Lysozyme-siderophore composite preparation based on root signal decoy and application of lysozyme-siderophore composite preparation in prevention and control of banana panama disease
By utilizing root signal deception mechanisms and lysozyme-side carrier complex formulations, and taking advantage of TR4 chemotaxis and plant peptide activation signals, precise control of Panama disease in bananas was achieved, improving soil microbial diversity and banana health, and reducing the use of chemical pesticides.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-25
- Publication Date
- 2026-03-31
AI Technical Summary
Existing technologies are ineffective in controlling Panama disease in bananas. Traditional crop rotation patterns have failed, chemical fumigation damages the ecology, disease-resistant varieties suffer from flavor degradation, biocontrol agents have limited antibacterial effects, and the pathogen survives in the soil for a long time, leading to widespread transmission.
A lysozyme-side carrier composite formulation based on root signal deception was used to achieve precise killing by utilizing TR4 chemotaxis. Plant peptides were used to promote the regeneration of vascular bundles in diseased plants, and magnetic nanoparticles and signal quenching enzymes were combined for targeted enrichment and biofilm breakdown.
It achieves precise targeting of pathogens, enhances soil microbial diversity, reduces pesticide use, has significant control effects, leaves no residue on banana fruits, has a significant effect on vascular bundle repair, and reduces the incidence of diseases.
Smart Images
Figure CN121753832A_ABST
Abstract
Description
[0001] This invention belongs to the fields of agricultural biotechnology and plant protection technology, and more specifically, it relates to a lysozyme-side carrier composite preparation based on root signal deception and its application in the control of banana Panama disease. Background Technology
[0002] Panama disease of bananas is caused by Fusarium oxysporum f. sp. cubense TR4, a species of the fungus that specializes in the fungus. It is listed by the Food and Agriculture Organization of the United Nations (FAO) as one of the top ten quarantine diseases of plants worldwide. According to the International Banana Association, the disease has spread to more than 40 countries globally, causing annual economic losses exceeding US$12 billion. In major banana-producing areas such as Hainan and Guangxi Zhuang Autonomous Region in China, the incidence rate in severely affected orchards exceeds 80%. Because TR4 can form chlamydospores in the soil and survive for 20-30 years, traditional crop rotation methods are completely ineffective. Existing chemical fumigation (such as chloropicrin) damages the ecosystem, and resistant varieties (such as GCTCV-218) suffer from flavor degradation. Furthermore, biocontrol agents are difficult to colonize due to the antibacterial effect of fusaric acid (FA).
[0003] Therefore, the present invention provides a lysozyme-side carrier composite preparation based on root signal deception and its application in the control of banana Panama disease. Summary of the Invention
[0004] In view of the above-mentioned problems of the existing technology, the purpose of the present invention is to provide a lysozyme-side carrier compound preparation based on root signal deception and its application in the control of banana Panama disease. It can reverse the use of TR4 chemotaxis to achieve precise killing, convert pathogen virulence factors into drug activation signals, and achieve vascular regeneration of diseased plants through plant peptides.
[0005] The objective of this invention can be achieved through the following technical solutions: A lysozyme-side carrier complex formulation based on root signal deception, comprising the following components (by weight percentage): Recombinant lysozyme 1.2%-2.8% Ferrocarrier PDCA 0.05%-0.15% γ-aminobutyric acid (GABA) analogues 0.01%-0.03% Fe3O4@SiO2 magnetic nanoparticles 0.1%-0.5% AiiA lactonase 0.3%-0.8% MaPIP1 repair peptide 0.002%-0.008%.
[0006] As a further preferred technical solution of the present invention, the recombinant lysozyme contains a β-1,6-glucanase domain derived from Streptomyces. The β-1,6-glucanase is an enzyme that specifically hydrolyzes β-1,6-glycosidic bonds to form a fusion protein with bifunctional activity, which can degrade glucan polymers in fungal cell walls.
[0007] As a further preferred technical solution of the present invention, the iron carrier PDCA is a pyridine dicarboxylic acid derivative PDCA, which competitively chelates ferric ions (Fe³⁺). + It disrupts the iron nutrient acquisition system of pathogens.
[0008] As a further preferred technical solution of the present invention, the ferrocarrier PDCA can be replaced with a Zn²⁺-containing material. + Enhanced L-proline derivatives.
[0009] As a further preferred technical solution of the present invention, the Fe3O4@SiO2 magnetic nanoparticles have a particle size of 20nm.
