Biological control preparation for inhibiting growth of fusarium graminearum and controlling wheat scab as well as preparation method and application of biological control preparation
By scientifically combining various probiotics and plant active ingredients, optimizing the extraction process, and constructing a multi-level, multi-target synergistic prevention and control system, the instability and drug resistance problems of existing biocontrol agents in the control of wheat scab have been solved, achieving efficient, safe, and sustainable disease control.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
- Filing Date
- 2026-01-28
- Publication Date
- 2026-05-05
AI Technical Summary
Existing biocontrol agents are not very effective in controlling wheat scab, have insufficient antibacterial activity, and the long-term use of chemical pesticides has led to resistance and environmental pressure. There is a lack of effective strategies to block the synthesis and accumulation of Fusarium graminearum toxin.
This product scientifically combines various probiotics, such as Myxococcus orange, Pseudomonas aeruginosa, Bacillus pumilus, and Penicillium micropurpurum, with active plant ingredients such as rose geranium essential oil, thistle extract, and Cnidium monnieri extract. It synergistically inhibits Fusarium graminearum by competing for nutrients, secreting antibacterial metabolites, and disrupting cell membranes. Furthermore, the purity of the active ingredients is improved through optimized extraction processes, and chitin oligosaccharides and pullulan polysaccharides are combined to promote the colonization and activity of beneficial bacteria.
It significantly enhances the inhibitory effect on Fusarium graminearum, reduces toxin synthesis and accumulation, improves the stability and duration of action of the formulation, and achieves green, efficient and multi-dimensional control effect. The field control effect is better than that of chemical agents.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a biological control agent for inhibiting the growth of Fusarium graminearum and preventing wheat scab, as well as its preparation method and application. Background Technology
[0002] Fusarium head blight, a major global fungal disease caused by Fusarium graminearum, severely threatens wheat yield and quality. This pathogen not only infects the ears, directly reducing yield, but also accumulates mycotoxins such as deoxynivalenol in the grains, posing a serious threat to human and animal health. Current control mainly relies on chemical fungicides such as benzimidazoles and sterol demethylation inhibitors; however, long-term single-use has led to increasingly prominent drug resistance in the pathogen, and also poses problems such as pesticide residues and environmental pressure. Developing efficient, green, and sustainable alternative control strategies has become an urgent need to ensure food security and high-quality agricultural development.
[0003] Biological control, due to its environmental friendliness and low likelihood of resistance development, is considered an effective way to solve the aforementioned problems. Current research focuses primarily on the development and application of single antagonistic strains (such as Bacillus and Trichoderma) or single plant extracts. However, the complex field environment means that single-mode biocontrol agents often exhibit bottlenecks such as unstable efficacy, short duration of action, and limited ability to control toxins. Therefore, how to construct a multi-level, multi-target synergistic control system through scientific component design has become a key scientific issue in improving the effectiveness of biological control products.
[0004] In existing technologies, the formulation of biocontrol agents is mostly based on simple combinations of common bacterial species and known plant active ingredients, such as the combination of Bacillus subtilis and flavonoid extracts. Its mechanism of action is relatively simple, and it lacks effective strategies to block the synthesis and accumulation of Fusarium graminearum toxin. Meanwhile, plant extracts obtained through conventional extraction processes (such as hot reflux and impregnation) may have problems such as low extraction rates of active ingredients and high levels of impurities, affecting their compatibility and stability in compound formulations. More importantly, when plant extracts with strong antibacterial activity are directly compounded with live probiotics, the potential antagonistic effects between the two are often overlooked, leading to a decrease in the survival rate of live bacteria and a significant reduction in the overall efficacy of the formulation.
[0005] Therefore, there is an urgent need to develop a novel biocontrol agent. The core of this agent lies in screening atypical probiotics with unique ecological niches and mechanisms of action, and combining them with optimized extraction of specific plant-derived active ingredients. This agent can synergistically exert multiple functions, including inhibiting pathogen growth, interfering with toxin synthesis, inducing plant resistance, and promoting crop growth, thereby achieving green, efficient, and sustainable control of wheat scab. Summary of the Invention
[0006] The purpose of this invention is to provide a biological control agent for inhibiting the growth of Fusarium graminearum and preventing wheat scab, as well as its preparation method and application.
