Bacillus licheniformis-derived peptide-3 and application thereof in prevention and control of root-knot nematode disease

Abstract

This invention discloses a Bacillus licheniformis-derived nematicide Peptide-3 and its application in the control of root-knot nematodes. The amino acid sequence of the Bacillus licheniformis-derived nematicide Peptide-3 is shown in SEQ ID NO.1. The agent includes the aforementioned Bacillus licheniformis-derived nematicide Peptide-3. The nematicide Peptide-3 provided by this invention, as a naturally active peptide derived from microorganisms, can be used to develop green biological agents for the control of root-knot nematodes. Compared with chemical nematicides, it has higher ecological safety and meets the requirements of green agricultural development.

Description

Technical Field

[0001] This invention relates to the field of root-knot nematode control, specifically to a Bacillus licheniformis-derived nematicide Peptide-3 and its application in the control of root-knot nematode diseases. Background Technology

[0002] Root-knot nematodes are a class of obligate, sessile endoparasitic plant pathogenic nematodes, including common species such as the southern root-knot nematode, the northern root-knot nematode, the peanut root-knot nematode, and the Javan root-knot nematode. Their larvae invade plant roots, stimulating abnormal cell proliferation to form nodular root knots, thus disrupting root absorption. Affected plants become weak, stunted, and yellow, and in severe cases, wither and die. Root-knot nematodes have a wide host range and often trigger combined infections of soil-borne diseases, leading to significant crop yield reductions and causing substantial economic losses to vegetable, fruit, and cash crop production.

[0003] Currently, the control of root-knot nematodes relies heavily on chemical nematicides such as thiazophos and abamectin. Long-term, single-use of these nematicides leads to problems such as pesticide residues in the soil, increased nematode resistance, and soil microecological imbalance. Breeding disease-resistant varieties is the most sustainable control strategy, but resources for root-knot nematode-resistant genes are scarce, and existing resistance genes suffer from thermal instability.

[0004] Bioactive peptides are a class of short peptides, typically composed of 5-100 amino acid residues, whether natural or synthetic, possessing various biological functions such as nematicidal, antibacterial, and antiviral activity. Due to their amphiphilic nature, abundance of positively charged amino acids, and predominantly α-helical or β-sheet conformations, they can act on pathogens through various mechanisms. Furthermore, nematicidal peptides exhibit relatively conserved target sites compared to NB-LRR resistance genes, demonstrating significant potential in plant disease control. Bioactive peptides are widely available, and current screening primarily utilizes three major technological systems: bioinformatics prediction, experimental verification, and machine learning mining. Research is trending towards a closed-loop model integrating multiple technologies, providing a reference for high-throughput screening and optimization of nematicidal peptides.

[0005] In summary: root-knot nematode resistance gene resources are scarce, and existing resistance genes suffer from thermal instability; chemical nematicides easily cause pesticide residues, resistance, and ecological pollution, which do not meet the requirements of green agriculture; there is an urgent need to explore new highly active microbial nematicide gene resources that can be used for the development of biocontrol agents for root-knot nematodes and the creation of new germplasm resources for root-knot nematode-resistant crops. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a Bacillus licheniformis-derived nematicide Peptide-3 and its application in the control of root-knot nematodes. This invention utilizes bioinformatics technology combined with key physicochemical properties such as the net charge, hydrophobicity, and amphiphilicity of active peptides to directionally optimize the nematicide sequence, obtaining a nematicide with high lethal activity against root-knot nematodes, which can be used for green control of root-knot nematodes and the creation of resistant germplasm. To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a Bacillus licheniformis-derived nematicidal peptide Peptide-3, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The aforementioned nematicidal peptide Peptide-3, derived from Bacillus licheniformis, is derived from the Bacillus licheniformis protein WP_009328013.1.

[0008] Furthermore, the Bacillus licheniformis-derived nematicidal peptide Peptide-3 was obtained by introducing an N-terminal methionine into the Bacillus licheniformis-derived nematicidal peptide and optimizing the sequence based on physicochemical properties; the total hydrophobicity of the Bacillus licheniformis-derived nematicidal peptide Peptide-3 is 0.33, the total net positive charge ratio is +7.00, and the Wimley-White total residue hydrophobicity is 5.11 kcal / mol.