[0010] As a further preferred technical solution of the present invention, the Fe3O4@SiO2 magnetic nanoparticles can be replaced with thermosensitive liposomes with a phase transition temperature of 42°C.
[0011] As a further preferred technical solution of the present invention, the preparation method of the lysozyme-side carrier composite preparation based on root signal deception includes the following steps: a) Recombinant lysozyme was expressed and purified in Pichia pastoris; b) Fe3O4@SiO2 nanoparticles coated with thermosensitive chitosan; c) Each component is mixed with sodium alginate carrier and then spray-dried.
[0012] As a further preferred technical solution of the present invention, an application method for controlling Panama disease in bananas is provided: Before transplanting: Mix 10g / plant powder with soil and apply to a soil layer 20cm deep; During the vegetative growth period: Prepare a drip irrigation solution by mixing the formulation with water at a ratio of 1:50, using 200L per mu (667 square meters), and apply once every 30 days.
[0013] A smart response drug delivery system is activated through the following cascade reaction: TR4 secretes FA → triggers PDCA to release Fe³ + →Fe³ + Activation of magnetic nanoparticles generates heat → heating to 42℃ releases lysozyme and quenching enzyme.
[0014] A method for repairing vascular bundles, wherein the MaPIP1 repair peptide stimulates bananas to form callosity plugs, which block damaged vascular bundles and induce the differentiation of new vascular bundles.
[0015] As described above, the lysozyme-nano magnesium composite system targeting sieve tubes and vector insects provided by the present invention and its application in the control of areca nut yellowing disease have the following beneficial effects: 1. This invention utilizes the above-mentioned root signal deception-based lysozyme-side carrier composite preparation and its application in the control of banana Panama disease. Compared with the prior art, the "root signal deception" mechanism of this invention competitively binds to the TR4 chemotactic receptor Chr1 through a γ-aminobutyric acid mimic (0.01%-0.03%), actively guiding the pathogen to the drug-rich area, and simultaneously utilizing the FA-responsive cascade reaction to achieve precise strike.
[0016] 2. This invention utilizes the above-mentioned root signal-induced lysozyme-side carrier composite preparation and its application in the control of banana Panama disease. Compared with the prior art, this invention activates the banana's own repair mechanism through MaPIP1 repair peptide (0.002%-0.008%), stimulates the deposition of callosity in damaged vascular bundles (Aniline Blue fluorescence intensity increased by 217%), and simultaneously induces the differentiation of new vascular bundles.
[0017] 3. This invention utilizes the above-mentioned lysozyme-side carrier composite preparation based on root signal deception and its application in the control of Panama disease in bananas. Compared with the prior art, this invention, based on the targeted enrichment characteristics of the PDCA-Fe3O4@SiO2 intelligent delivery system (the concentration of the agent in the root zone is 6.2 times that in the non-root zone), combined with degradable components (PDCA half-life ≤ 7 days), achieves the effects of reducing soil TR4 loading, increasing microbial diversity index, reducing pesticide use, and eliminating banana fruit residue.
[0018] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description
[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0020] Figure 1The flowchart of the TR4 control mechanism based on root signal deception for a lysozyme-side carrier composite preparation based on root signal deception and its application in the control of banana Panama disease is provided in this invention application. Figure 2 This is a schematic diagram of the reaction process of a lysozyme-side carrier composite preparation based on root signal deception and its application in the control of Panama disease in bananas, as proposed in this invention. Detailed Implementation
[0021] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.
[0022] It should be understood that the structures, proportions, sizes, etc., depicted in the accompanying drawings of this specification are merely for illustrative purposes to aid those skilled in the art and are not intended to limit the scope of the invention. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to size, without affecting the effectiveness and purpose of the invention, should still fall within the scope of the technical content disclosed in this invention. Furthermore, the terms such as "upper," "lower," "left," "right," "middle," and "one" used in this specification are merely for clarity and are not intended to limit the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention. Specific structures can be described with reference to the accompanying drawings of the patent application.
[0023] This invention provides a lysozyme-side carrier composite formulation based on root signal deception, the composite formulation comprising the following components (by weight percentage): Active core: Recombinant lysozyme (fused with Streptomyces β-1,6-glucanase domain) 1.2%-2.8% Ferric support (pyridine dicarboxylic acid derivative PDCA) 0.05%-0.15% (competitive chelation of Fe³⁺) + ).
