Meloidogyne-resistant oligopeptide and application thereof

Abstract

The invention provides an oligopeptide for resisting root-knot nematode and application of the oligopeptide. The amino acid sequence of the oligopeptide is HIIPNPFHDFA or SIKRWPLII. The anti-root-knot nematode oligopeptide comes from biocontrol bacteria with an inhibiting effect on root-knot nematode, can provide an effective anti-nematode effect at the concentration level of mu g / mL, is remarkable in effect and environment-friendly, and is suitable for being used as a biopesticide.

Description

Field of the Invention

[0001] The present application belongs to the field of proteins and pesticides. Specifically, the present application provides short peptides for resisting root-knot nematodes and applications thereof. Background Art

[0002] Root-knot nematode disease is a soil-borne disease caused by root-knot nematodes. Infected plants are prone to forming giant cells in their root tissue, leading to root knots and severely impacting nutrient transport and other plant functions. Tobacco is one of the crops most severely affected by root-knot nematodes, resulting in significant economic losses and environmental pollution each year. The dominant root-knot nematode species affecting tobacco in southern Taiwan is the southern root-knot nematode.

[0003] Currently, the main means of controlling root-knot nematode disease is chemical pesticides. However, the damage caused by chemical pesticides to the environment and soil ecology has led to increasingly restricted application.

[0004] Developing biopesticides such as metabolites and peptides from plants and microorganisms is the main way to greenly control root-knot nematodes. Summary of the Invention

[0005] The applicant screened short peptides against root-knot nematodes in the Citrobacter freundii strain, which has a good biocontrol effect on root-knot nematodes. The applicant screened peptides in the bacterial cell hydrolysis products and fermentation broth, and identified two short peptides against root-knot nematodes with good practicality in the bacterial cell hydrolysis products.

[0006] On the one hand, the present application provides a short peptide for resisting root-knot nematodes, wherein the amino acid sequence of the short peptide is HIIPNPFHDFA or SIKRWPLII.

[0007] On the other hand, the present application provides the use of the above short peptide in the preparation of pesticides for controlling root-knot nematodes.

[0008] Furthermore, the root-knot nematode is Meloidogyne incognita.

[0009] Furthermore, the pesticide is a liquid preparation.

[0010] Furthermore, the pesticide also contains auxiliary ingredients acceptable to pesticides.

[0011] Those skilled in the art can select suitable excipients for the polypeptide based on factors such as solubility and stability, including but not limited to solvents, cosolvents, surfactants, antioxidants, preservatives, etc.

[0012] In another aspect, the present application provides a method for inhibiting tobacco root-knot nematode disease, comprising applying a liquid formulation containing the above-mentioned short peptide to tobacco plants.

[0013] Furthermore, the method comprises applying a liquid preparation containing the short peptide to the tobacco plants by root irrigation.

[0014] Furthermore, the root-knot nematode is Meloidogyne incognita.

[0015] Furthermore, the liquid preparation contains 20-100 μg / mL of the short peptide.

[0016] On the other hand, the present application provides a method for preparing the above-mentioned short peptide, which is a solid-phase synthesis method.

[0017] The solid-phase synthesis method is the method used when the synthesis is commissioned by the company in the examples of this application. In order to further expand the production scale / reduce costs, those skilled in the art can use other synthesis or production methods, such as using genetic engineering methods to express in host cells.

[0018] The above preferred technical solutions do not limit the scope of protection of this application. For example, due to the obvious killing effect on root-knot nematodes themselves, in addition to tobacco, the short peptide of this application can also be used for other plants suffering from root-knot nematode disease. After appropriate verification, the short peptide of this application can also be used to kill other types of root-knot nematodes besides southern root-knot nematodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The secondary mass spectrum and corresponding sequence of HIIPNPFHDFA.

[0020] Figure 2 The secondary mass spectrum and corresponding sequence of SIKRWPLII.

[0021] Figure 3 Representative root conditions of plants treated with peptide (right) and distilled water (left). DETAILED DESCRIPTION

[0022] The Citrobacter freundii used in the examples was derived from CGMCC No. 1.12830, and was propagated and preserved by the applicant.