[0009] Sequence-directed optimization based on physicochemical properties enabled the nematicidal peptide Peptide-3 to satisfy the following characteristics: The introduction of methionine (M) at the N-terminus provides a start codon for subsequent overexpression in plants; b. The two ends of the molecule exhibit hydrophilic and hydrophobic characteristics, respectively, presenting a typical amphiphilic conformation; c. Retain the original sequence of α-helical or β-sheet core secondary structure to maintain its membrane permeability; d. Increase the number of positively charged residues (Lys, Arg) to enhance their electrostatic interaction with the negatively charged membrane of nematodes; e. Retain the necessary cysteine ​​(Cys) residues to maintain the stability of the potential disulfide bond.

[0010] The present invention also provides the application of the above-mentioned nematicide-3 in the control of root-knot nematodes and / or the creation of root-knot nematode-resistant plant germplasm resources.

[0011] The present invention also provides the application of the above-mentioned nematicide-3 in the preparation of products for the prevention and control of root-knot nematodes.

[0012] The present invention also provides an agent for controlling root-knot nematodes, the agent comprising the above-mentioned Bacillus licheniformis-derived nematicide Peptide-3.

[0013] Furthermore, the concentration of the nematicide-3 in the agent is 0.33 mmol / L to 1.00 mmol / L.

[0014] Furthermore, the solvent of the agent is a NaCl solution with a mass fraction of 0.9%.

[0015] The present invention also provides an application of the above-mentioned agent in the control of root-knot nematodes.

[0016] The present invention also provides a method for controlling root-knot nematodes, the method comprising soaking the above-mentioned agent with second-instar larvae of root-knot nematodes. Alternatively, the method may involve directly applying the aforementioned agent to the plant roots via root irrigation.

[0017] Furthermore, the specific steps of the method are as follows: hatch and collect second-instar larvae of root-knot nematodes, and incubate them together with the above-mentioned agent for 48 hours.

[0018] The beneficial effects of this invention are: 1. The nematicidal peptide Peptide-3 provided by this invention is derived from the natural protein WP_009328013.1 of Bacillus licheniformis. Through the introduction of N-terminal methionine and sequence orientation optimization based on physicochemical properties, the total hydrophobicity and / or total net positive charge ratio of the optimized nematicidal peptide molecule are improved to varying degrees, and the AntiBP Server prediction score is significantly improved.

[0019] 2. The nematicidal peptide Peptide-3 provided by this invention has a clear lethal effect on second-instar larvae of root-knot nematodes. At a final concentration of 1.00 mmol / L, the 24-hour (24hpi) corrected mortality rate of root-knot nematodes is as high as 88.0%, and the 48hpi corrected mortality rate is similar to that at 24hpi, demonstrating a significant nematicidal effect.

[0020] 3. The nematicidal peptide Peptide-3 provided by this invention contains a methionine residue at its N-terminus, providing a start codon that facilitates subsequent overexpression in plants. It can be used to create new germplasm resources of crops resistant to root-knot nematodes and has broad application prospects.

[0021] 4. The nematicide-3 provided by this invention, as a natural active peptide of microbial origin, can be used to develop green control biological agents for root-knot nematodes. Compared with chemical nematicides, it has higher ecological safety and meets the requirements of green agricultural development. Attached Figure Description

[0022] Figure 1 The image shows the three-dimensional structure and surface physicochemical properties of the nematicidal peptide Peptide-3.

[0023] In the figure, the first row shows the three-dimensional structure of Peptide-3 and its electrostatic potential distribution from different perspectives. The second row shows the hydrophilic and hydrophobic surface distribution of Peptide-3 from different perspectives.

[0024] Figure 2 The graph shows the lethal activity of the nematicide Peptide-3 at a concentration of 1.00 mmol / L for 48 h (48 hpi) against second instar larvae (J2s) of root-knot nematodes.

[0025] Figure 3 A schematic diagram showing the corrected mortality rate of root-knot nematodes J2s under different concentrations of nematicide-3 treatment. In the figure, 24 hpi indicates that the drug stock solution and root-knot nematodes J2s were incubated together for 24 hours (hours post-incubation). 48 hpi indicates that the drug stock solution was incubated with second-instar larvae of root-knot nematodes for 48 hours. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0027] Example 1: Screening and optimization of nematicidal peptides derived from Bacillus licheniformis 1. Experimental materials: Software and tools: ChimeraX, AntiBP Server, DeepSeek AI platform.