[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a biological control agent for inhibiting the growth of Fusarium graminearum and preventing wheat scab, comprising the following components in parts by weight: 18-23 parts of Myxococcus orange, 10-18 parts of Pseudomonas aeruginosa, 12-20 parts of Bacillus pumilus, 5-10 parts of halophilic Bacillus, 6-12 parts of Penicillium micropurpurum, 3-8 parts of rose geranium essential oil, 5-12 parts of Cirsium japonicum extract, 4-10 parts of Cnidium monnieri extract, 0.4-0.7 parts of matrine, 2-6 parts of licorice extract, 5-10 parts of chitin oligosaccharide, and 5-10 parts of pullulan polysaccharide.
[0008] Preferably, the bacterial counts of *Myxococcus orangeii*, *Pseudomonas aeruginosa*, *Bacillus pumilus*, and *Bacillus halophilus* are all 400-600 million / g; and the spore content of *Penicillium purpureus* is (6-9) × 10⁻⁶. 9 CFU / g.
[0009] Preferably, the preparation method of the small thistle extract is as follows: (1) Take the whole plant of thistle and crush it through a 10-30 mesh sieve; (2) Add the raw material obtained in step (1) to a 60% to 75% ethanol solution at a material-to-liquid ratio of 1:8 to 1:12 (w / v), and perform hot reflux extraction at 70 to 80°C for 1.5 to 2.5 hours each time, for a total of 2 to 3 extractions; (3) Combine the extracts obtained in step (2), filter, concentrate the filtrate under reduced pressure to a relative density of 1.05~1.15, and elute; (4) The eluent obtained in step (3) is concentrated and dried into powder to obtain thistle extract.
[0010] Preferably, the elution method in step (3) is as follows: the concentrate is loaded onto an HPD-100 macroporous adsorption resin column at a flow rate of 1-3 column bed volumes / h, washed sequentially with 3-5 column bed volumes of water, 2-4 column bed volumes of 10%-30% ethanol, and finally eluted with 4-6 column bed volumes of 60%-75% ethanol, and the eluent is collected.
[0011] Preferably, the preparation method of the Cnidium monnieri extract is as follows: a. Crush the fruit of Cnidium monnieri and pass it through a 30-50 mesh sieve; b. Add cyclohexane to the raw material obtained in step a at a material-to-liquid ratio of 1:10 to 1:15 (w / v) and extract at 40 to 60°C for 30 to 60 minutes, repeating the extraction 2 to 3 times. c. Combine the extracts obtained in step b, filter, concentrate the filtrate and dry it into powder to obtain Cnidium monnieri extract.
[0012] Preferably, the extraction process in step b requires ultrasound, with an ultrasound power of 300~500W and a frequency of 22~28kHz.
[0013] Preferably, the licorice extract is prepared by adding licorice to a 70-75% ethanol solution at a material-to-liquid ratio of 1:8 to 1:12 (w / v), refluxing at 80-85°C for 1.5-2.5 hours, extracting 2-3 times, combining the extracts, concentrating under reduced pressure to recover ethanol, water precipitation at low temperature, filtering, concentrating the filtrate, and drying to obtain the licorice extract.
[0014] Preferably, the low-temperature water immersion is performed by standing at 4~6℃ for 20~28 hours.
[0015] This invention provides a method for preparing a biological control agent for inhibiting the growth of Fusarium graminearum and preventing wheat scab. The method involves mixing Myxococcus orange, Pseudomonas aeruginosa, Bacillus pumilus, halophilic Bacillus, Penicillium micropurpurum (all strains are from Shanghai Xuanke Biotechnology Co., Ltd.), rose geranium essential oil, Cirsium japonicum extract, Cnidium monnieri extract, matrine, licorice extract, chitin oligosaccharide, and pullulan polysaccharide to obtain the biological control agent for inhibiting the growth of Fusarium graminearum and preventing wheat scab.
[0016] This invention provides the application of the aforementioned biological control agent for inhibiting the growth of Fusarium graminearum and preventing wheat scab in the preparation of wheat scab control agents.
[0017] The biological control agent disclosed in this invention exhibits a significant synergistic effect through the scientific compatibility of multiple components, and its beneficial effects are mainly reflected in the following aspects: First, the compound microorganisms and plant-derived active ingredients in the formulation form a multiple synergistic mechanism in inhibiting the growth of *Fusarium graminearum*. Probiotics such as *Myxococcus orange*, *Pseudomonas aeruginosa*, *Bacillus pumilus*, and halophilic *Bacillus* can directly inhibit the pathogen by competing for nutrients and ecological niches and secreting antimicrobial metabolites. Simultaneously, *Penicillium micropurpurum*, as a fungal antagonist, can parasitize or competitively inhibit *Fusarium graminearum*. When these microorganisms are combined with plant-derived active substances such as rose geranium essential oil, *Cirsium japonicum* extract, *Cnidium monnieri* extract, matrine, and licorice extract, not only is the antimicrobial spectrum broadened, but also, through different targets (such as cell membrane disruption, enzyme activity inhibition, and mycelial growth interference), they form synergistic and complementary effects, significantly enhancing the inhibitory ability on *Fusarium graminearum* mycelial growth, spore production, and germination, and effectively reducing the synthesis and accumulation of DON toxin.