[0023] The southern root-knot nematode (Meloidogyne incognita) used in the examples was collected and identified by the applicant and its cooperating units. Common long-necked yellow tobacco plants were used for indoor breeding and preservation.

[0024] Example 1: Bacteria fragmentation and enzymolysis

[0025] After the preserved Citrobacter freundii strain was revived and activated, it was cultured in liquid LB medium until the logarithmic phase. The bacterial solution was centrifuged to obtain the precipitate, resuspended in physiological saline, and centrifuged again to obtain the precipitate (repeat twice to remove the medium components). An appropriate amount of distilled water was added to resuspend the cells to a cell density of approximately 1×10 8 cells / mL.

[0026] 50 mL of bacterial suspension was placed in an ice bath for ultrasonic disruption. The ultrasonic disruptor used a 6 mm probe, a power of 400 W, and a cycle of 10 s after 5 s of operation. The total disruption time was 20 min.

[0027] Add 5 w / v% composite protease (400,000 U / g trypsin and 100,000 U / g papain in a mass ratio of 1:2) to the disrupted bacterial solution, adjust the pH to 7.0, and shake in a 50°C water bath for 60 minutes for enzymatic hydrolysis.

[0028] After cooling, the mixture was filtered through filter paper for use in the following experiments.

[0029] Example 2 Nematicidal Effects of Enzymatic Hydrolysates of Bacteria with Different Molecular Weights

[0030] The enzymatic hydrolysate obtained in Example 1 was fractionated into three parts: 10-30 kDa, 3-5 kDa, and below 3 kDa using an Amicon Ultra ultrafiltration device (equipped with a 15 mL centrifugal filter) with a molecular weight cut-off, and freeze-dried into powder for the following nematicidal activity experiments.

[0031] Basic experimental methods for nematicidal activity:

[0032] A large number of second-instar root-knot nematode larvae were obtained by selecting appropriate egg cysts and then hatching them uniformly. The larvae were prepared into a worm suspension of about 200 larvae / mL.

[0033] Approximately 100 larvae were added to each well of a plate along with various concentrations of the enzymatic hydrolysate components or a distilled water control (triplicate experiments). The cells were incubated at 25°C for 48 hours and the mortality rate was calculated (instability was considered as death). The mortality rate was corrected by subtracting the control group's mortality rate to determine the actual drug effect:

[0034] (Mortality rate of drug group - mortality rate of control group) / (1 - mortality rate of control group) × 100%

[0035] The nematicidal activities of the three fractions are shown in Table 1. The 3 kDa fraction with the lowest molecular weight was significantly more effective than the two fractions with higher molecular weights, indicating that the main component of the nematicidal activity should be a peptide or a protein with a low molecular weight.

[0036] Table 1 Evaluation of nematicidal activity of each component of enzymatic hydrolysis product

[0037] Corrected mortality % 10-30 kDa 3-5kD Below 3kD 5mg / mL 5.14 7.13 18.95 10 mg / mL 12.37 15.67 26.92 20 mg / mL 18.53 21.42 34.59 50mg / mL 28.25 23.83 43.28 .

[0038] Example 3 Identification of polypeptides in fractions below 3 kDa

[0039] The best-performing fraction below 3 kD in Example 2 was prepared into a solution at a concentration of 10 mg / mL using mobile phase, and filtered for RPLC-MS:

[0040] Reverse phase RPLC-MS was performed using a WATERS 2685-WATERS ACQUITY Q Da II system: the chromatographic column was an XSelect Premier Peptide CSH C18 column (2.5 μm, 2.1×150 mm); the mobile phase was acetonitrile / water / trifluoroacetic acid (50:50:0.1); the flow rate was 0.2 mL / min; the detection wavelength was 220 nm; the spray voltage was 1.5 kV; the gas pressure was 20 psi; the collision gas was argon; and the collision energy was 35 volts.

[0041] The RPLC results were separated into seven components at different retention times. Considering the peak area percentages and peak properties (component 6 is a free amino acid), peaks 2-4 were selected for further analysis of nematicidal activity at 10 mg / mL according to the method of Example 2. Based on the results, components 3 and 4 were further selected for peptide analysis (Table 2).