[0028] 2. Experimental Procedure Based on 4167 proteins from the entire genome of Bacillus licheniformis, candidate nematicide prediction was performed. 209 molecules with the top 5% prediction scores were selected for 3D structure prediction, and α-molecules were screened. Helical or β Molecules with folded domains comprising more than 50% of the total sequence were identified as 114 candidate nematicides. The DeepSeek AI platform was used to optimize the sequences of these candidate nematicides, strictly adhering to the following constraints: the introduction of a methionine (M) at the amino terminus to provide a start codon for subsequent overexpression in plants; the presence of distinct hydrophilic and hydrophobic characteristics at both ends of the molecule; maintaining the α-helix or β-sheet core secondary structure of the original sequence to preserve its membrane permeability; and increasing the number of positively charged residues to obtain optimized products of the candidate molecules.

[0029] 3. Optimized nematicide structure AntiBP Server was used to predict the scores of the optimized nematicidal peptides. Molecules with high predicted scores that retained N-terminal methionine residues were selected. Through comprehensive evaluation of multiple indicators (hydrophilicity / hydrophobicity distribution, positive charge density, disulfide bond stability, and secondary structure integrity), nematicidal peptide molecules that simultaneously met all optimization indicators were screened out, and the optimized product Peptide-3 was obtained. Its amino acid sequence is shown in SEQ ID NO.1: MRIKCYGKKATKKCK.

[0030] The charge distribution, polarity, and 3D structure of the nematicidal peptide Peptide-3 were characterized using ChimeraX software.

[0031] Table 1. Amino acid sequence and key physicochemical properties of Peptide-3 The results showed that the optimized nematicidal peptide Peptide-3 improved the total hydrophobicity ratio and / or total net positive charge ratio, increased the AntiBP Server prediction score, and retained the N-terminal methionine residue. Figure 1 (See Table 1). The three-dimensional structure of Peptide-3 exhibits a typical amphiphilic conformation, with the positive potential mainly distributed on the molecular surface and the hydrophobic residues concentrated on the other side of the molecule, consistent with the structural characteristics of typical nematicidal / antimicrobial peptides.

[0032] Example 2 Synthesis of Peptide-3 (a nematicidal peptide) The nematicidal peptide Peptide-3 was synthesized by Genscript Biotech Inc.; Peptide-3 is derived from protein WP_009328013.1, and its amino acid sequence is shown in SEQ ID NO.1: MRIKCYGKKATKKCK.

[0033] Example 3: Stock solution of pesticide for controlling root-knot nematodes 1 The preparation of stock solution 1 for controlling root-knot nematodes is as follows: Chemically synthesized nematicide Peptide-3 (purity ≥95%) was dissolved in a 0.9% NaCl solution to prepare a 2.00 mmol / L solution, which is the mother liquor 1.

[0034] Example 4: Stock solution of pesticide for controlling root-knot nematodes 2 The preparation of stock solution 2 for controlling root-knot nematodes is as follows: Chemically synthesized nematicide Peptide-3 (purity ≥95%) was dissolved in a 0.9% NaCl solution to prepare a 0.66 mmol / L solution, which is the mother liquor 2.

[0035] Example 5: In vitro nematicidal activity assay of nematicidal peptides 1. Experimental materials: (1) Compounds: Mother liquor 1 prepared in Example 3 and mother liquor 2 prepared in Example 4.

[0036] (2) Biological material: second-instar larvae (J2s) of root-knot nematodes of the Poaceae family.

[0037] 2. Experimental Procedure Second-instar larvae (J2s) of root-knot nematodes were collected after hatching. 100 μL of nematode solution + 100 μL of stock solution were added to each well of a 24-well plate to obtain experimental groups 1 and 2 (i.e., the final concentrations of nematicide-3 in experimental groups 1 and 2 were 1.00 mmol / L and 0.33 mmol / L, respectively, forming agent 1 and 2). Each treatment had three replicate wells. A 0.9% NaCl solution was used as a control. After incubation at room temperature for 48 h, the number of live / dead nematodes was counted at 24 hpi (24 hpi) and 48 hpi (48 hpi), and the corrected mortality rate was calculated. Corrected mortality rate (%) = (treatment group mortality rate - control group mortality rate) ÷ (1 - control group mortality rate) × 100%.

