characterized by high efficiency, low toxicity, and easy degradation in nature. There are many plant-derived fungicides on the market, such as matrine, eugenol, allicin, and kasugamycin, which have been widely used and achieved significant results in the prevention and control of various crop diseases. However, no relevant research reports have been found on the prevention and control of black rot in broccoli. Summary of the Invention

[0005] This invention provides a combination of plant-derived fungicides for the prevention and control of black rot in broccoli, offering a more effective and less toxic method for controlling black rot in broccoli and increasing yield.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A plant-derived fungicide combination for controlling black rot in broccoli, the active ingredients of which are composed of 0.2% benzoyl mycotoxin and 0.5% matrine.

[0008] Preferably, the mass ratio of the 0.2% benzoyl mycelium to the 0.5% matrine is 3:7 to 6:4.

[0009] Furthermore, as the most preferred option, the mass ratio of the 0.2% benzoyl mycelium to the 0.5% matrine is 3:7.

[0010] The technical effects achieved by this invention are as follows: Indoor fungicide toxicity tests show that both 0.2% benzoyl mycelium and 0.5% matrine, two plant-derived fungicides, have strong inhibitory effects on black rot of broccoli; when the mass ratio of 0.2% benzoyl mycelium to 0.5% is 3:7~6:4, the co-toxicity coefficient against the broccoli black rot pathogen Xcc is greater than 120, showing a synergistic effect. Among them, when the mass ratio of benzoyl mycelium to matrine is 3:7, a significant synergistic effect is shown; Indoor pot experiments show that when the mass ratio of 0.2% benzoyl mycelium to 0.5% matrine is 3:7~6:4, the control effect is much higher than that of 200 mg / L 15% agricultural streptomycin. Detailed Implementation

[0011] The present invention will be further illustrated below with reference to embodiments. These embodiments are merely illustrative examples and do not limit the scope of the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0012] Example 1: Antibacterial activity of different plant-derived fungicides

[0013] 1. Experimental Methods

[0014] 1.1 Test strains

[0015] The pathogen Xcc, which causes black rot in broccoli, was isolated by our research group.

[0016] 1.2 Tested plant-derived fungicides

[0017] The test reagents and related information are shown in Table 1.

[0018] The test reagent was diluted with a 0.1% Tween-80 aqueous solution to prepare a single-agent stock solution.

[0019] Table 1. Test reagents, manufacturers, and concentrations

[0020]

[0021] 1.3Xcc activation and colony counting

[0022] Xcc was streaked onto potato dextrose agar (PDA) and incubated at 30°C for 1-2 days until colonies were established. In a sterile test tube, 2 ml of LB medium was added, and a single colony was picked and placed into the LB medium. The mixture was then incubated at 30°C for 10 hours using a shaker at 110 rpm. The bacterial suspension was then diluted to 10⁻¹⁰ using a serial dilution method. -8 Take 10 -6 -10 -8 Spread 10 μl of bacterial suspension from the corresponding concentration test tubes onto LB agar medium, incubate at 30 ℃ for 16-18 h, and then calculate the colony count.

[0023] 1.4 Determination of the inhibitory effect of a single agent on Xcc growth

[0024] The Oxford cup inhibition zone method was used to determine the antibacterial activity. Sterile PDA medium was heated to melt, cooled to approximately 55–50°C, poured into petri dishes, and after solidification and cooling, 50 μl of a 1.2 × 10⁻⁶ solution was added. 7 The pathogen was tested at CFU / ml. Four Oxford cups were inserted into the culture medium at equal intervals, with 250 μl of plant-derived fungicide added to each cup. Sterile water was used as a blank control. Three replicates were set up for each concentration of each fungicide. The cups were incubated at 30 ℃ in the dark until the control colony diameter reached 2 / 3 to 3 / 4 of the culture dish diameter. The diameter of each colony was measured using a ruler, employing a cross-sectional method, and the average value was calculated to determine the Xcc growth inhibition rate. The mycelial growth inhibition rate was converted to a biostatistical probability value (y), and the fungicide concentration was converted to a logarithm (x). A linear regression equation was obtained using the logarithm-probability regression method, and the correlation coefficient and EC of Xcc under the tested fungicide treatment were calculated. 50 The formula for calculating the growth inhibition rate of Xcc is: Xcc growth inhibition rate = [(colony diameter of pathogen in control group - colony diameter of pathogen in treatment group) / colony diameter of pathogen in control group] × 100%.

