Bactericidal composition for preventing and treating tobacco bacterial wilt and application thereof

Abstract

The invention provides a bactericidal composition for preventing and treating tobacco bacterial wilt and application of the bactericidal composition, and belongs to the technical field of pesticides. The bactericidal composition comprises the following components: a rheum officinale extract and a black fungus extract in a mass ratio of (0.5-10): (0.5-10). The rhubarb extract and the black fungus extract in different mass ratios are compounded to obtain the bactericidal composition, and the antibacterial performance of the bactericidal composition on Ralstonia solanacearum is researched. Results show that the bactericidal composition in a specific proportion shows a significant synergistic antibacterial effect, and the inhibitory effect of the bactericidal composition on the growth of pathogenic bacteria is significantly superior to that of a single component, indicating that the bactericidal composition has outstanding prevention and control potential on tobacco bacterial wilt caused by Ralstonia solanacearum. A promising candidate scheme is provided for developing a green prevention and control strategy of the tobacco bacterial wilt, and the method has important significance for guaranteeing the safety production of tobacco.

Description

Technical Field

[0001] This invention belongs to the field of pesticide technology, and in particular relates to a fungicide composition for controlling tobacco bacterial wilt and its application. Background Technology

[0002] Plant bacterial pathogens, second only to fungi as the leading cause of plant pathogens, have become a significant biological stressor affecting agricultural production. These diseases are caused by more than 40 genera of pathogenic bacteria, including *Pseudomonas*, *Ralstonia*, and *Xanthomonas*, exhibiting characteristics of outbreaks, epidemics, and devastation in plants and various crops. They often lead to tissue necrosis, vascular system dysfunction, and even overall plant death, resulting in severe yield reductions. Statistics show that the average annual affected area of ​​bacterial diseases reaches approximately 8 million hectares, making effective control a core issue for ensuring agricultural production and food security. Research data shows that bacterial wilt in tobacco is particularly severe in tropical and subtropical regions, with a field incidence rate typically ranging from 10% to 30%, and sometimes exceeding 80%, leading to widespread plant death and a very high risk of total crop failure. Bacterial leaf streak and bacterial blight in rice typically affect 330,000 to 660,000 hectares annually, causing yield reductions of approximately 10%. Bacterial blight in soybeans causes about 20% yield loss. Black shank in potatoes can cause an incidence rate of 40% to 50%, resulting in yield reductions of about 20%. Severe bacterial angular leaf spot in cucumbers can lead to seedling destruction rates exceeding 30%. Furthermore, long-term reliance on chemical control has led to a series of prominent problems, including increased pathogen resistance, pesticide residues, and environmental pollution, necessitating the development of new, efficient, and environmentally friendly control methods.

[0003] Bio-derived fungicides are a class of naturally occurring active substances derived directly from nature. They are effective and degrade in the natural environment, making them environmentally friendly pesticides. The main varieties are divided into two categories: microbial pesticides (such as bacteriophages and Bacillus subtilis) and plant-derived pesticides (mostly extracted from traditional Chinese medicinal herbs). Bio-derived fungicides have attracted much attention and favor due to their high specificity, abundant resources, low resistance to pesticide resistance, strong compatibility, and multifunctionality. As of the end of 2023, biopesticides accounted for 90% of the market, with over 80% being microbial pesticides, making them the main force of innovation in the industry, and their research and development are explicitly supported by national policies. However, while developing rapidly, this field still faces many challenges, such as insufficient timeliness of control, high research and development difficulty, limited large-scale production, strong environmental dependence, and poor storage stability. These problems restrict the widespread application of bio-derived fungicides and urgently need to be addressed through technological innovation and process optimization.