[0038] 3. Activity verification analysis and statistical results of killing effect: like Figure 2 As shown, after co-incubating the optimized nematicide-3 with root-knot nematodes at a concentration of 1 mmol / L for 48 h, the nematodes exhibited obvious rigidity and death (the nematodes were straightened, stiff, and showed no movement response), indicating that the optimized nematicide has the activity of killing root-knot nematodes.

[0039] like Figure 3 As shown: the corrected mortality rate of root-knot nematodes after 24 h (24 hpi) was 88.0% in experimental group 1 with a final concentration of 1.00 mmol / L of nematicide-3, and 24.24% in experimental group 2 with a final concentration of 0.33 mmol / L. The corrected mortality rate at 48 hpi was similar to that at 24 hpi.

[0040] The results showed that treatment with the nematicidal peptide Peptide-3 had a significant nematicidal effect, and the higher the concentration, the better.

[0041] As shown above, the agent containing nematicide-3 (experimental group) exhibited a significant nematicidal effect after treatment, and the nematicidal effect was concentration-dependent, with higher concentrations resulting in better nematicidal effects. Compared to experimental group 2 (nematicide-3 concentration of 0.33 mmol / L), experimental group 1 (1.00 mmol / L nematicide-3 concentration) showed a better nematicidal effect against root-knot nematodes.

[0042] Depending on the actual situation, the nematicide Peptide-3 is formulated as an agent for controlling root-knot nematodes at a concentration of 0.33 mmol / L to 1.00 mmol / L. Its specific application methods include, but are not limited to: in vitro immersion treatment with second-instar larvae of root-knot nematodes for nematicide killing; or direct application of the agent to the roots of plants for irrigation (the optimal concentration, dosage and frequency of root irrigation can be determined by routine experiments conducted by those skilled in the art based on the crop species and the severity of the disease).

[0043] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A nematicidal peptide Peptide-3 derived from Bacillus licheniformis, characterized in that: The amino acid sequence of the Bacillus licheniformis-derived nematicidal peptide Peptide-3 is shown in SEQ ID NO.

1.

2. The Bacillus licheniformis-derived nematicidal peptide Peptide-3 according to claim 1, characterized in that: The Bacillus licheniformis-derived nematicidal peptide Peptide-3 was obtained by introducing an N-terminal methionine into the Bacillus licheniformis-derived nematicidal peptide and optimizing the sequence based on physicochemical properties. The total hydrophobicity of the Bacillus licheniformis-derived nematicidal peptide Peptide-3 is 0.33, the total net positive charge ratio is +7.00, and the Wimley-White total residue hydrophobicity is 5.11 kcal / mol.

3. The application of the nematicide Peptide-3 as described in claim 1 or 2 in the control of root-knot nematodes and / or the creation of root-knot nematode-resistant plant germplasm resources.

4. The use of the nematicide-3 as described in claim 1 in the preparation of products for controlling root-knot nematodes.

5. An agent for controlling root-knot nematodes, characterized in that: The agent includes the Bacillus licheniformis-derived nematicidal peptide Peptide-3 as described in claim 1.

6. The pharmaceutical preparation according to claim 5, characterized in that: The concentration of the nematicide-3 in the agent is 0.33 mmol / L to 1.00 mmol / L.

7. The pharmaceutical agent according to claim 5 or 6, characterized in that: The solvent for the reagent is a 0.9% NaCl solution.

8. The use of the agent according to any one of claims 5 to 7 in the control of root-knot nematodes.

9. A method for controlling root-knot nematodes, characterized in that: The method includes soaking the agent according to any one of claims 5 to 7 with second-instar larvae of root-knot nematodes. Alternatively, the method may involve directly applying the agent according to any one of claims 5 to 7 to the plant through root irrigation.

10. The method according to claim 9, characterized in that: The specific steps of the method are as follows: hatch and collect second-instar larvae of root-knot nematodes, and incubate them together with the agent described in any one of claims 5 to 7 for 48 hours.

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