Microbial bactericide and application thereof
By combining functional microbial compound agents, targeted bacteriophages, and pH-responsive nanogel carriers, we have solved many limitations of existing microbial fungicides, achieving efficient control of complex diseases and promoting crop growth.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HEILONGJIANG GOLDEN PHOENIX AGRICULTURAL DEVELOPMENT CO LTD
- Filing Date
- 2026-02-04
- Publication Date
- 2026-05-12
AI Technical Summary
Existing microbial fungicides have limited functions, short duration of action, poor stability, slow effect, and lack of intelligent response capabilities, making them difficult to effectively control complex diseases.
A multi-mechanism synergistic bactericide system is formed by combining functional microbial compound agents, targeted bacteriophage compositions, and pH-responsive nanogel carriers. This system leverages the environmental adaptability of deep-sea strains and the targeting ability of bacteriophages, combined with the intelligent release technology of nanocarriers.
It achieves highly efficient control of target diseases, has a long-lasting effect, and is widely applicable. It can quickly respond to pathogens by releasing active ingredients, improving control efficacy by 70%-85% and promoting crop growth.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural biological control technology, specifically referring to a microbial bactericide and its application. Background Technology
[0002] Plant diseases, especially those caused by soil-borne pathogens (such as *Ralstonia solanacearum* and *Fusarium spp.*) and foliar pathogens (such as *Pseudomonas syringae* and *Erwinia amylovora*), are a major cause of reduced crop yields and lower quality worldwide. Currently, disease control relies primarily on chemical pesticides, but long-term use can lead to increased pathogen resistance, excessive pesticide residues, and environmental pollution.
[0003] Biological control has become a research hotspot due to its environmental friendliness and sustainability. In existing technologies, microbial fungicides mostly employ single strains or combinations of homologous strains, for example: Bacillus subtilis preparations: inhibit pathogens by secreting lipopeptide antibiotics, but have limited effect on established pathogen populations, and their efficacy is greatly affected by environmental factors.
[0004] Trichoderma preparations: have competitive and hyperparasitic effects, but act slowly and are ineffective against bacterial diseases.
[0005] Single phage preparations: can specifically lyse target pathogens, but have a narrow host range, are easily inactivated by ultraviolet light, and have weak diffusion ability in soil.
[0006] The limitations of existing technologies mainly include: Single function: Most products rely on only one mechanism of action (such as antagonism or competition), making it difficult to deal with complex diseases.
[0007] Short duration of effect: Live bacteria preparations are easily inactivated by environmental factors such as temperature, ultraviolet radiation, and dryness in the field.
[0008] Slow to take effect: Biocontrol bacteria need time to colonize and reproduce, and cannot control outbreaks of diseases in a timely manner.
[0009] Poor stability: Bacteriophages are easily inactivated in the environment, and their activity is greatly lost during formulation processing.
[0010] Lack of intelligent response: The release of active ingredients is uncontrollable and cannot be accurately released when pathogens are active.
[0011] Therefore, there is an urgent need to develop a novel composite microbial bactericide with multi-mechanism synergy, strong environmental adaptability, long-lasting effect, and intelligent response capability. Summary of the Invention
[0012] In order to meet the needs and problems mentioned in the background above, the present invention provides a microbial bactericide and its application to at least partially solve the above problems.
[0013] According to the technical solution of the present invention, a microbial bactericide is provided, wherein the microbial bactericide is composed of the following components in weight percentage: Functional microbial compound inoculant: 30%-50% (based on a total viable count ≥1.0×10⁻⁶) 10 CFU / g); Targeted phage composition: 10%-20% (≥1.0×10⁻⁶ phage titer) 9 PFU / g); pH-responsive nanogel carriers: 20%-30%; Excipients (including dispersants, UV protectants, and lyophilization protectants): Balance.
[0014] 1. Functional microbial compound inoculant It is a compound of the following three types of bacterial strains screened from deep-sea sediments, in proportion to their viable cell count: Bacillus strain: Marine Bacillus with accession number PD20142272.
[0015] Pseudomonas strain: Fluorescent Pseudomonas, catalog number CICC10282.
[0016] Streptomyces strain: Marine Streptomyces with product number HZB269487.