[0004] Tobacco bacterial wilt is caused by Ralstonia solanacearum (Ralstonia solanacearum). Ralstonia solanacearumBiological fungus (BNF) is a devastating soil-borne vascular disease caused by the growth of tobacco plants. It is characterized by rapid onset, widespread transmission, and difficulty in control, and is often referred to as "plant cancer." This disease primarily damages the vascular system of tobacco plants, leading to wilting, browning of vascular tissue, and overall death. In severely affected areas, it can cause yield losses exceeding 60%, posing a serious threat to the tobacco industry. Currently, chemical control remains the dominant strategy, with copper-based pesticides and antibiotics widely used in production. However, long-term reliance on chemical pesticides has led to significantly increased resistance in pathogens, resulting in a continuous decline in control efficacy and secondary problems such as pesticide residues and environmental pollution. Although breeding resistant varieties is an important control approach, its application is limited by bottlenecks such as poor resistance stability and narrow regional adaptability. Therefore, developing novel control methods with novel mechanisms of action and good environmental compatibility, especially constructing a green integrated pest management system centered on biological control, has become a key research direction for the management of bacterial diseases. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a bactericidal composition for the prevention and control of tobacco bacterial wilt and its application. This invention uses rhubarb extract and black fungus extract in different mass ratios to obtain a bactericidal composition. This composition exhibits a significant synergistic antibacterial effect and has outstanding control potential against tobacco bacterial wilt caused by *Ralstonia solanacearum*. This invention provides a promising candidate for developing green control strategies for tobacco bacterial wilt and is of great significance for ensuring safe tobacco production.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a bactericidal composition for preventing and controlling tobacco bacterial wilt, comprising the following components in the following mass ratio: rhubarb extract: black fungus extract in a ratio of 0.5~10:0.5~10.

[0007] As a preferred embodiment, the components include the following mass ratio: rhubarb extract: black fungus extract in a ratio of 4~9.5:0.8~6.

[0008] As a preferred option, the components include the following mass ratio: rhubarb extract: black fungus extract in a ratio of 6~9:1~4.

[0009] Preferably, the rhubarb extract is a rhubarb alcohol extract, and the black fungus extract is a black fungus alcohol extract.

[0010] Preferably, the preparation method of the rhubarb extract and black fungus extract is as follows: using rhubarb or black fungus as raw material, using ethanol as extraction agent, ultrasonic extraction is performed to obtain an extract, the extract is centrifuged, the supernatant is collected, filtered, the filtrate is obtained, concentrated, a crude extract is obtained, and freeze-dried to obtain rhubarb extract or black fungus extract.

[0011] The present invention provides a method for using the aforementioned bactericidal composition, wherein the bactericidal composition is prepared into a solution and then applied to the roots of the plants for root irrigation.

[0012] Preferably, the concentration of the bactericidal composition is 0.1~0.7 mg / L, and the amount of the bactericidal composition used is 20~40 mL / plant.

[0013] The present invention also provides the application of the bactericidal composition in the prevention and control of bacterial diseases in crops.

[0014] Preferably, the bacterial disease is tobacco bacterial wilt.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) The bactericidal composition of the present invention, after being combined with rhubarb extract and black fungus extract, has a synergistic effect on the target pathogen of tobacco bacterial wilt, especially the 8:2 ratio, which significantly improves the prevention and control effect.

[0016] (2) The two active ingredients in the bactericidal composition of the present invention have different mechanisms of action and can act on different physiological processes of pathogens, effectively reducing the screening pressure of a single agent, thereby delaying the generation and development of drug resistance in pathogens.

[0017] (3) The bactericidal composition of the present invention can significantly reduce the amount of pesticides applied per unit area in the field, thereby reducing pesticide residues and environmental pollution, which meets the requirements of green and sustainable development of tobacco agriculture.

[0018] (4) This invention uses rhubarb extract and black fungus extract in different mass ratios to obtain a bactericidal composition, and studies the antibacterial properties of this composition against Ralstonia solanacearum. The results show that the specific ratio of the compound combination exhibits a significant synergistic antibacterial effect, and its inhibitory effect on the growth of pathogens is significantly better than that of the single component, indicating that the compound combination has outstanding control potential against tobacco bacterial wilt caused by Ralstonia solanacearum. This provides a promising candidate for developing a green control strategy for tobacco bacterial wilt, which is of great significance for ensuring safe tobacco production. Attached Figure Description