[0025] 2. Experimental Results

[0026] The inhibitory effects of five fungicides on Xcc growth are shown in Table 2. Among them, benzoyl mycelium showed the strongest inhibitory effect on Xcc growth. When the mass concentration of benzoyl mycelium was 10 mg / L, its inhibitory effect on Xcc was the lowest at 40.02%, and when the mass concentration of benzoyl mycelium was 80 mg / L, the inhibitory effect was the highest at 68.54%. Matrine was less effective, with the highest inhibitory effect on Xcc at 67.42%. Kasugamycin had the worst inhibitory effect, with an inhibitory rate of only 39.20% on Xcc growth when its mass concentration was 80 mg / L.

[0027] The toxicity regression equations, correlation coefficients, and EC50 of five fungicides to Xcc were calculated using standard curves. 50 The results are shown in Table 3. The correlation coefficients of the toxicity regression equations for the five fungicides were all higher than 0.96, indicating a positive correlation between fungicide concentration and the inhibitory effect of Xcc. Among them, the EC50 of benzoyl propiconazole was the highest. 50 The concentration was 22.18 mg / L, the lowest among other fungicides, indicating that benzoylprop-ethyl had the best inhibitory effect on Xcc growth; matrine had the second best antibacterial effect, with an EC50 of 22.18 mg / L. 50 The concentration was 39.71 mg / L. The EC50 of five fungicides against Xcc was referenced. 50 It can be seen that the inhibitory effect of the active ingredients of the five fungicides on Xcc from strong to weak is as follows: benzyl benzoate > matrine > osthol > eugenol > kasugamycin.

[0028] Table 2. Inhibitory effects of five plant-derived fungicides on Xcc growth

[0029]

[0030] Note: Different lowercase letters in the same column of data indicate the significance of differences between different treatment groups at the 0.05 level. The same applies below.

[0031] Table 3. Toxicity analysis of five plant-derived fungicides against Xcc

[0032]

[0033] Example 2 Screening of compound formulations of plant-derived fungicides

[0034] 1. Experimental Methods

[0035] In the preliminary experiment, based on the toxicity test results of the above five fungicides and referring to the different mechanisms of action of single agents, EC formulations of two single agents, 0.2% benomyl and 0.5% matrine, were prepared. 50Solutions were mixed in different volume ratios of 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0. The growth of Xcc in plates containing these different compound formulations was observed, and the average inhibition rate was calculated (results are shown in Table 4). Based on the preliminary experimental results, 0.2% benzylpyridinium and 0.5% matrine were compounded in mass ratios of 3:7, 4:6, 5:5, 6:4, and 7:3. The concentrations of the experimental mixed agents were 10, 20, 40, 60, and 80 mg / L. The inhibition zone method was used to determine the antibacterial activity; the specific method is described in Example 1. The diameter of the inhibition zone was measured, a standard curve was plotted, and the regression equation, correlation coefficient, and EC50 of the compound agents for different treatment groups on Xcc were calculated. 50 The results were used to evaluate the combined toxicity of the two agents based on the Co-toxicity Coefficient (CTC) method proposed by Sun Yunpei et al. A CTC less than 80 indicates an antagonistic effect, greater than 120 indicates a synergistic effect, greater than 200 indicates a significant synergistic effect, and between 80 and 120 indicates an additive effect. The Co-toxicity Coefficient was calculated using the following formula:

[0036]

[0037]

[0038] Theoretical Toxicity Index (TTI) of a Mixture = Toxicity Index of Agent A × Proportion of Agent A in the Mixture (%) + Toxicity Index of Agent B × Proportion of Agent B in the Mixture (%)

[0039]

[0040] In this experiment, matrine was used as the standard reagent.

[0041] 2. Experimental Results

[0042] The toxicity test results of the above five fungicides showed that benzoyl propiconazole and matrine had good inhibitory effects on Xcc. Benzoyl propiconazole achieves its fungicidal purpose by destroying the cell wall membrane system of plant pathogens and interfering with cell metabolism; matrine has good inhibitory effects on both bacteria and fungi. Based on the above reasons, this experiment further conducted a compound experiment of benzoyl propiconazole and matrine to clarify whether there is a synergistic effect between the two. The inhibitory effects of different compound ratios on Xcc growth are shown in Table 5. When 0.2% benzoyl propiconazole and 0.5% matrine are compounded at a mass ratio of 3:7 to 7:3, the inhibitory effect on Xcc is obvious. In addition, when the mass ratio of 0.2% benzoyl propiconazole and 0.5% matrine is 3:7 or 6:4, the toxicity to Xcc is stronger, and its EC50 is higher. 50The concentrations were 10.71 and 10.79 mg / L, respectively (Table 6). Further calculations of the co-toxicity coefficients (CTC) of different combinations of the two fungicides were performed. Table 6 shows that when the mass ratio of 0.2% benzylpyrrolidone to 0.5% matrine was 3:7 to 6:4, both exhibited synergistic effects, with the synergistic effect against Xcc being strongest at a ratio of 3:7.