[0017] Screening and cultivation conditions: The strains were isolated from deep-sea sediments at depths >1000m and temperatures of 4-10°C. They were initially screened using oligotrophic culture media, and then strains with inhibition zones ≥15mm on the target pathogens were screened using the plate confrontation method.
[0018] The fermentation medium was a modified seawater-based medium with 0.5% corn flour and 0.1% yeast extract added. The pH was 7.2-7.5, the fermentation temperature was 28-30°C, the aeration rate was 1:1 vvm, and the fermentation time was 48-72 h.
[0019] 2. Targeted phage composition A mixture containing two or more of the following bacteriophages in equal proportions: Lysogenic phage 1: Xiaoqingning, a bacterial wilt phage preparation produced by Wuhan Greenong Biotechnology Co., Ltd.
[0020] Lysogenic phage 2: GoldenEco, a Pseudomonas phage produced by EcoPhage, Israel.
[0021] Lysogenic phage 3: EcoFire, a bacteriophage preparation for fire blight produced by EcoPhage, Israel.
[0022] Bacteriophage preparation: Isolate the bacteria from diseased plant tissue using the double-layer agar plate method, and proliferate them through co-culture fermentation with a host bacterium, with a fermentation endpoint titer ≥1.0×10⁻⁶. 11 PFU / mL.
[0023] Purification was achieved by PEG precipitation combined with CsCl gradient centrifugation to obtain high-purity phage particles.
[0024] 3. pH-responsive nanogel carriers Material composition: Gelatin-pectin crosslinked polymer, particle size 100-200nm, encapsulation rate ≥85%.
[0025] Response characteristics: When pathogens infect plant leaves, the organic acids produced by their metabolism will cause the local microenvironment pH of the leaves to drop to 5.0-6.5. Under the conditions of pH 5.0-6.5, the slightly acidic environment caused by pathogen infection swells and releases active ingredients, which remain stable at pH ≥ 7.0.
[0026] Preparation method: Gelatin and pectin are mixed at 40°C, and after adding the active ingredient, it is passed through Ca... 2+ Ionic cross-linking forms nanoparticles, which are then freeze-dried for later use.
[0027] 4. Adjuvants Dispersant: Polyoxyethylene sorbitan monooleate (Tween80), accounting for 0.5%-1%.
[0028] UV protectant: a combination of trehalose (2%-3%) and nano titanium dioxide (0.1%-0.2%).
[0029] Freeze-drying protectants: skim milk powder (5%) and glycerin (3%).
[0030] The preparation process of the microbial bactericide of this invention is as follows: Step 1: Functional microbial fermentation and harvesting: The three strains were inoculated into seed culture medium and cultured at 30°C and 200 rpm for 24 h with shaking.
[0031] Transfer 5% inoculum to the fermenter and control the following parameters: temperature 30°C, pH 7.2-7.5, dissolved oxygen ≥30%.
[0032] At the end of fermentation, the spore formation rate of Bacillus fermentation broth was ≥90%.
[0033] The bacterial powder (viviculture count ≥1.0 × 10⁻⁶) was obtained by centrifugation concentration (8000 rpm, 15 min) combined with vacuum freeze-drying. 11 CFU / g).
[0034] Step 2: Phage proliferation and purification The host pathogen was inoculated into LB liquid medium and cultured to mid-log (OD600≈0.6).
[0035] Add the phage stock solution (MOI=0.1) and continue culturing until the bacterial culture is clear (lysis is complete).
[0036] Bacterial cell fragments were removed by filtration (0.22 μm), PEG8000 precipitation was used for concentration, and CsCl gradient centrifugation was used for purification.
[0037] The purified phage was resuspended in SM buffer and the titer was adjusted to ≥1.0×10⁻⁶. 11 PFU / mL.
[0038] Step 3: Nanogel encapsulation and formulation The functional bacterial powder, bacteriophage suspension and auxiliary agent are mixed to form a uniform slurry.
[0039] The slurry was mixed with gelatin-pectin prepolymer at 40°C and nanoparticles were formed using a microfluidic device.
[0040] Spray drying (inlet temperature 120°C, outlet temperature 60°C) or freeze drying is used to produce dispersible granules.
[0041] Final product specifications: viable count ≥ 1.0 × 10⁻⁶ 10 CFU / g, phage titer ≥1.0×10 9 PFU / g, nanoparticle encapsulation rate ≥85%.