[0019] Figure 1 The antibacterial effects of rhubarb extract, black fungus extract and their compound preparations on Ralstonia solanacearum are shown. Column A contains the stock extract (red arrow), negative control (black arrow) and positive control (blue arrow). Columns B and E contain the extracts at dilutions of 2 to 4096 times (the concentrations are arranged clockwise). The black asterisk indicates the dilution factor corresponding to the minimum inhibitory concentration (MIC). Detailed Implementation

[0020] This invention provides a bactericidal composition for preventing and controlling tobacco bacterial wilt, comprising the following components in the following mass ratio: rhubarb extract: black fungus extract in a ratio of 0.5~10:0.5~10, preferably 4~9.5:0.8~6, more preferably 6~9:1~4, and even more preferably 8:2.

[0021] In this invention, the rhubarb extract is a rhubarb ethanol extract, and the black fungus extract is a black fungus ethanol extract. The preparation methods for the rhubarb extract and black fungus extract are as follows: Rhubarb or black fungus is pulverized through an 80-120 mesh sieve and added to a 75%-95% ethanol solution at a material-to-liquid ratio of 1g:20-40mL. The mixture is ultrasonicated to obtain an extract. The extract is centrifuged to obtain a supernatant, filtered to obtain a filtrate, and the filtrate is concentrated to obtain a crude extract. This extract is then freeze-dried to obtain the rhubarb extract or black fungus extract. The ultrasonic power is 200W-400W, preferably 250W-350W, and more preferably 300W. The ultrasonic time is 20-40min / time, preferably 25-35min / time, and more preferably 30min / time.

[0022] The present invention provides a method for using the aforementioned bactericidal composition, wherein the bactericidal composition is prepared into a solution and then applied to the roots of a plant, wherein the plant is tobacco.

[0023] In this invention, the concentration of the bactericidal composition is 0.1~0.7 mg / L, preferably 0.15~0.5 mg / L, and more preferably 0.19~0.4 mg / L; the amount of the bactericidal composition used is 20~40 mL / plant, preferably 25~35 mL / plant, and more preferably 30 mL / plant.

[0024] The present invention also provides the application of the bactericidal composition in the prevention and control of bacterial diseases in crops.

[0025] In this invention, the bacterial disease is tobacco bacterial wilt.

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1: Preparation of rhubarb extract and black fungus extract

[0028] Rhubarb (purchased from Nanning Yishengyuan Traditional Chinese Medicine Development Co., Ltd.) and black fungus (purchased from Chengyou Smart Agriculture Technology Co., Ltd.) were pulverized separately and passed through a 100-mesh sieve to obtain rhubarb powder and black fungus powder, which were used as samples.

[0029] Weigh 100g of sample and add 85% ethanol solution at a material-to-liquid ratio of 1:30 (g / mL), then extract using an ultrasonic-assisted method. The extraction process is repeated three times, each time for 30 minutes. The volume ratio of 85% ethanol is 2:1:1, i.e., 1500mL of 85% ethanol for the first extraction, 750mL for the second, and 750mL for the third. Combine the extracts from the three extractions and centrifuge at 8000 rpm for 15 minutes. Collect the supernatant and filter it through a 1.2μm microporous membrane to remove precipitate, obtaining the filtrate. Subsequently, concentrate the filtrate under reduced pressure to obtain a crude extract. Finally, evenly spread the extract on a lyophilization tray and freeze-dry under vacuum for 12–24 hours to obtain a loose extract powder.

[0030] Example 2: Indoor bioactivity test of the compound preparation of rhubarb extract and black fungus extract

[0031] 1. Test strains and reagents

[0032] Ralstonia solanacearum ( Ralstonia solanacearum The extract was isolated and purified from tobacco plants infected with bacterial wilt disease in Fang County, Shiyan City, Hubei Province in 2021. The rhubarb extract and black fungus extract were prepared using the method described in Example 1.

[0033] 2. Test Methods

[0034] 2.1 Preparation of test bacterial suspension

[0035] In a clean bench, a single colony of *Ralstonia solanacearum* was picked up using a sterile inoculation loop and inoculated into an Erlenmeyer flask containing NA liquid medium. The flask was incubated at 28°C and 1200 rpm with shaking for 24 hours. The bacterial concentration was adjusted to 1×10⁻⁶ cells / ml using sterile NA liquid medium. ~1×1 CFU / mL, store at 4℃ in a sealed container for later use (storage time ≤2h).