[0018] Secondly, the optimized extraction processes of each plant extract improved the purity and bioavailability of active ingredients, further enhancing the overall preventative and therapeutic effects of the formulation. The *Cirsium japonicum* extract, after purification with macroporous adsorption resin, accumulated a higher concentration of antibacterial active substances; the *Cnidium monnieri* extract, using cyclohexane combined with ultrasonic extraction, effectively extracted fat-soluble antibacterial components; and the licorice extract, after low-temperature water precipitation to remove impurities, retained more effective components with anti-inflammatory and immunomodulatory effects. These high-purity extracts not only possess antibacterial functions in the compound system but also act as prebiotics, promoting the colonization and activity of beneficial bacteria, forming a positive "bacteria-plant" interaction, thereby improving the stability and duration of efficacy of the formulation.
[0019] Furthermore, chitin oligosaccharides and pullulan, as bio-polysaccharide excipients, not only act as carriers and stabilizers but also stimulate the systemic resistance of wheat. Chitin oligosaccharides can act as elicitors to induce defensive responses in plants, enhancing cell wall strength and antimicrobial protein expression; pullulan, on the other hand, possesses excellent film-forming and slow-release properties, facilitating the adhesion and long-term release of active ingredients on crop surfaces. Together with microorganisms and plant extracts, these two components construct a multi-dimensional control network of "pathogen inhibition—resistance induction—growth promotion," thus maintaining high disease control efficacy even in complex field environments.
[0020] Finally, comparative experiments with the examples and comparative examples fully verified the rationality and synergistic necessity of the formulation's component design. Experimental results showed that Example 5, with its complete formulation, was significantly superior to the comparative examples lacking or replacing any key component in terms of inhibition zone diameter, mycelial inhibition rate, spore germination inhibition, and DON toxin control. Furthermore, its field control effect was comparable to, or even better than, conventional chemical agents. This indicates that the components are not simply additive, but rather achieve deep functional integration and synergistic effects through scientific compatibility, thus providing an efficient, safe, and sustainable solution for the green control of wheat scab. Detailed Implementation
[0021] 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.
[0022] Example 1
[0023] A method for preparing a biological control agent for inhibiting the growth of Fusarium graminearum and preventing wheat scab, specifically comprising mixing 18 parts of Myxococcus orange, 10 parts of Pseudomonas aeruginosa, 12 parts of Bacillus pumilus, 5 parts of halophilic Bacillus, 6 parts of Penicillium purpureus, 3 parts of rose geranium essential oil, 5 parts of Cirsium japonicum extract, 4 parts of Cnidium monnieri extract, 0.4 parts of matrine, 2 parts of licorice extract, 5 parts of chitin oligosaccharide, and 5 parts of pullulan polysaccharide; The bacterial counts of *Myxococcus orangeii*, *Pseudomonas aeruginosa*, *Bacillus pumilus*, and *Bacillus halophilus* were all 400 million / g; the spore content of *Penicillium purpureus* was 6 × 10⁻⁶. 9 CFU / g; The preparation method of thistle extract is as follows: (1) Take the whole plant of thistle and crush it through a 10-mesh sieve; (2) Add the raw material obtained in step (1) to a 60% ethanol solution at a material-to-liquid ratio of 1:8 (w / v) and perform hot reflux extraction at 70°C for 1.5 h each time, for a total of 2 extractions; (3) Combine the extracts obtained in step (2), filter, concentrate the filtrate under reduced pressure to a relative density of 1.05, and elute; load the concentrate onto an HPD-100 macroporous adsorption resin column at a flow rate of 1 column bed volume / h, wash with 3 column bed volumes of water, 2 column bed volumes of 10% ethanol, and finally elute with 4 column bed volumes of 60% ethanol, and collect the eluent.
[0024] (4) The eluent obtained in step (3) is concentrated and dried into powder to obtain thistle extract.