[0042] On the other hand, the present invention also provides a method for applying the above-mentioned microbial bactericide. 1. Application method and dosage: Seed treatment: Dilute the preparation 200 times and mix it with the seeds, using 10-15 mL / kg of seeds.
[0043] Root drenching / root dipping: Dilute the preparation 500-800 times, and drench each plant with 100-200 mL at transplanting time.
[0044] Foliar spray: Dilute the formulation 800-1000 times and spray 30-50L per acre.
[0045] 2. Target groups for prevention and control Soil-borne diseases: bacterial wilt of tomatoes, bacterial scab of peppers, wilt of cucumbers, etc.
[0046] Leaf diseases: cucumber angular leaf spot, pear fire blight, tobacco wildfire disease, etc.
[0047] 3. Expected Results Control efficacy: 70%-85% field control efficacy against target diseases, with a duration of 14-21 days.
[0048] Growth-promoting effects: Increases crop root vitality by 15%-30% and leaf chlorophyll content by 10%-20%.
[0049] Beneficial effects: Multi-mechanism synergistic prevention and control system: It integrates four mechanisms: competitive site occupation, antimicrobial substance inhibition, phage lysis, and induction of systemic resistance, breaking through the limitations of single-action mode.
[0050] Extreme environment strain resources: Deep-sea strains have stronger environmental adaptability and unique metabolite profiles, which improve the field stability of formulations.
[0051] Intelligent response release technology: pH-responsive nanocarriers enable precise matching between pathogen activity and bactericide release, improving utilization efficiency.
[0052] Wide applicability: It can be used to control soil-borne diseases as well as foliar diseases, reducing the types of pesticides farmers need to use. Detailed Implementation
[0053] The technical solutions in the embodiments will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection.
[0054] The general preparation method for the following embodiments is described below: Preparation of inoculum: Each strain was fermented separately. Bacillus was cultured in a nutrient broth seawater medium at 30°C for 48 hours until the spore formation rate was >90%; Pseudomonas and Streptomyces were cultured in Gao's No. 1 seawater medium at 28°C with shaking for 72 hours. The fermentation broth was centrifuged, concentrated, and then freeze-dried to obtain inoculum powder.
[0055] Phage preparation: The phages were proliferated using the corresponding host bacteria double-layer plate culture method, concentrated with PEG8000, purified by CsCl density gradient centrifugation, and stored in SM buffer.
[0056] Formulation: Bacterial powder, phage suspension, and excipients were mixed in a specific ratio. The excipients included 1% Tween 80, 2.5% trehalose, 0.1% nano-TiO2, and the remainder being inert filler. This mixture was then combined with 10% gelatin and 15% pectin at 40°C and added dropwise to a 1% CaCl2 solution using a microfluidic device to form nanogel particles. These particles were collected by centrifugation and freeze-dried to obtain dispersible granules.
[0057] Example 1 The components and proportions of functional microbial compound agents and targeted bacteriophage compositions in bacterial powder, bacteriophage suspension, and bacterial powder are shown below: The preparation method consisted of 40% Bacillus strains and 15% lytic phage 1, and was carried out according to general methods.
[0058] Example 2 The difference from Example 1 is that the components and proportions of the functional microbial compound agent and the targeted bacteriophage composition in the bacterial powder and bacteriophage suspension are as follows: The mixture contained 30% Bacillus strains, 10% Pseudomonas strains, 7.5% lytic phage 1 and 7.5% lytic phage 2, and the pH-responsive nanogel carrier was a mixture of 15% gelatin and 15% pectin.
[0059] Example 3 The difference from Example 1 is that the components and proportions of the functional microbial compound agent and the targeted bacteriophage composition in the bacterial powder and bacteriophage suspension are as follows: 20% Bacillus strains, 20% Streptomyces strains and 15% lytic bacteriophages.
[0060] Example 4 The difference from Example 2 is that the components and proportions of the functional microbial compound agent and the targeted bacteriophage composition in the bacterial powder and bacteriophage suspension are as follows: 15% Bacillus strains, 10% Pseudomonas strains, 15% Streptomyces strains, 7.5% lytic phage 1 and 7.5% lytic phage 2.