[0042] Table 2 RPLC results grouping and nematicidal activity

[0043]

[0044] The mass spectrometry results were filtered and quality controlled, and Mzcloud62 was used for scoring to screen peptides with a false discovery rate of less than 1%, resulting in the identification of a total of 376 peptides.

[0045] GO analysis was performed on the 376 identified peptides, excluding peptides related to development, movement, reproduction, monitoring, organelle proteins, intracellular proteins, catalysis, electron carrier transfer, and epigenetic structural molecules. Only peptides related to antioxidant, binding, membrane proteins, and immunity were retained. A total of 17 potential peptides were identified. After analysis of the difficulty / cost of synthesis and the basic properties of the peptides, NewPu Bio was commissioned to synthesize 5 of them using the solid-phase synthesis method to verify their anti-nematode activity. It was found that two of the peptides had significant anti-nematode activity. Their sequences and secondary mass spectra are shown in the figure. Figure 2 , the specific activity is shown in Table 3:

[0046] Table 3 Nematicidal effects of CF-M-2 and CF-M-3

[0047]

[0048] CF-M-2 (SIKRWPLII, SEQ ID NO. 1) and CF-M-3 (HIIPNPFHDFA, SEQ ID NO. 2) exhibit excellent anti-nematode activity, both providing significant anti-nematode efficacy at concentrations as low as μg / mL. CF-M-2 exhibits a faster onset of action, producing moderate insecticidal activity within 24 hours, while CF-M-3 exhibits a slightly slower onset of action, reaching similar levels of activity after 48 hours. However, its hydrophilicity makes formulation relatively easy.

[0049] Example 4 Anti-nematode effect of peptides in actual tobacco plants

[0050] Considering the high price of solid-phase synthetic peptides (although a peptide with a lower synthesis price was selected for synthesis research in Example 4, the price was still around 50 yuan / mg), the nematicidal effects of CF-M-2 and CF-M-3 were first verified on potted tobacco plants.

[0051] Tobacco seedlings were cultivated in sterilized soil and inoculated with approximately 1,000 second-instar larvae of the southern root-knot nematode (Meloidogyne incognita) per pot. Seven days after inoculation, 200 mL of 50 μg / mL CF-M-2 or CF-M-3, distilled water, and 2% avermectin were administered to the roots of 10 pots per treatment. Eight weeks after administration, tobacco survival was observed, and the root knot inhibition rate was calculated (number of root knots in the control group minus the number of knots in the treatment group / number of root knots in the control group).

[0052] Representative root system conditions and root knot inhibition rate results are as follows Figure 3 As shown in Table 4:

[0053] Table 4 Root knot inhibition rate of different groups

[0054] Group Root knot inhibition rate% 50 μg / mL CF-M-2 65.35±4.31 50 μg / mL CF-M-3 71.29±7.94 2% avermectin 83.17±5.85

[0055] like Figure 3 As shown, the root system of the treated group was significantly better than that of the distilled water control, with fewer root knots, larger root volume, and more abundant capillary roots. CF-M-2 or 3 at a concentration of 50 μg / mL can provide root knot inhibition comparable to 2% avermectin, while also having better environmental effects, showing potential as a biopesticide.

Claims

1. A short peptide for resisting root-knot nematodes, characterized in that: The amino acid sequence of the short peptide is HIIPNPFHDFA or SIKRWPLII.

2. Use of the short peptide according to claim 1 in the preparation of pesticides for controlling root-knot nematodes.

3. The use according to claim 2, wherein the root-knot nematode is Meloidogyne incognita.

4. The use according to claim 2, wherein the pesticide is a liquid preparation.

5. The use according to claim 3, wherein the pesticide further comprises an adjuvant component acceptable in pesticides.

6. A method for inhibiting tobacco root knot nematode disease, characterized in that: The method comprises applying a liquid formulation containing the short peptide according to claim 1 to tobacco plants.

7. The method according to claim 6, comprising applying a liquid formulation containing the short peptide according to claim 1 to tobacco plants by root irrigation. The method according to claim 6 , wherein the root-knot nematode is Meloidogyne incognita.

9. The method according to claim 6 or 7, wherein the liquid preparation contains 20-100 μg / mL of the short peptide according to claim 1.

10. The method for preparing the short peptide according to claim 1, characterized in that: The method is a solid phase synthesis method.

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