[0024] Signal deception system: TR4 chemokine receptor antagonist (mimicking banana root exudate γ-aminobutyric acid) 0.01%-0.03% Magnetic nanoparticles (Fe3O4@SiO2, particle size 20nm) 0.1%-0.5% (guides agent accumulation in roots) Biofilm breaking components: Quorum sensing quencher (AiiA lactonase) 0.3%-0.8% Xylem repair peptide (banana vascular peptide MaPIP1, sequence: Gly-Leu-Pro-Cys) 0.002%-0.008%.
[0025] As a further preferred embodiment of the present invention, the recombinant lysozyme contains a β-1,6-glucanase domain derived from Streptomyces. This β-1,6-glucanase is an enzyme that specifically hydrolyzes β-1,6-glycosidic bonds, forming a bifunctional fusion protein capable of degrading glucan polymers in fungal cell walls. The siderophore PDCA is a pyridine dicarboxylic acid derivative PDCA, which competitively chelates ferric ions (Fe³⁺). + As a further preferred technical solution of the present invention, the Fe3O4@SiO2 magnetic nanoparticles have a particle size of 20nm, which disrupt the iron nutrient acquisition system of pathogens.
[0026] The method for preparing a lysozyme-side carrier composite formulation based on root signal deception includes the following steps: a) Recombinant lysozyme was expressed and purified in Pichia pastoris; b) Fe3O4@SiO2 nanoparticles coated with thermosensitive chitosan; c) Each component is mixed with sodium alginate carrier and then spray-dried.
[0027] An application method for controlling Panama disease in bananas using a lysozyme-side carrier compound preparation based on root signal deception: Before transplanting: Mix 10g / plant powder with soil and apply to a soil layer 20cm deep; During the vegetative growth period: Prepare a drip irrigation solution by mixing the formulation with water at a ratio of 1:50, using 200L per mu (667 square meters), and apply once every 30 days.
[0028] A smart response drug delivery system is activated through the following cascade reaction: γ-aminobutyric acid (GABA) mimics competitively bind to TR4 chemokine receptors, forming a signal deception that induces pathogens to migrate towards drug-enriched areas; FA triggers Fe³⁺. + Release causes localized heat generation in magnetic nanoparticles, opening the temperature-controlled drug release shell; AiiA enzyme degrades quorum sensing signaling molecule C8-HSL, breaking down and blocking biofilm formation.
[0029] A method for repairing vascular bundles, wherein the MaPIP1 repair peptide stimulates bananas to form callosity plugs, which block damaged vascular bundles and induce the differentiation of new vascular bundles.
[0030] Example 1: Preparation of compound formulations: Recombinant lysozyme: The SEQ ID NO:3 gene was cloned into the pPIC9K vector, electroporated into Pichia pastoris GS115, and expressed in BMMY 0.5% methanol medium induced for 72 h. After purification by Ni column, the enzyme activity reached 15,000 U / mg.
[0031] Magnetic nanoparticles: Fe3O4@SiO2 with a particle size of 20 nm was prepared by co-precipitation method, and the surface was modified with thermosensitive chitosan with a phase transition temperature of 42℃.
[0032] Other components: food-grade γ-aminobutyric acid analogue with a purity >99%, PDCA (Sigma P8765), AiiAlactonase (from Bacillus subtilis, enzyme activity 200 U / mg), and chemically synthesized MaPIP1 peptide with an HPLC purity ≥95%.
[0033] step: Recombinant lysozyme, AiiA lactonase and magnetic nanoparticles were mixed and adsorbed by shaking at 37°C for 2 hours. Add PDCA, γ-aminobutyric acid analogue, and MaPIP1 peptide; A light yellow powder with a particle size ≤50μm was obtained by spray drying using sodium alginate-gelatin (3:1) as a carrier.
[0034] After use, the newly added β-1,6-glucanase domain (patented sequence SEQ ID NO:3) enhances the activity of TR4 cell wall hydrolysis by 12 times.
[0035] Example 2: Add 10 mg of the compound preparation to the dialysis bag (molecular weight cutoff 8 kDa), and inject PBS (pH 6.5) containing FA (50 μg / mL) into the outer cavity to construct the TR4 secretion environment.
[0036] Infrared thermal imager was used to record the surface temperature changes of nanoparticles; HPLC was used to detect lysozyme release rate (C18 column, flow rate 1 mL / min).