[0043] Table 4. Different proportions of benzo[a]pyrethrum EC in the preliminary experiment 50 Dosage and matrine EC 50 Average inhibition rate of Xcc by dose

[0044]

[0045] Table 5. Inhibitory effect of compound agents on Xcc growth

[0046]

[0047] Table 6. Toxicity determination of compound drugs against Xcc

[0048]

[0049] Example 3: Pot experiment on black rot in broccoli

[0050] 1. Experimental Methods

[0051] The pot experiment to assess the protective effect of the agent against black rot in broccoli was conducted in a greenhouse. Xcc was inoculated into LB liquid medium and cultured at 30°C with shaking for 3 hours. The bacterial suspension was then diluted with physiological saline to a concentration of 3.9 × 10⁻⁶. 7 CFU / mL, potted broccoli seedlings with uniform growth at the two-leaf-one-heart stage were selected, and 20 μL of bacterial suspension was injected into the base of the seedling stem. Different concentrations of azoxystrobin and matrine compound were sprayed onto the roots and leaves of the broccoli seedlings using a constant-volume sprayer. The dosage was determined by wetting both sides of the leaves without dripping, with approximately 300 mL sprayed per plant. The control group was treated with sterile physiological saline, and 15% agricultural streptomycin wettable powder, a fungicide for controlling broccoli black rot, was used as a commercial fungicide control. Each treatment was replicated three times. The treated broccoli seedlings were cultured in a greenhouse (room temperature 24±1℃, relative humidity 90%, L / D = 12 h / 12 ​​h), and the experimental results were investigated after 7 days of culture.

[0052] Disease grading standards:

[0053] Grade 0: No lesions;

[0054] Grade 1: The lesion area accounts for less than 5% of the total leaf area;

[0055] Grade 3: Lesions cover 6%-10% of the total leaf area;

[0056] Level 5: Lesions cover 11%-25% of the total leaf area;

[0057] Grade 7: Lesions cover 26%-50% of the total leaf area;

[0058] Level 9: The lesion area accounts for more than 50% of the total leaf area.

[0059] The disease severity index and prevention efficacy are calculated based on the grading standards. The calculation formula is as follows:

[0060]

[0061]

[0062] 2. Experimental Results

[0063] As shown in Table 7, at a dosage of 800 mg / L, the control efficacy of 0.2% benzoyl mycelium and 0.5% matrine in a mass ratio of 3:7 to 6:4 was significantly higher than that of 200 mg / L 15% agricultural streptomycin (70.25%). In particular, the control efficacy was best when the mass ratio of benzoyl mycelium and matrine was 3:7, with a control efficacy of 90.31%.

[0064] Table 7. Control efficacy of the tested agents against black rot in broccoli

[0065]

[0066] In summary, the results of indoor fungicide toxicity tests showed that both 0.2% benzoyl mycelium and 0.5% matrine, two plant-derived fungicides, had strong inhibitory effects on black rot of broccoli. When the mass ratio of 0.2% benzoyl mycelium to 0.5% matrine was 3:7 to 6:4, the co-toxicity coefficients against the broccoli black rot pathogen Xcc were all greater than 120, indicating a synergistic effect. Among them, when the mass ratio of benzoyl mycelium to matrine was 3:7, a significant synergistic effect was observed. Indoor pot experiments showed that when the mass ratio of benzoyl mycelium to matrine was 3:7 to 6:4, the control effect was much higher than that of 200 mg / L 15% agricultural streptomycin (70.25%).

[0067] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent transformations made using the content of the present invention under the concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. The use of a plant-derived fungicide combination in the control of black rot of broccoli, characterized in that, The active ingredient of the plant-derived fungicide combination is composed of 0.2% benzoyl mycelium and 0.5% matrine. The pathogen of black rot is Xanthomonas Campestris Pv. Campestris, and the mass ratio of 0.2% benzoyl mycelium to 0.5% matrine is 3:7 to 6:

4.

2. Use according to claim 1, characterized in that, The mass ratio of 0.2% benzyl ketone to 0.5% matrine is 3:7.

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