[0036] 2.2 Preparation of test samples

[0037] The rhubarb extract and the black fungus extract were uniformly mixed at 11 mass ratios (rhubarb: black fungus = 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10) to obtain the crude extract of the sample. Using a 12.5% (v / v) aqueous solution of dimethyl sulfoxide (DMSO) as the solvent, the 11 crude extracts of the samples were respectively prepared into stock solutions with a concentration of 200 mg / L. The two-fold dilution method was used to perform serial dilutions on each stock solution. The specific operation was as follows: taking the stock solution as the initial concentration, diluting it successively according to the two-fold dilution principle, with the dilution factor range of 2 - 4096 times, and finally obtaining a total of 12 concentration gradient solutions including 100.000, 50.000, 25.000, 12.500, 6.250, 3.125, 1.563, 0.781, 0.391, 0.195, 0.098, 0.049 mg / L. The diluents were stored in the dark at -20 °C for subsequent experiments.

[0038] 2.3 Determination of the antibacterial activity of the sample by the Oxford cup method

[0039] In the ultra-clean workbench, the sterilized Oxford cups were gently placed in the petri dish in a triangular distribution with sterile forceps; subsequently, the NA solid medium was heated and dissolved and kept warm in a water bath at 48 - 52 °C. 60 μL of the bacterial suspension was aspirated and added to 20 mL of the warm NA medium, mixed well and then poured into the petri dish, and left to stand at 26 °C to make the bacteria-containing plate; then, 100 μL of the sample to be tested was accurately added to each Oxford cup, and 100 μL of 12.5% DMSO was set as the negative control and 100 μL of 100 mg / L streptomycin sulfate was set as the positive control; after adding the samples, the petri dish was left to stand horizontally for 1.5 h to facilitate the full diffusion of the samples in the agar.

[0040] 2.4 Detection and data analysis

[0041] After culturing the plate at 28 °C for 28 h, a 0.02 mm vernier caliper was used to measure the diameter of each inhibition zone and take the average value. Each group was set with 10 replicates, and the inhibition rate was calculated. The antibacterial effect is shown in Figure 1 .

[0042] .

[0043] 2.4 Data analysis

[0044] Further, a probit regression model between the logarithm of the concentration and the probit value of the inhibition rate was fitted through SPSS software. Based on this, a toxicity regression equation was established and EC ,

[0043] ,

[0042] , , Figure 1 , ,

[0044] , 50 , was calculated, and the co-toxicity coefficient (CTC) was calculated using the Sun Yunpei method to evaluate the combined action effect. The judgment criteria are as follows: CTC ≤ 80 indicates antagonistic action, 80 < CTC < 120 indicates additive action, and CTC ≥ 120 indicates synergistic action.

[0045] Cotoxicity coefficient (CTC) = ATI / TTI × 100; The measured toxicity index (ATI) of the compound combination is calculated as M / S × 100. In the formula: S is the EC of the standard reagent. 50 The unit is mg / L; M is the EC of the tested compound combination. 50 The unit is mg / L; Theoretical toxicity index (TTI) of compound combination = TIA × P a +TIB×P b ; In the formula: TIA is the toxicity index of drug A; TIB represents the percentage content of agent A in the compound combination, expressed as %; TIB represents the toxicity index of agent B; P b The percentage content of drug B in the compound combination is expressed in units of %.

[0046] 3. Indoor toxicity test

[0047] To systematically evaluate the combined effect of rhubarb extract and black fungus extract, 11 treatments with mass ratios of 1:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:1 were set up for toxicity determination. The results are shown in Table 1.