[0061] Example 5 The difference from Example 2 is that the components and proportions of the functional microbial compound agent and the targeted bacteriophage composition in the bacterial powder and bacteriophage suspension are as follows: 15% Bacillus strains, 10% Pseudomonas strains, 15% Streptomyces strains and 10% lytic bacteriophages.
[0062] Example 6 The difference from Example 2 is that the components and proportions of the functional microbial compound agent and the targeted bacteriophage composition in the bacterial powder and bacteriophage suspension are as follows: 15% Bacillus strains, 10% Pseudomonas strains, 15% Streptomyces strains, 7.5% lytic phage 1 and 7.5% lytic phage 3.
[0063] Example 7 The difference from Example 4 is that the pH-responsive nanogel carrier is a mixture of 15% chitosan and 15% sodium alginate. The chitosan-sodium alginate carrier is cross-linked by calcium ions and swells in an environment of pH 5.0-6.5. The particle size is 80-250nm and the encapsulation rate is ≥80%.
[0064] Example 8 The difference from Example 4 is that the pH-responsive nanogel carrier is a mixture of 15% gelatin and 15% pectin, and the mass percentage of trehalose is increased to 5% by adjusting the auxiliary agent.
[0065] Example 9 The difference from Example 4 is that the pH-responsive nanogel carrier is a mixture of 10% gelatin and 10% pectin.
[0066] Example 10 The difference from Example 1 is that the components and proportions of the functional microbial compound agent and the targeted bacteriophage composition in the bacterial powder and bacteriophage suspension are as follows: 25% Bacillus strains, 15% Pseudomonas strains and 15% lytic bacteriophages 2.
[0067] Comparative Example 1 The commercially available biocontrol bacterium, Bacillus subtilis (GB03) powder (40%), was used without the addition of bacteriophages. Ordinary soluble starch (30%) was used as the carrier, and the adjuvants were the same as in Example 1.
[0068] Comparative Example 2 The functional microbial compound inoculant is the same as in Example 4 (40%), without the addition of any bacteriophages. The pH-responsive nanogel carrier and auxiliary agents are the same as in Example 4.
[0069] Comparative Example 3 The functional microbial compound agent and bacteriophage composition are the same as in Example 4. The pH-responsive nanogel carrier is replaced with an equal amount of ordinary sodium carboxymethyl cellulose (CMC), forming a viscous solution instead of smart particles.
[0070] The application methods and test data of Examples 1-10 and Comparative Examples 1-3 are described below: Test method: Target diseases for prevention and control: Examples 1-5, 9, Comparative Examples 1 and 2 tested for bacterial wilt of tomato (soil-borne); Examples 6-10 and Comparative Example 3 tested for bacterial angular leaf spot of cucumber (foliar).
[0071] Application method: Soil-borne diseases (tomato): Dilute the preparation 500 times and apply 100 mL to the roots of each tomato seedling during transplanting. Inoculate with Ralstonia solanacearum (1×10⁻⁶) 7 days after transplanting. 8 CFU / mL bacterial suspension. Water treatment was used as a blank control group.
[0072] Foliar diseases (cucumber): Dilute the preparation 800 times and spray the leaves of cucumber seedlings at the three-leaf-one-heart stage until both sides of the leaves are evenly moistened. Inoculate with *Pseudomonas syringae* (1×10⁻⁶) 3 days after spraying. 7 CFU / mL bacterial suspension. Water treatment was used as a blank control group.
[0073] Data collection: Disease incidence was investigated 14 days (tomato) or 10 days (cucumber) after inoculation. Disease index and control effect were calculated. Simultaneously, plant height, root length, and root activity were measured in tomatoes, and leaf chlorophyll content (SPAD value) in cucumbers.
[0074] Formula for calculating the prevention and control effect: Prevention and control effect (%) = [(disease index of corresponding blank control - disease index of treatment group) / disease index of corresponding blank control] × 100.
[0075] The test results are shown in Table 1 below: Table 1. Field application effect data of Examples 1-10
[0076] As shown in Table 1 above, Example 4 exhibited the best and most stable preventive efficacy (83.0%) and growth-promoting effect in all tests. Comparison with Comparative Example 1 and Comparative Example 2 reveals that its superior performance stems from: the complementary nature of deep-sea multi-genera strains in terms of stress resistance and metabolites; the combination of preventative action by biocontrol bacteria and immediate treatment by bacteriophages; and the protection and intelligent release by nanocarriers. All three are indispensable.