[0025] The preparation method of the Cnidium monnieri extract is as follows: a. Crush the fruit of Cnidium monnieri and pass it through a 30-mesh sieve; b. Add cyclohexane to the raw material obtained in step a at a material-to-liquid ratio of 1:10 (w / v) and extract at 40°C for 30 min, and extract twice; the extraction process requires ultrasound, the ultrasound power is 300W and the frequency is 22kHz. c. Combine the extracts obtained in step b, filter, concentrate the filtrate and dry it into powder to obtain Cnidium monnieri extract; The preparation method of the licorice extract is as follows: licorice is added to a 70% ethanol solution at a material-to-liquid ratio of 1:8 (w / v), and extracted by reflux at 80°C for 1.5 h. The extraction is repeated twice, and the extracts are combined. The ethanol is recovered by vacuum concentration, and the extract is subjected to low-temperature water precipitation (standing at 4°C for 20 h), filtered, and the filtrate is concentrated and dried to obtain the licorice extract.
[0026] Example 2
[0027] A method for preparing a biological control agent for inhibiting the growth of Fusarium graminearum and preventing wheat scab, specifically comprising mixing 23 parts of Myxococcus orange, 18 parts of Pseudomonas aeruginosa, 20 parts of Bacillus pumilus, 10 parts of halophilic Bacillus, 12 parts of Penicillium micropurpurum, 8 parts of rose geranium essential oil, 12 parts of Cirsium japonicum extract, 10 parts of Cnidium monnieri extract, 0.7 parts of matrine, 6 parts of licorice extract, 10 parts of chitin oligosaccharide, and 10 parts of pullulan polysaccharide; The bacterial counts of *Myxococcus orangeii*, *Pseudomonas aeruginosa*, *Bacillus pumilus*, and *Bacillus halophilus* were all 600 million / g; the spore content of *Penicillium purpureus* was 9 × 10⁻⁶. 9 CFU / g; The preparation method of thistle extract is as follows: (1) Take the whole plant of thistle and crush it through a 30-mesh sieve; (2) Add the raw material obtained in step (1) to a 75% ethanol solution at a material-to-liquid ratio of 1:12 (w / v) and perform hot reflux extraction at 80°C for 2.5 h each time, for a total of 2 extractions; (3) Combine the extracts obtained in step (2), filter, concentrate the filtrate under reduced pressure to a relative density of 1.15, and elute; load the concentrate onto an HPD-100 macroporous adsorption resin column at a flow rate of 3 column bed volumes / h, wash with 5 column bed volumes of water, 4 column bed volumes of 30% ethanol, and finally elute with 6 column bed volumes of 75% ethanol, and collect the eluent.
[0028] (4) The eluent obtained in step (3) is concentrated and dried into powder to obtain thistle extract.
[0029] The preparation method of the Cnidium monnieri extract is as follows: a. Crush the fruit of Cnidium monnieri and pass it through a 50-mesh sieve; b. Add cyclohexane to the raw material obtained in step a at a material-to-liquid ratio of 1:15 (w / v) and extract at 60°C for 60 min, repeating the extraction 3 times; the extraction process requires ultrasound, with an ultrasound power of 500W and a frequency of 28kHz. c. Combine the extracts obtained in step b, filter, concentrate the filtrate and dry it into powder to obtain Cnidium monnieri extract; The preparation method of the licorice extract is as follows: licorice is added to a 75% ethanol solution at a material-to-liquid ratio of 1:12 (w / v), and extracted by reflux at 85°C for 2.5 h. The extraction is repeated 3 times. The extracts are combined, and the ethanol is recovered by vacuum concentration. After water precipitation at low temperature (standing at 6°C for 28 h), the extract is filtered, and the filtrate is concentrated and dried to obtain the licorice extract.
[0030] Example 3
[0031] A method for preparing a biological control agent for inhibiting the growth of Fusarium graminearum and preventing wheat scab, specifically comprising mixing 19 parts of Myxococcus orange, 15 parts of Pseudomonas aeruginosa, 14 parts of Bacillus pumilus, 6 parts of halophilic Bacillus, 7 parts of Penicillium micropurpurum, 4 parts of rose geranium essential oil, 6 parts of Cirsium japonicum extract, 5 parts of Cnidium monnieri extract, 0.5 parts of matrine, 3 parts of licorice extract, 6 parts of chitin oligosaccharide, and 6 parts of pullulan polysaccharide; The bacterial counts of *Myxococcus orangeii*, *Pseudomonas aeruginosa*, *Bacillus pumilus*, and *Bacillus halophilus* were all 500 million / g; the spore content of *Penicillium purpureus* was 7 × 10⁻⁶. 9 CFU / g; The preparation method of thistle extract is as follows: (1) Take the whole plant of thistle and crush it through a 15-mesh sieve; (2) The raw material obtained in step (1) was added to a 65% ethanol solution at a material-to-liquid ratio of 1:9 (w / v) and subjected to hot reflux extraction at 72°C for 2 hours each time, for a total of 3 extractions. (3) Combine the extracts obtained in step (2), filter, concentrate the filtrate under reduced pressure to a relative density of 1.08, and elute; load the concentrate onto an HPD-100 macroporous adsorption resin column at a flow rate of 2 column bed volumes / h, wash with 4 column bed volumes of water, 3 column bed volumes of 15% ethanol, and finally elute with 4 column bed volumes of 68% ethanol, and collect the eluent.