[0048] Table 1. Indoor bioactivity assays of different combinations against Ralstonia solanacearum

[0049] Table 1 shows that bactericidal compositions obtained by mixing rhubarb extract and black fungus extract at different mass ratios exhibited significant differences in their inhibitory effects on *Ralstonia solanacearum*. Within the ratio range of 9:1 to 6:4, the co-toxicity coefficients of all combinations exceeded 120, confirming a synergistic effect. Particularly noteworthy was the peak co-toxicity coefficient of 1111.9306 at a mass ratio of 8:2, demonstrating the strongest synergistic effect. This indicates a significant positive interaction between the two components at this specific ratio, with their actual antibacterial activity far exceeding the simple sum of their individual single-agent activities. When the mass ratio was 5–4:5–6, the co-toxicity coefficients of the combinations against *Ralstonia solanacearum* were between 80 and 120, exhibiting an additive effect. This suggests that the combinations functioned independently within this range without interference. When the mass ratio was 3–1:7–9, the co-toxicity coefficients of the combinations against *Ralstonia solanacearum* were less than 80, exhibiting an antagonistic effect.

[0050] In summary, rhubarb and black fungus exhibited significant synergistic effects within a certain ratio range, with the 8:2 ratio showing the highest activity and EC50. 50At concentrations as low as 0.38 mg / L, its antibacterial activity is significantly superior to that of any single agent.

[0051] Comparative Example 1: Indoor bioactivity assay of various combinations of rhubarb extract and black fungus extract against *Pseudomonas syringae*.

[0052] The experiment was conducted using the same method as in Example 2, the only difference being that the test bacterium used was *Pseudomonas syringae* (a pathogenic strain of *Pseudomonas syringae* in kiwifruit). Pseudomonas syringae pv. actinidiae (Psa) was isolated and purified from kiwifruit canker diseased plants in Shiyan City, Hubei Province in 2021. The results are shown in Table 2.

[0053] Table 2. Indoor bioactivity assays of different combinations against *Pseudomonas syringae*.

[0054] Table 2 shows that when the mass ratio of rhubarb extract to black fungus extract is 7:3 and 6:4, the co-toxicity coefficients of the compound combination against *Pseudomonas syringae* are 107.9823 and 80.6415, respectively, falling between 80 and 120, indicating an additive effect. This suggests that the compound combination works independently at these ratios without interference. When the mass ratio of rhubarb extract to black fungus extract is 9:1, 8:2, and 5:5–1:9, the co-toxicity coefficients of the compound combination against *Pseudomonas syringae* are all less than 80, indicating an antagonistic effect.

[0055] Therefore, it can be concluded that the inhibitory effect of rhubarb extract and black fungus extract on *Pseudomonas syringae* is mainly antagonistic, with an additive effect only observed in the 7:3 and 6:4 ratio ranges, showing no synergistic effect. Specifically, the EC50-EC50-C ... 50 The concentration was as low as 191.90 mg / L, but its antibacterial activity was still not superior to that of rhubarb alone.

[0056] Comparative Example 2: Indoor bioactivity assay of various combinations of rhubarb extract and black fungus extract against Erwinia.

[0057] The experiment was conducted using the same method as in Example 2, the only difference being that the test bacteria used were Erwinia (Erwinia amyloliquefaciens). Erwinia amylovora (The results were obtained from the Shiyan Academy of Agricultural Sciences in Hubei Province). See Table 3 for the results.

[0058] Table 3. Indoor bioactivity assays of different combinations against Erwinia.

[0059] Table 3 shows that when the mass ratio of rhubarb extract to black fungus extract was 7:3 and 6:4, the co-toxicity coefficients (CTCs) of the compound combinations against Erwinia were 104.5119 and 95.8343, respectively, falling between 80 and 120, indicating an additive effect. The CTCs of other compound combinations were all below 80, showing stable antagonistic effects. Among them, the CTC value was highest at a mass ratio of 7:3, with the highest EC50 value. 50 The concentration was as low as 9.3 mg / L, but its antibacterial activity was still not superior to that of rhubarb alone.

[0060] In summary, the combined composition of rhubarb extract and black fungus extract did not exhibit broad-spectrum antibacterial activity against the various plant pathogens tested. Its synergistic effect showed high specificity and selectivity only against *Ralstonia solanacearum*. This characteristic further highlights the unique application value and targeted nature of this invention in the prevention and control of plant diseases caused by *Ralstonia solanacearum*.