[0077] Comparing Example 4 with Comparative Example 2, the efficacy differed by nearly 20 percentage points. This directly demonstrates that in the composite system of the present invention, the bacteriophage is not an optional component, but a key technical means to solve the bottleneck of slow effectiveness of biocontrol bacteria and achieve rapid suppression of pathogen numbers, producing a significant synergistic effect.
[0078] The comparison of Examples 6 and 8 with Comparative Example 3 shows that the formulation using the pH-responsive nanogel carrier has an efficacy improvement of more than 10 percentage points. This demonstrates that the carrier can effectively protect bacteriophages and live bacteria from environmental degradation and can precisely release active ingredients during the initial acidification stage of the microenvironment in disease occurrence, thereby significantly improving utilization efficiency and duration of effectiveness.
[0079] Examples 1-10 demonstrate that, within the scope of the claims, by adjusting the combination and ratio of strains, bacteriophages, and formulations, the focus of prevention and control can be flexibly shifted from soil-borne diseases (as in Examples 1-5) or foliar diseases (as in Examples 6 and 10), or environmental adaptability can be optimized (as in Example 8), while maintaining a core control efficacy of over 70%. This illustrates the universality and adjustability of the technical solution of the present invention.
[0080] In summary, the composite microbial bactericide provided by this invention creatively integrates three major elements: multi-source functional bacteria in special habitats, targeted bacteriophages, and intelligent responsive nanocarriers, forming a multi-stage, multi-target synergistic disease control system.
[0081] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A microbial bactericide, characterized in that, It contains the following components: Functional microbial compound inoculants, targeted bacteriophage compositions, pH-responsive nanogel carriers and adjuvants; The functional microbial compound agent includes strains isolated from deep-sea sediments and screened by the plate confrontation method that have an inhibition zone diameter ≥15mm against at least one target pathogen; the targeted phage composition includes at least two lytic phages, which specifically lyse different species of pathogens including Ralstonia solanacearum, Pseudomonas syringae, and Erwinia.
2. The microbial bactericide according to claim 1, characterized in that, The strains include at least two different genera from Bacillus, Pseudomonas, and Streptomyces.
3. The microbial bactericide according to claim 2, characterized in that, The functional microbial compound agent is composed of live bacteria of Bacillus, Pseudomonas, and Streptomyces strains in a live bacteria ratio of (1-3):(0.5-1.5):(0.5-1.5), with a total live bacteria count of not less than 1.0 × 10⁻⁶. 10 CFU / g.
4. The microbial bactericide according to claim 1, characterized in that, The total titer of the targeted phage composition is not less than 1.0 × 10⁻⁶. 9 PFU / g.
5. The microbial bactericide according to claim 1, characterized in that, The pH-responsive nanogel carrier is a biopolymer cross-linked polymer that swells or degrades in an environment of pH 5.0-6.5 to release active ingredients; the active ingredients include the functional microbial compound agent and / or the targeted bacteriophage composition.
6. The microbial bactericide according to claim 5, characterized in that, The pH-responsive nanogel carrier comprises a gelatin-pectin complex cross-linked by calcium ions, with a particle size of 50-300 nm, and an encapsulation rate of not less than 80% for the active ingredient.
7. The microbial bactericide according to claim 1, characterized in that, Based on the total weight of the bactericide, the mass percentage of each component is as follows: The composition consists of 30%-50% functional microbial compound inoculant, 10%-20% targeted bacteriophage composition, 20%-30% pH-responsive nanogel carrier, and the remainder being auxiliary agents.
8. The microbial bactericide according to claim 1, characterized in that, The auxiliary agent includes at least one of a dispersant, an ultraviolet protectant, and a lyophilization protectant; the ultraviolet protectant includes a mixture of trehalose and nano-titanium dioxide.
9. The application of a microbial bactericide, characterized in that, The use of the microbial fungicide according to any one of claims 1-8 in the preparation of an agent for the prevention and control of plant diseases, wherein the plant disease is a soil-borne disease or a bacterial foliar disease.
10. The application according to claim 9, characterized in that, The soil-borne diseases mentioned are bacterial wilt of tomatoes or wilt of cucumbers, and the bacterial foliar diseases mentioned are angular leaf spot of cucumbers or wildfire of tobacco.