[0032] (4) The eluent obtained in step (3) is concentrated and dried into powder to obtain thistle extract.
[0033] The preparation method of the Cnidium monnieri extract is as follows: a. Crush the fruit of Cnidium monnieri and pass it through a 35-mesh sieve; b. Add cyclohexane to the raw material obtained in step a at a material-to-liquid ratio of 1:11 (w / v) and extract at 45°C for 35 min, repeating the extraction 3 times; the extraction process requires ultrasound, with an ultrasound power of 350W and a frequency of 23kHz. c. Combine the extracts obtained in step b, filter, concentrate the filtrate and dry it into powder to obtain Cnidium monnieri extract; The preparation method of the licorice extract is as follows: licorice is added to a 70% ethanol solution at a material-to-liquid ratio of 1:9 (w / v), and extracted by reflux at 81°C for 2 hours. The extraction is repeated 3 times. The extracts are combined, and the ethanol is recovered by vacuum concentration. After water precipitation at low temperature (standing at 5°C for 22 hours), the extract is filtered, and the filtrate is concentrated and dried to obtain the licorice extract.
[0034] Example 4
[0035] A method for preparing a biological control agent for inhibiting the growth of Fusarium graminearum and preventing wheat scab, specifically comprising mixing 22 parts of Myxococcus orange, 17 parts of Pseudomonas aeruginosa, 18 parts of Bacillus pumilus, 9 parts of halophilic Bacillus, 10 parts of Penicillium micropurpurum, 7 parts of rose geranium essential oil, 10 parts of Cirsium japonicum extract, 9 parts of Cnidium monnieri extract, 0.6 parts of matrine, 5 parts of licorice extract, 9 parts of chitin oligosaccharide, and 9 parts of pullulan polysaccharide; The bacterial counts of *Myxococcus orangeii*, *Pseudomonas aeruginosa*, *Bacillus pumilus*, and *Bacillus halophilus* were all 500 million / g; the spore content of *Penicillium purpureus* was 8 × 10⁻⁶. 9 CFU / g; The preparation method of thistle extract is as follows: (1) Take the whole plant of thistle and crush it through a 25-mesh sieve; (2) The raw material obtained in step (1) was added to a 70% ethanol solution at a material-to-liquid ratio of 1:11w / v and subjected to hot reflux extraction at 78°C for 2 hours each time, for a total of 3 extractions. (3) Combine the extracts obtained in step (2), filter, concentrate the filtrate under reduced pressure to a relative density of 1.13, and elute; load the concentrate onto an HPD-100 macroporous adsorption resin column at a flow rate of 3 column bed volumes / h, wash with 5 column bed volumes of water, 3 column bed volumes of 25% ethanol, and finally elute with 6 column bed volumes of 72% ethanol, and collect the eluent.
[0036] (4) The eluent obtained in step (3) is concentrated and dried into powder to obtain thistle extract.
[0037] The preparation method of the Cnidium monnieri extract is as follows: a. Crush the fruit of Cnidium monnieri and pass it through a 45-mesh sieve; b. Add cyclohexane to the raw material obtained in step a at a material-to-liquid ratio of 1:13 (w / v) and extract at 55°C for 55 min, repeating the extraction 3 times; the extraction process requires ultrasound, with an ultrasound power of 450W and a frequency of 27kHz. c. Combine the extracts obtained in step b, filter, concentrate the filtrate and dry it into powder to obtain Cnidium monnieri extract; The preparation method of the licorice extract is as follows: licorice is added to a 73% ethanol solution at a material-to-liquid ratio of 1:11 (w / v), and extracted by reflux at 83°C for 2 hours. The extraction is repeated 3 times. The extracts are combined, and the ethanol is recovered by vacuum concentration. After water precipitation at low temperature (standing at 5°C for 24 hours), the extract is filtered, and the filtrate is concentrated and dried to obtain the licorice extract.
