Application of microbial natural product GameXPeptide-A in improving pest control effect of insecticide

Abstract

The invention discloses application of a microbial natural product GameXPeptide-A in improving the pest control effect of an insecticide, and belongs to the technical field of biological control. Experiments prove that after the GameXPeptide-A is combined with insecticides such as metarhizium anisopliae, beauveria bassiana, nuclear polyhedrosis virus, 4% of abamectin acetamiprid, 10% of spinosad emamectin benzoate, 30% of etoxazole nitrile, 45% of biphenyl etoxazole, 5% of emamectin benzoate, 5% of chlorantraniliprole, 5% of abamectin and the like for use, the insecticidal effect is obviously improved; the insecticidal activity of the insecticide on agricultural pests such as thrips, ostrinia nubilalis and prodenia litura can be effectively improved, so that the substance can be combined with different insecticides for use, and the insecticidal activity of the insecticide on crop pests in agricultural production such as lepidoptera, diptera, coleoptera, orthoptera, hemiptera and homoptera is improved; the compound can be used for development and application of novel pest immunosuppressants or insecticide synergists.

Description

Technical Field

[0001] The present application belongs to the field of biological control technology, and in particular to the application of a natural microbial product GameX Peptide-A in improving the control effect of insecticides on pests. Background Art

[0002] Pests are extremely harmful to food production, causing losses of nearly $100 billion each year. Pesticides are the most commonly used plant protection method in modern agriculture, which can prevent and control pests and increase crop yields. However, the long-term overuse of chemical pesticides has led to negative problems such as pest resistance. Natural active products from microbial sources have many advantages, such as fast decomposition, less residue, and no pollution to the environment. They are effective alternatives to chemical pesticides and have great potential in agricultural production, meeting the requirements of sustainable development.

[0003] Insects have a powerful immune system that relies on humoral and cellular immune responses, including phagocytosis, nodulation and encapsulation, melanization, release of antimicrobial peptides, complex enzyme cascades, and activation of immune adaptive signaling pathways, for their own immune defense. By inhibiting the function of the pest immune system, reducing the pest's immune adaptability, and putting it in a sub-healthy state, the probability of pest death can be effectively increased. The toxins of many parasitic natural enemies, such as black egg wasps, ichneumon wasps, braconid wasps, golden wasps, etc., can interfere with the host's immune system and can be used in the development of specific biological pesticides, but they are difficult to develop industrially due to limited sources.

[0004] Symbionts of insect pathogenic nematodes are Gram-negative bacteria of the family Enterobacteriaceae that parasitize the intestines of insect pathogenic nematodes. They include the genera Xenorhabdus and Photorhabdus, which coexist with Steinernema and Heterorhabditis, respectively. When juvenile insect pathogenic nematodes infect the host through natural openings such as the mouth, anus, and spiracles, they release symbiotic bacteria into the host's hemocoel, causing the insect to die within 24 to 48 hours after infection. In order to survive in the insect body and complete its life cycle, suppressing the host immune system is essential for successful infection and host death. Insect pathogenic nematodes mainly rely on secondary metabolites produced by symbiotic bacteria with insect immunosuppressive activity to suppress the host's immune response.

[0005] GameXPeptide-A is a cyclic pentapeptide compound composed of 1 valine, 3 leucine and 1 phenylalanine amino acid residues, which can effectively inhibit insect immune responses, such as the production of antimicrobial peptides, phospholipase PLA2 activity and nodule formation. However, there are no reports on the effect of GameXPeptide-A combined with green anisopliae, beauveria bassiana, insect viruses and chemical pesticides in controlling agricultural pests. Summary of the invention

[0006] In order to overcome the above-mentioned defects existing in the prior art, the present application provides the use of a microbial natural product GameX Peptide-A in improving the insecticide's pest control effect. The GameX Peptide-A is obtained by separation and purification from the fermentation product of the insect pathogenic nematode symbiotic bacteria strain XBD101. The insect pathogenic nematode symbiotic bacteria strain XBD101 is deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration, and the deposit number is CGMCC No: 22056. The study found that GameX Peptide-A, when used in combination with insecticides such as Metarhizium anisopliae, Beauveria bassiana, nuclear polyhedrosis virus, chlorantraniliprole, acetamiprid, emamectin benzoate and avermectin, can effectively improve the insecticidal activity of insecticides against agricultural pests such as Spodoptera litura, thrips, citrus red spider, corn borer and diamondback moth. This indicates that the substance can be used in combination with contact insecticides, stomach poison insecticides, fumigation insecticides, systemic insecticides, biological insecticides, etc. to effectively improve the insecticidal activity of insecticides against Lepidoptera, Diptera, Coleoptera, Orthoptera, Hemiptera, Homoptera and other agricultural pests that harm crops, and can be used for the development and application of new pest immunosuppressants or insecticide enhancers.