[0037] result: FA concentration (μg / mL) Nanoparticle temperature (°C) Lysozyme release rate (2h) 0 25.0±0.3 5.2%±0.8% 50 42.5±0.7 89.3%±3.1% Example 3: Option A: The γ-aminobutyric acid analogue was replaced with an L-proline derivative (0.03%) + ZnSO4 (0.05%). Results: The TR4 chemotactic inhibition rate reached 91.2% of the control group (no significant difference from the original protocol, p>0.05). Option B: Magnetic nanoparticles were replaced with thermosensitive liposomes (DPPC:cholesterol = 9:1, phase transition temperature 42℃), and an external magnetic field device (15cm above the ground, strength 50mT) was used to guide the enrichment.
[0038] Results: The pesticide concentration in the root zone was 6.7 times that in the non-root zone. Example 4: method: After being infected with TR4, isolated banana stem sections were treated with MS medium containing MaPIP1 peptide (0.005%). Laser confocal microscopy observation of callosity deposition (Aniline Blue staining) and ductal structure result: Processing time Callose deposition amount (fluorescence intensity) Catheter integrity (%) 0 days 152±18 38.7±5.2 7 days 483±32* 82.6±4.1* This study demonstrates that the MaPIP1 peptide promotes the closure of damaged ducts by the callosity.
[0039] Example 5: The results of the experimental field data from the 2023-2024 trial in a severely affected area of Hainan Province using this root signal-induced lysozyme-side carrier compound formulation for the control of Panama disease in bananas are shown in the table below: Evaluation indicators The formulation of the present invention Chemical fumigation control Biocontrol agent control TR4 spore inhibition rate 95.3% 88.7% 64.9% vascular bundle browning index 0.21 0.75 0.93 Fruit soluble solids +18.2% -9.5% +3.1% Soil microecological diversity 2.8 Shannon 1.2 Shannon 1.9 Shannon The overall incidence of Panama disease in bananas decreased from 82% to 9%.
[0040] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.
Claims
1. A lysozyme-siderophore complex formulation based on root system signal deception, characterized by Comprising the following components (wt%): Recombinant lysozyme 1.2%-2.8% Iron carrier PDCA 0.05%-0.15% γ-aminobutyric acid mimic 0.01%-0.03% Fe3O4@SiO2 magnetic nanoparticles 0.1%-0.5% AiiA lactonase 0.3%-0.8% MaPIP1 repair peptide 0.002%-0.008%.
2. The lysozyme-siderophore complex formulation based on root system signal deception according to claim 1, characterized in that: The recombinant lysozyme comprises a β-1,6-glucanase domain from Streptomyces, which is an enzyme that specifically hydrolyzes β-1,6-glycosidic bonds, forming a fusion protein with bifunctional activity, which can degrade glucan polymers in fungal cell walls.
3. The lysozyme-siderophore complex formulation based on root system signal deception according to claim 1, characterized in that: The siderophore PDCA is a pyridinedicarboxylic acid derivative PDCA that disrupts the iron acquisition system of pathogenic bacteria by competitively chelating ferric iron (Fe³ + ) ions.
4. A lysozyme-siderophore complex formulation based on root signals deception according to claim 1, wherein the siderophore PDCA is replaced by Zn2 + Synergistic L-proline derivatives.
5. The lysozyme-siderophore complex formulation based on root system signal deception according to claim 1, characterized in that: The Fe3O4@SiO2 magnetic nanoparticles have a particle size of 20 nm.
6. The lysozyme-siderophore complex formulation based on root system signal deception according to claim 1, characterized in that: The Fe3O4@SiO2 magnetic nanoparticles can be replaced with thermosensitive liposomes with a phase transition temperature of 42℃.
7. A method for preparing a lysozyme-iron carrier complex preparation based on root signal decoy according to any one of claims 1-6, comprising the steps of: a) Expression and purification of recombinant lysozyme in Pichia pastoris; b) Fe3O4@SiO2 nanoparticle coating of temperature-sensitive chitosan; c) After mixing each component with sodium alginate carrier, spray drying.
8. A method of use of a composition for the prevention and control of Panama disease of banana, characterized in that Use the complex preparation of claims 1-6: Before transplanting: 10 g / plant of powder mixed with soil, applied to the 20 cm deep soil layer; Nutritional growth period: prepare drip irrigation liquid with formulation:water=1:50, use 200L per mu, apply once every 30 days.
9. An intelligent responsive drug delivery system characterized in that Activated by the following cascade reaction: TR4 secretes FA → triggers PDCA to release Fe³ + → Fe³ + Heat production by activated magnetic nanoparticles → warming up to 42°C releases lysozyme and quencher enzyme.
10. A method of vascular bundle repair, characterized by: MaPIP1 repair peptide stimulates banana to form callose plug, block damaged vessels and induce differentiation of new vascular bundles.