[0038] Example 5
[0039] A method for preparing a biological control agent for inhibiting the growth of Fusarium graminearum and preventing wheat scab, specifically comprising mixing 20 parts of Myxococcus orange, 14 parts of Pseudomonas aeruginosa, 15 parts of Bacillus pumilus, 7 parts of halophilic Bacillus, 10 parts of Penicillium micropurpurum, 5 parts of rose geranium essential oil, 8 parts of Cirsium japonicum extract, 8 parts of Cnidium monnieri extract, 0.6 parts of matrine, 4 parts of licorice extract, 8 parts of chitin oligosaccharide, and 8 parts of pullulan polysaccharide; The bacterial counts of *Myxococcus orangeii*, *Pseudomonas aeruginosa*, *Bacillus pumilus*, and *Bacillus halophilus* were all 500 million / g; the spore content of *Penicillium purpureus* was 7 × 10⁻⁶. 9 CFU / g; The preparation method of thistle extract is as follows: (1) Take the whole plant of thistle and crush it through a 20-mesh sieve; (2) Add the raw material obtained in step (1) to a 65% ethanol solution at a material-to-liquid ratio of 1:10 (w / v) and perform hot reflux extraction at 75°C for 2 hours each time, for a total of 3 extractions; (3) Combine the extracts obtained in step (2), filter, concentrate the filtrate under reduced pressure to a relative density of 1.13, and elute; load the concentrate onto an HPD-100 macroporous adsorption resin column at a flow rate of 2 column bed volumes / h, wash with 4 column bed volumes of water, 3 column bed volumes of 20% ethanol, and finally elute with 5 column bed volumes of 70% ethanol, and collect the eluent.
[0040] (4) The eluent obtained in step (3) is concentrated and dried into powder to obtain thistle extract.
[0041] The preparation method of the Cnidium monnieri extract is as follows: a. Crush the fruit of Cnidium monnieri and pass it through a 40-mesh sieve; b. Add cyclohexane to the raw material obtained in step a at a material-to-liquid ratio of 1:13 w / v and extract at 50°C for 50 min, repeat 3 times; the extraction process requires ultrasound, the ultrasound power is 400W and the frequency is 25kHz. c. Combine the extracts obtained in step b, filter, concentrate the filtrate and dry it into powder to obtain Cnidium monnieri extract; The preparation method of the licorice extract is as follows: licorice is added to a 75% ethanol solution at a material-to-liquid ratio of 1:10 (w / v), and extracted by reflux at 85°C for 2 hours. The extraction is repeated 3 times. The extracts are combined, and the ethanol is recovered by vacuum concentration. After water precipitation at low temperature (standing at 4°C for 24 hours), the extract is filtered, and the filtrate is concentrated and dried to obtain the licorice extract.
[0042] Comparative Example 1
[0043] The other methods are the same as in Example 5, except that the orange myxococcus is replaced with an equal amount of Pseudomonas aeruginosa.
[0044] Comparative Example 2
[0045] The other methods are the same as in Example 5, except that the addition of Pseudomonas aeruginosa and Penicillium micropurpurum is omitted.
[0046] Comparative Example 3
[0047] The other methods are the same as in Example 5, except that the addition of thistle extract is omitted.
[0048] Comparative Example 4
[0049] The other methods are the same as in Example 5, except that cyclohexane in step b of the preparation process of Cnidium monnieri extract is replaced with an equal volume of 75% ethanol solution.
[0050] Comparative Example 5
[0051] The other methods are the same as in Example 5, except that the addition of thistle extract, Cnidium monnieri extract, matrine, and licorice extract is omitted.
[0052] Experimental Example 1
[0053] The biocontrol agents prepared in Example 5 and Comparative Examples 1-5 were respectively prepared into solutions with a concentration of 3.5 mg / mL. 180 μL of each solution was added to an Oxford cup containing Fusarium graminearum coated with PDA medium. After incubation at 26±2℃ for 5 days, the diameter of the inhibition zone was determined by the cross-cross method. Five replicates were set for each group, and the average value of the results was taken, as shown in Table 1.