[0007] In order to achieve the above-mentioned invention object, this application provides the following technical solutions:

[0008] On the one hand, the present application provides the use of a microbial natural product GameX Peptide-A in improving the efficacy of pesticides against pests.

[0009] Optionally, the GameX Peptide-A is separated and purified from the fermentation product of the insect pathogenic nematode symbiotic bacteria XBD101 strain.

[0010] Optionally, the fermentation product of the insect pathogenic nematode symbiotic bacteria strain XBD101 is obtained by placing the insect pathogenic nematode symbiotic bacteria strain XBD101 in a culture medium for fermentation.

[0011] Optionally, the culture medium includes separation medium, seed medium and fermentation medium in sequence.

[0012] Optionally, the separation medium comprises NBTA agar medium.

[0013] Optionally, the seed culture medium includes LB medium.

[0014] Optionally, the fermentation medium includes LB liquid medium.

[0015] Optionally, during the application process, GameX Peptide-A needs to be dissolved and diluted with dimethyl sulfoxide to 10 to 100 ppm.

[0016] Optionally, during the application process, GameX Peptide-A needs to be dissolved and diluted with dimethyl sulfoxide to any value of 10ppm (μg / mL), 20ppm, 30ppm, 40ppm, 50ppm, 60ppm, 70ppm, 80ppm, 90ppm, 100ppm or any range between two values.

[0017] Optionally, the insecticide includes one or more of contact insecticides, stomach poison insecticides, fumigation insecticides, systemic insecticides, and biological insecticides.

[0018] Optionally, the insecticide includes one or more of Metarhizium anisopliae, Beauveria bassiana, acetamiprid, emamectin benzoate, ethoxachlor, biphenyl-etoxazole, emamectin benzoate, nuclear polyhedrosis virus, chlorantraniliprole, and avermectin.

[0019] Optionally, the pests include one or more of Lepidoptera pests, Diptera pests, Coleoptera pests, Orthoptera pests, Hemiptera pests, and Homoptera pests.

[0020] Optionally, the pests include one or more of thrips, citrus red spider, corn borer, Spodoptera litura, and diamondback moth.

[0021] Optionally, the application is to use GameX Peptide-A in combination with an insecticide.

[0022] Optionally, the application is to use GameX Peptide-A in combination with a dilution of an insecticide.

[0023] Optionally, the final concentration of the GameX Peptide-A after being used in combination with a diluted solution of the insecticide is 10 to 50 μg / mL.

[0024] Optionally, the final concentration of the GameX Peptide-A after being used in combination with a dilution of the insecticide is independently selected from any value among 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, or any range value therebetween.

[0025] Optionally, the diluted solution of the insecticide is obtained by diluting the insecticide 1000 to 20000 times with water.

[0026] Compared with the prior art, this application has the following beneficial effects:

[0027] The present application provides the use of a microbial natural product GameX Peptide-A in improving the insecticide's pest control effect. The GameX Peptide-A is separated and purified from the fermentation product of the insect pathogenic nematode symbiotic bacteria XBD101 strain. The insect pathogenic nematode symbiotic bacteria XBD101 strain is a symbiotic bacteria of the pseudo-bicornutus Steinernema nematode Xenorhabdus budapestensis isolated from a soil sample collected in the northeast by the inventor's laboratory, and the deposit number is CGMCC No: 22056 (the strain has been disclosed in the patent CN113265364A text previously applied for by the inventor). Experiments have shown that the combined use of GameXPeptide-A with contact insecticides, stomach poison insecticides, fumigation insecticides, systemic insecticides, biological insecticides and other insecticides can effectively improve the insecticidal activity of insecticides against Lepidoptera, Diptera, Coleoptera, Orthoptera, Hemiptera, Homoptera and other agricultural pests that harm crops, and can be used for the development and application of new pest immunosuppressants or insecticide synergists. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 The experimental results of the compound GameX Peptide-A of the present application in combination with 4% acetamiprid and 10% emamectin benzoate insecticides to kill thrips (Note: the values ​​represent the mean ± SD of three repeated measurements from three independent experiments: *** indicates p < 0.001; **** indicates p < 0.0001; no mark indicates insignificance);