[0061] Comparative Example 3: Indoor bioactivity assay of various combinations of rhubarb extract and shiitake mushroom extract against Ralstonia solanacearum.

[0062] Shiitake mushroom extract was prepared using the method described in Example 1. The experiment was conducted using the same method as in Example 2, except that the compound combination used was replaced with rhubarb extract and black fungus extract instead of rhubarb extract and shiitake mushroom extract. The results are shown in Table 4.

[0063] Table 4. Indoor bioactivity assays of different combinations against Ralstonia solanacearum

[0064] Table 5 shows that the inhibitory effect of the rhubarb extract and shiitake mushroom extract combination on *Ralstonia solanacearum* was generally poor under different mass ratios. When the mass ratios were 9:1 and 8:2, the co-toxicity coefficients (CTCs) against *Erwinia* were 103.5560 and 99.3929, respectively, falling between 80 and 120, indicating an additive effect. The CTCs of other ratio combinations were all below 80, showing stable antagonistic effects. Among these, the CTC value was highest at a mass ratio of 9:1, with the highest EC50 value. 50 The concentration was as low as 3.62 mg / L, comparable to that of rhubarb alone, indicating that no synergistic effect was produced when rhubarb extract and shiitake mushroom extract were combined. In conclusion, the combination of rhubarb extract and shiitake mushroom extract is not suitable for controlling plant diseases caused by *Ralstonia solanacearum*. Therefore, this invention further reveals that the significant synergistic effect produced by the combination of effective active ingredients in rhubarb extract and black fungus extract is highly specific and not universally present in other edible fungi.

[0065] Experimental example: Greenhouse pot experiment

[0066] 1. Test materials

[0067] The tobacco variety tested was Yunyan 87.

[0068] 2. Preparation of the test reagent

[0069] In vitro antibacterial test results showed that ( Figure 1 The minimum inhibitory concentrations (MICs) of rhubarb extract and black fungus extract were 256 and 4 times dilution, respectively. When the two were combined at a mass ratio of 8:2, the MIC of the extract increased to 2048 times dilution, indicating a significant enhancement in antibacterial activity. Based on the optimal antibacterial effect exhibited by this ratio in vitro, this invention combines rhubarb extract and black fungus extract at a mass ratio of 8:2 to obtain a bactericidal composition. The bactericidal composition was then dissolved in 12.5% ​​DMSO to a concentration of 200 mg / L to obtain a stock solution. The stock solution was then diluted at three gradients (low, medium, and high) for subsequent pot experiment control.

[0070] Compound treatment group 1: The compound stock solution was diluted to 2048 times for root irrigation. Compound treatment group 2: The compound stock solution was diluted to 1024 times and used for root irrigation. Compound treatment group 3: The compound stock solution was diluted 512 times and applied as a root drench; Rhubarb single-agent treatment group: Rhubarb extract was obtained using the method in Example 1, and dissolved in 12.5% ​​DMSO to 200 mg / L to obtain rhubarb extract stock solution. The stock solution was diluted 256 times for root irrigation treatment. Black fungus single-agent treatment group: Black fungus extract was obtained using the method in Example 1, and dissolved in 12.5% ​​DMSO to 200 mg / L to obtain the black fungus extract stock solution. The stock solution was diluted 4 times for root irrigation treatment. Blank control group: sprayed with only an equal amount of water; Positive control group: Spray with 72% streptomycin sulfate wettable powder diluted 1500 times for root irrigation (a commonly used antibiotic for the prevention and control of bacterial wilt).

[0071] 2.2 Determination of the control efficacy of compound agents (fungicide composition) against tobacco bacterial wilt

[0072] Prepare seedling cups of the same size and material (13cm×14cm). The potting soil should be mainly peat moss. When the tobacco variety Yunyan 87 has been cultivated for about 45 days, transplant healthy and uniformly growing tobacco seedlings into the seedling cups. Transplant one tobacco seedling into each seedling cup and manage it as usual.