[0054] Table 1. Survey of Antibacterial Zone Diameter
[0055] As can be seen from the inhibition zone diameter data in Table 1, the biocontrol formulation of this invention exhibits the strongest antibacterial effect, with an inhibition zone diameter of 30.57 mm, significantly higher than that of the comparative groups. The antibacterial effects of Comparative Example 1 (replacing *Myxococcus spp.*) and Comparative Example 4 (changing the extraction solvent) decreased, indicating that *Myxococcus spp.* and the cyclohexane extraction process play a crucial role in maintaining high-efficiency antibacterial activity. The antibacterial abilities of Comparative Example 2 (omitting *Pseudomonas aeruginosa* and *Penicillium micropurpurum*) and Comparative Example 3 (omitting *Cirsium japonicum* extract) were further reduced, indicating that the synergistic combination of multiple microorganisms and plant extracts is essential for enhancing the antibacterial effect. Comparative Example 5 (omitting all plant extracts) showed the weakest antibacterial ability, highlighting the core contribution of plant active ingredients in this formulation.
[0056] Experimental Example 2
[0057] PDA solid culture medium was heated to form a liquid, and the biocontrol agent prepared in Example 5 was added. Different addition amounts were set to achieve final concentrations of 0 (without any reagents added as a blank control group), 1, 3, 5, 7, and 10 μg / mL of the biocontrol agent in the PDA solid culture medium for different groups. After cooling, plates were prepared, and one disc of Fusarium graminearum was inoculated onto each plate with the mycelial side facing up. Each concentration plate was inoculated three times. The plates were incubated at 25°C until the colonies of the blank control group covered more than 2 / 3 of the plate. Relevant indicators were measured, and the average values of the results are shown in Table 2.
[0058] The diameter of mycelial growth was measured using the cross-cross method. The mycelial growth inhibition rate (%) was calculated using the following formula:
[0059] Spore production was determined using a hemocytometer method, and spore germination rate was determined using a hanging drop culture method. The determination of DON content followed GB / T 23503-2009, "Determination of Deoxynivalenols in Food: Immunoaffinity Chromatography Purification High Performance Liquid Chromatography".
[0060] Table 2. Survey on Antibacterial Effect
[0061] The concentration gradient experiment results in Table 2 show that this formulation has a significant dose-dependent inhibitory effect on mycelial growth, spore production, spore germination, and toxin synthesis of Fusarium graminearum. At a concentration of 7 μg / g, spore production and germination are completely inhibited, and the toxin content is reduced to zero. Its half-maximum effective concentration (WMC) performance is superior to conventional chemical agents, indicating that this formulation can achieve highly efficient control at low concentrations and has good application potential and economic efficiency.
[0062] Experimental Example 3
[0063] Wheat was planted in an area with a high incidence of wheat scab, and the area was divided into equal planting zones. Each experimental group was randomly assigned to three zones, ensuring that the zones did not interfere with each other. The biocontrol agents prepared in Example 5 and Comparative Examples 1-5 were sprayed directly onto the wheat ears and plants in the corresponding zones of each experimental group during the wheat flowering stage. The spraying method was as follows: after diluting 200 times, spray at a rate of 25 kg / mu / time, once every 5 days, for a total of 3 times, avoiding application on cloudy or rainy days.
[0064] Existing technology group: Apply thiophanate-methyl according to the instructions.
[0065] Blank group: No pesticides were sprayed.
[0066] Evaluation method for wheat resistance to Fusarium head blight: At the milk stage, the severity of wheat disease was investigated and statistically analyzed according to the disease grading standards of the following wheat resistance to Fusarium head blight evaluation method. The average value of the results is shown in the table below.
[0067] Grade 0: Disease-free spikelets
[0068] Level 1: Scattered cases of disease, accounting for less than 25% of the total spikelets.
[0069] Grade 2: Diseased spikelets account for 25%-50.0% of the total number of spikelets.
[0070] Grade 3: Diseased spikelets account for 50.1%-75.0% of the total number of spikelets.
[0071] Level 4: Diseased spikelets account for 75.1% or more of the total number of spikelets.
[0072] DI (Disease Index) = ∑(Disease grade number × Number of plants at that disease grade) / (Highest disease grade number × Total number of plants)
[0073] Table 3. Survey on the Control Effect of Wheat Fusarium Head Blight
[0074] Table 3 shows the field control effects, further validating the practical value of this invention. The disease index of Example 5 was 16.67%, comparable to the efficacy of the existing chemical agent thiophanate-methyl, and significantly superior to all comparative groups. This indicates that the present invention, through the synergistic effect of multiple fungal species and multiple plant active ingredients, can maintain a stable and highly effective control effect in real field environments. Particularly noteworthy is that Comparative Example 5, which removed all plant extracts, had a disease index as high as 34.25%, further demonstrating the irreplaceable role of plant extracts in enhancing the overall efficacy of the formulation, especially in toxin control.