[0030] Figure 2 The experimental results of the compound GameX Peptide-A of the present application being used in combination with 30% ethoxaclone and 45% biphenyl·ethoxaclone insecticides to kill citrus spider mites (Note: the values ​​represent the mean ± SD of three repeated measurements from three independent experiments: * indicates p < 0.1; ** indicates p < 0.01; **** indicates p < 0.0001; those not marked are not significant);

[0031] Figure 3The experimental results of the compound GameX Peptide-A of the present application being used in combination with 5% emamectin benzoate, nuclear polyhedrosis virus and 5% chlorantraniliprole to kill corn borer (Note: the values ​​represent the mean ± SD of three repeated measurements from three independent experiments: * indicates p < 0.1; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001; those not marked are not significant);

[0032] Figure 4 The experimental results of the compound GameX Peptide-A of the present application in combination with the biological insecticides nuclear polyhedrosis virus (active ingredient content: 1 billion PIB / mL), green anisopliae (active ingredient content: 15 billion spores / g) and white bassiana (active ingredient content: 10 billion spores / g) to kill Spodoptera litura (Note: the value represents the mean ± SD of three repeated measurements from three independent experiments: **** indicates p < 0.0001);

[0033] Figure 5 The experimental results of the combined use of the compound GameX Peptide-A of the present application and 5% avermectin insecticide to kill Plutella xylostella (Note: the values ​​represent the mean ± SD of three repeated measurements from three independent experiments: *** represents p < 0.001; **** represents p < 0.0001).

[0034] Figure 6 Information of the compound GameX Peptide-A of this application (Note: a represents the structure of GameX Peptide-A; b represents the structure of GameX Peptide-A in DMSO-d6 1 HNMR spectrum (500 MHz); c represents the secondary mass spectrum information of GameX Peptide-A). DETAILED DESCRIPTION

[0035] The present application is further described below in conjunction with specific embodiments. The following are only a few embodiments of the present application, and are not intended to limit the present application in any form. Although the present application discloses the following preferred embodiments, they are not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the above disclosed technical content to make some changes or modifications are equivalent to equivalent implementation cases and are within the scope of the technical solution.

[0036] Unless otherwise specified, the raw materials in the examples of the present application were purchased from commercial sources and used directly without any special treatment.

[0037] Unless otherwise specified, the analysis methods in the examples all adopt conventional settings and conventional analysis methods of instruments or equipment.

[0038] Example 1

[0039] Compound GameXPeptide-A purification and identification:

[0040] Pretreatment of GXP crude extract samples: Since the hfq gene is an RNA chaperone gene that regulates the production of natural products in nematode symbiotic bacteria, knocking it out will result in silencing the synthesis of most secondary metabolites, including natural products synthesized by gene clusters. The hfq gene is preferentially deleted in the original strain of the XBD101 strain of the present application by engineering modification, and a mutant strain that synthesizes the gxpS gene cluster controlled by the arabinose promoter is constructed based on the Δhfq mutant strain, named ΔP BAD -gxpS-hfq-XBD8, and the mutant strain was induced for fermentation. BAD -gxpS-hfq-XBD8 was cultured with the first-level seed liquid. A single colony with a smooth surface and uniform size was selected from the NBTA medium plate using a sterilized pipette tip. The culture was cultured in LB liquid medium containing 50μg / mL ampicillin at a temperature of 28°C and a rotation speed of 200rpm for 18-20h. A seed liquid was inoculated into 10L LB medium (1L×10), and 5% XAD16 adsorption resin was added after fermentation for 48 hours. Fermented in a shake flask at 28°C for 48h (1L / 2L; 200rpm.). The XAD16 resin was separated from the supernatant by sieving, washed twice with 2L ddH2O, and eluted with a total volume of 3L anhydrous methanol (1L×4) and then rotary evaporated to obtain 40.8g of crude extract. According to the different polarity and molecular size of the substance, ODS reverse silica gel column chromatography technology was used for separation. The Thermo Scientific TSQ Quantum Ultra triple quadrupole liquid-mass spectrometer was used to perform mass spectrometry analysis and tracking of the crude extracts of each component, and the target components were prepared by gradient separation using a Syprex semi-preparative high-performance liquid chromatography, and the purified compounds were identified by secondary mass spectrometry and nuclear magnetic resonance. Figure 6 As shown in the figure, the active compound GameX Peptide-A was successfully purified. LC-MS detection confirmed the main ion peak at m / z 586.3946 [M+H] +, corresponding to the molecular formula C 32 H 51 N5O5 (such as Figure 6 c) in 3p 1 H-NMR and 2D-NMR data (HSQC, HMBC and TOCSY) showed that it is composed of one valine (Val), three leucine (Leu) and one phenylalanine (Phe) amino acid residues (e.g. Figure 6a). H, COSY, HSQC and HMBC spectra were acquired on a Bruker AV400 [400 MHz ( 1 H) and 100MHz( 13 C)] recorded on the spectrometer (e.g. Figure 6 b) in the above. 13 C NMR spectra were acquired on a Bruker AV300 [300 MHz ( 1 H) and 75MHz( 13 Chemical shifts relative to the solvent signal are reported in ppm (δ) ([D4]-MeOD; 1 H-NMR: δ=3.14ppm, [D6]-DMSO; 1 H-NMR: δ = 2.50 ppm; 13 C-NMR: δ = 39.51 ppm and [D5]-pyridine 1 H-NMR: δ = 7.22 ppm; 13 C-NMR: δ = 123.87 ppm).

[0041] Example 2

[0042] Experiment on the use of GameX Peptide-A and insecticides in combination to kill insects:

[0043] For indoor tests on killing thrips and citrus red spiders: fresh citrus leaves and cucumber leaves are preferred for use. Weigh 10 mg of the pure compound GameXPeptide-A and dissolve it in 10 mL of DMSO solution to prepare a 1 mg / mL GameXPeptide-A solution for subsequent dilution. Soak fresh leaves in a mixture of 10 mL of GXP solution and different concentrations of aqueous solutions of various insecticides in different proportions for 10 seconds. After taking out and drying, inoculate 20 adult mites or thrips, place them in an incubator at 27°C and RH95%, and culture under natural light conditions to observe and statistically analyze the mortality rate. The statistical results are as follows: Figure 1 shown.

[0044] For indoor tests on killing Spodoptera litura, Plutella xylostella and Ostrinia furnacalis: Weigh 10 mg of pure GameXPeptide-A compound and dissolve it in 10 mL of DMSO solution to prepare a 1 mg / mL GameXPeptide-A solution for subsequent dilution. Add 10 mL of GXP solution and different concentrations of aqueous solutions of various insecticides in different proportions to 24 g of insect feed, mix well and divide evenly into 9 mm petri dishes to dry. Set up three replicates, with 10 insects in each group. The statistical results are as follows: Figure 2-Figure 5 shown.

[0045] Depend on Figure 1It can be seen that after 10ppm (μg / mL) and 50ppm (μg / mL) GameX Peptide-A were mixed with a 20,000-fold diluted acetamiprid (active ingredient content: 4%) and a 6,000-fold diluted multi-sulphate emamectin benzoate (active ingredient content: 10%) insecticide aqueous solution, the 5-day mortality rate of thrips was significantly increased. Among them, the mortality rate of the group treated with 50ppm GXP solution and 20,000-fold diluted 10% multi-sulphate emamectin benzoate increased by 41.7% compared with the control group using only 10% multi-sulphate emamectin benzoate insecticide;

[0046] Depend on Figure 2 It can be seen that after 10ppm and 50ppm were mixed with 4000 times diluted biphenyl·ethoxazole (active ingredient content: 45%) and 3000 times diluted ethoxaclofon (active ingredient content: 30%) insecticide aqueous solutions, the mortality rate of citrus red spider mites reached 90% 4 days after the application, which was 26.4% higher than that of the control group.