[0073] Tobacco seedlings of uniform growth were selected for root drenching treatment. The experiment included six treatments, each replicated three times, with ten seedlings per replicate. Each seedling was drenched with 30 mL of the solution, with water as a blank control and 72% streptomycin sulfate as a positive control. Forty-eight hours after root drenching, *Ralstonia solanacearum* was inoculated using the root-wound inoculation method, with 20 mL of a 1×10⁻⁶ concentration injected into the roots of each seedling. A bacterial suspension of CFU / mL was administered. A second root drenching treatment was carried out on the 5th day after inoculation. Disease occurrence was investigated on the 7th, 15th, and 23rd days after inoculation. The incidence rate, disease index, and control effect were calculated using the following formulas. The disease grading of tobacco bacterial wilt was carried out in accordance with GB / T 23222-2008 "Grading and Investigation Methods for Tobacco Diseases and Pests", with details shown in Table 5. Disease began to appear on the 7th day after inoculation, and the control effect of the compound agent on bacterial wilt is shown in Table 6.

[0074] Disease index = ∑ (number of diseased plants at each level × disease 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.

[0075] Table 5 Disease severity criteria for bacterial wilt

[0076] Table 6. Effects of different treatments on the control of tobacco bacterial wilt in potted plants.

[0077] Note: Different lowercase letters in the same column indicate significant differences at the p=0.05 level.

[0078] According to the disease survey results on days 7, 15, and 23 post-inoculation (Table 6), all tested agents showed some control effect against tobacco bacterial wilt, but the control effects varied significantly among treatments. In the three surveys, treatment groups 2 and 3 of the compound agent showed the most outstanding control effects, with disease indices below 16% and control effects exceeding 79%, with no significant difference between the two groups, and significantly better than the other treatments. In contrast, treatment group 1 of the compound agent showed a significantly reduced control effect, with a disease index above 37% and a control effect below 63%. In conclusion, diluting the rhubarb-black fungus compound agent to a concentration range of 512–1024 times provides the best control effect against tobacco bacterial wilt.

[0079] This invention demonstrates that a specific volume ratio of rhubarb extract and black fungus extract exhibits a significant synergistic antibacterial effect against *Ralstonia solanacearum*, the pathogen causing bacterial wilt of tobacco. This compound formulation significantly enhances the control of this major bacterial disease, which is of great significance for ensuring safe tobacco crop production, reducing reliance on chemical pesticides, and promoting green and sustainable agricultural development.

[0080] 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. A bactericidal composition for preventing and controlling tobacco bacterial wilt, characterized in that, The components include the following mass ratio: rhubarb extract: black fungus extract at 0.5~10:0.5~10.

2. The bactericidal composition according to claim 1, characterized in that, The components include the following mass ratio: rhubarb extract: black fungus extract in a ratio of 4~9.5:0.8~6.

3. The bactericidal composition according to claim 1, characterized in that, The components include the following mass ratio: rhubarb extract: black fungus extract in a ratio of 6~9:1~4.

4. The bactericidal composition according to any one of claims 1 to 3, characterized in that, The rhubarb extract is an alcoholic extract of rhubarb, and the black fungus extract is an alcoholic extract of black fungus.

5. The bactericidal composition according to claim 4, characterized in that, The preparation methods of the rhubarb extract and black fungus extract are as follows: using rhubarb or black fungus as raw materials, using ethanol as the extractant, ultrasonic extraction is performed to obtain an extract, the extract is centrifuged, the supernatant is collected, filtered, the filtrate is concentrated, a crude extract is obtained, and freeze-dried to obtain the rhubarb extract or black fungus extract.

6. The method of using the bactericidal composition according to any one of claims 1 to 5, characterized in that, The bactericidal composition was prepared into a solution and then applied to the roots of the plants.

7. The method of use according to claim 6, characterized in that, The concentration of the bactericidal composition is 0.1~0.7 mg / L, and the amount of the bactericidal composition used is 20~40 mL / plant.

8. The use of the bactericidal composition according to any one of claims 1 to 5 in the prevention and control of bacterial diseases of crops.

9. The application according to claim 8, characterized in that, The bacterial disease mentioned is tobacco bacterial wilt.

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