[0075] In summary, the experimental data systematically demonstrate the comprehensive advantages of the biocontrol agent of this invention in inhibiting the growth of Fusarium graminearum, controlling toxin synthesis, and preventing wheat scab. Through a multi-target synergistic mechanism between microorganisms and plant active ingredients, it achieves efficient, green, and sustainable disease control, providing a feasible technical solution to replace traditional chemical pesticides.
[0076] 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 biological control agent for inhibiting the growth of Fusarium graminearum and controlling wheat scab, characterized in that, The product comprises the following components in parts by weight: 18-23 parts of Myxococcus orange, 10-18 parts of Pseudomonas aeruginosa, 12-20 parts of Bacillus pumilus, 5-10 parts of halophilic Bacillus, 6-12 parts of Penicillium purpureus, 3-8 parts of rose geranium essential oil, 5-12 parts of Cirsium japonicum extract, 4-10 parts of Cnidium monnieri extract, 0.4-0.7 parts of matrine, 2-6 parts of licorice extract, 5-10 parts of chitin oligosaccharide, and 5-10 parts of pullulan polysaccharide.
2. The biological control agent according to claim 1, characterized in that, The bacterial counts of *Orange Myxococcus*, *Pseudomonas aeruginosa*, *Bacillus pumilus*, and *Bacillus halophilus* were all 400-600 million / g; the spore content of *Penicillium purpureus* was (6-9) × 10⁻⁶. 9 CFU / g.
3. The biological control agent according to claim 1, characterized in that, The preparation method of the small thistle extract is as follows: (1) Take the whole plant of thistle and crush it through a 10-30 mesh sieve; (2) Add the raw material obtained in step (1) to a 60% to 75% ethanol solution at a material-to-liquid ratio of 1:8 to 1:12 (w / v), and perform hot reflux extraction at 70 to 80°C for 1.5 to 2.5 hours each time, for a total of 2 to 3 extractions; (3) Combine the extracts obtained in step (2), filter, concentrate the filtrate under reduced pressure to a relative density of 1.05~1.15, and elute; (4) The eluent obtained in step (3) is concentrated and dried into powder to obtain thistle extract.
4. The biological control agent according to claim 3, characterized in that, The elution method described in step (3) is as follows: the concentrate is loaded onto an HPD-100 macroporous adsorption resin column at a flow rate of 1-3 column bed volumes / h, and washed sequentially with 3-5 column bed volumes of water, 2-4 column bed volumes of 10%-30% ethanol, and finally eluted with 4-6 column bed volumes of 60%-75% ethanol, and the eluent is collected.
5. The biological control agent according to claim 1, characterized in that, The preparation method of the Cnidium monnieri extract is as follows: a. Crush the fruit of Cnidium monnieri and pass it through a 30-50 mesh sieve; b. Add cyclohexane to the raw material obtained in step a at a material-to-liquid ratio of 1:10 to 1:15 (w / v) and extract at 40 to 60°C for 30 to 60 minutes, repeating the extraction 2 to 3 times. c. Combine the extracts obtained in step b, filter, concentrate the filtrate and dry it into powder to obtain Cnidium monnieri extract.
6. The biological control agent according to claim 5, characterized in that, The extraction process described in step b requires ultrasound, with an ultrasound power of 300~500W and a frequency of 22~28kHz.
7. The biological control agent according to claim 1, characterized in that, The method for preparing the licorice extract is as follows: licorice is added to a 70-75% ethanol solution at a material-to-liquid ratio of 1:8 to 1:12 (w / v), and extracted by reflux at 80-85°C for 1.5-2.5 hours. The extraction is repeated 2-3 times. The extracts are combined, and the ethanol is recovered by vacuum concentration. After low-temperature water precipitation, the solution is filtered, and the filtrate is concentrated and dried to obtain the licorice extract.
8. The biological control agent according to claim 7, characterized in that, The low-temperature water immersion is performed by standing at 4~6℃ for 20~28 hours.
9. A method for preparing the biological control agent for inhibiting the growth of Fusarium graminearum and controlling wheat scab according to any one of claims 1 to 8, characterized in that, A biological control agent for inhibiting the growth of Fusarium graminearum and preventing wheat scab was prepared by mixing Pseudomonas aeruginosa, Bacillus pumilus, Bacillus halophilus, Penicillium purpureus, rose geranium essential oil, Cirsium japonicum extract, Cnidium monnieri extract, matrine, licorice extract, chitin oligosaccharide, and pullulan polysaccharide.
10. The application of the biological control agent according to any one of claims 1 to 8 for inhibiting the growth of Fusarium graminearum and controlling wheat scab in the preparation of wheat scab control agents.