[0047] Depend on Figure 3 It can be seen that after 10ppm and 50ppm were mixed with 20,000 times diluted emamectin benzoate (active ingredient content: 5%), 5,000 times diluted nuclear polyhedrosis virus (active ingredient content: 1 billion PIB / mL) and 10,000 times diluted chlorantraniliprole (active ingredient content: 5%), the mortality rate of corn borers was significantly increased. Among them, the mortality rate of the 50ppm GXP treatment group increased most significantly, which was about 23.4% higher than that of the control group;

[0048] Depend on Figure 4 It can be seen that after 10ppm and 50ppm were mixed with a 5000-fold diluted nuclear polyhedrosis virus (active ingredient content: 1 billion PIB / mL) insecticide aqueous solution, a 3000-fold diluted green anisopliae (active ingredient content: 15 billion spores / g) insecticide aqueous solution, and a 1000-fold diluted white bassiana (active ingredient content: 10 billion spores / g) insecticide aqueous solution, the mortality rate of Spodoptera litura was significantly increased. Among them, the mortality rate of the 50ppm GXP treatment group increased most significantly, and the 5-day mortality rate increased by about 30% compared with the control group;

[0049] Depend on Figure 5 It can be seen that after 50 ppm was mixed with 20,000 times diluted avermectin (active ingredient content: 5%), the mortality rate of diamondback moth was significantly increased. The mortality rate increased most significantly in 3 days, which was about 37.7% higher than that of the control group.

[0050] From the above results, it can be seen that the GameX Peptide-A solutions of different concentrations provided in the present application can be mixed with different pesticides to effectively enhance the insecticidal activity of the pesticides, with the mortality rate increased by up to 40% or more, and the enhancement effect is very significant.

[0051] The above experiments prove that the compound GameX Peptide-A provided in the present application can effectively improve the insecticidal activity against lepidopteran pests of corn borer, diamondback moth and Spodoptera litura when used in combination with insecticides such as green muscardine, beauveria bassiana, nuclear polyhedrosis virus, chlorantraniliprole, emamectin benzoate and avermectin, indicating that the compound can be used in combination with contact insecticides, stomach poison insecticides, fumigation insecticides, systemic insecticides, biological insecticides, etc. to improve the insecticidal activity of insecticides against agricultural pests that harm crops in agricultural production such as Lepidoptera, Diptera, Coleoptera, Orthoptera, Hemiptera, Homoptera, etc., and can be used for the development and application of new pest immunosuppressants or insecticides.

[0052] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. Application of microbial natural product GameX Peptide-A in improving the efficacy of pesticides against pests.

2. The use according to claim 1, characterized in that: The GameX Peptide-A is separated and purified from the fermentation product of the insect pathogenic nematode symbiotic bacteria XBD101 strain.

3. The use according to claim 2, characterized in that: The fermentation product of the insect pathogenic nematode symbiotic bacteria strain XBD101 is obtained by placing the insect pathogenic nematode symbiotic bacteria strain XBD101 in a culture medium for fermentation culture; Preferably, the culture medium comprises a separation medium, a seed medium and a fermentation medium in sequence; Preferably, the separation medium comprises NBTA agar medium; Preferably, the seed culture medium comprises LB medium; Preferably, the fermentation medium comprises LB liquid medium.

4. The use according to claim 1, characterized in that: During the application process, GameX Peptide-A needs to be dissolved and diluted with dimethyl sulfoxide to 10-100 ppm.

5. The use according to claim 1, characterized in that: The insecticide includes one or more of contact insecticide, stomach poison insecticide, fumigation insecticide, systemic insecticide and biological insecticide; Preferably, the insecticide comprises one or more of Metarhizium anisopliae, Beauveria bassiana, acetamiprid, emamectin benzoate, ethoxachlor, biphenyl-etoxazole, emamectin benzoate, nuclear polyhedrosis virus, chlorantraniliprole, and avermectin.

6. The use according to claim 1, characterized in that: The pests include one or more of Lepidoptera pests, Diptera pests, Coleoptera pests, Orthoptera pests, Hemiptera pests, and Homoptera pests; Preferably, the pests include one or more of thrips, citrus red spider, corn borer, Spodoptera litura, and diamondback moth.

7. The use according to claim 1, characterized in that: The application is to use GameX Peptide-A in combination with insecticides.

8. The use according to claim 7, characterized in that: The application is to use GameX Peptide-A in combination with a diluted solution of an insecticide.

9. The use according to claim 8, characterized in that: The final concentration of the GameX Peptide-A after being used in combination with the dilution of the insecticide is 10 to 50 μg / mL; Preferably, the diluted solution of the pesticide is obtained by diluting the pesticide 1000 to 20000 times with water.

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