Application of microbial natural product GameXPeptide-A in improving the control effect of insecticides on pests

Abstract

The application discloses application of a microbial natural product GameXPeptide-A in improving the control effect of insecticides on pests, and belongs to the technical field of biological control. Experiments prove that GameXPeptide-A can effectively improve the insecticidal activity of insecticides on agricultural pests such as thrips, corn borer and prodenia litura when GameXPeptide-A is combined with insecticides such as Metarrhizium anisopliae, Beauveria bassiana, nuclear polyhedrosis virus, 4% abamectin and pyridaben, 10% dimethoate and spinosad, 30% ethoxazole, 45% bifenthrin and etoxazole, 5% spinosad, 5% chlorantraniliprole, 5% abamectin and the like, and it is indicated that the substance can be combined with different insecticides to improve the insecticidal activity of insecticides on pests such as Lepidoptera, Diptera, Coleoptera, Orthoptera, Hemiptera and Homoptera in agricultural production, and can be used for the development and application of a new type of pest immunosuppressant or insecticide synergist.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological control, and particularly relates to application of a microorganism natural product GameXPeptide-A in improving the control effect of insecticides on pests. BACKGROUND

[0002] Pests cause great harm to grain production, resulting in nearly 100 billion US dollars of loss each year. Pesticides are the most commonly used plant protection method in modern agriculture, which can prevent and control pests and improve crop yield. However, long-term overuse of chemical pesticides leads to negative problems such as pest resistance. Microbial natural active products have many advantages, such as fast decomposition, less residue, and no environmental pollution, and are effective substitutes for chemical pesticides, which have great potential in agricultural production and meet the requirements of sustainable development.

[0003] Insects have a strong immune system, which relies on humoral and cellular immune responses, including phagocytosis, nodule and cyst formation, melanization, release of antimicrobial peptides, complex enzyme cascade reactions, and activation of immune adaptive signaling pathways for self-immune defense. By inhibiting the function of the immune system of pests, the immune adaptability of pests is reduced, and they are in a sub-healthy state, which can effectively increase the probability of pest death. The toxins of many parasitic natural enemies such as black tachinid fly, parasitic wasp, braconid wasp, and chalcid wasp can interfere with the immune system of the host, which can be used for the development of specific biological pesticides, but it is difficult to develop industries due to limited sources.

[0004] Insect pathogenic nematode symbiotic bacteria is a kind of gram-negative bacteria of enterobacteriaceae, which parasitizes in the intestinal tract of insect pathogenic nematode, including Xenorhabdus and Photorhabdus, which are symbiotic with Steinernema and Heterorhabditis respectively. When the juvenile insect pathogenic nematode invades the host through natural openings such as the mouth, anus and spiracles, they will release symbiotic bacteria into the hemocoel of the host, leading to the death of the insect within 24-48 hours after infection. In order to survive in the insect body and complete its life cycle, it is essential to inhibit the immune system of the host for successful infection and host death, and the insect pathogenic nematode mainly relies on the secondary metabolites produced by symbiotic bacteria with insect immune inhibitory activity to inhibit the immune response of the host.

[0005] GameXPeptide-A is a cyclic 5-peptide compound composed of 1 valine, 3 leucines and 1 phenylalanine amino acid residues, which can effectively inhibit the immune response of insects, such as the production of antimicrobial peptides, phospholipase PLA2 activity and nodule formation. However, there is no report on the effect of GameXPeptide-A combined with Metarhizium anisopliae, Beauveria bassiana and insect viruses and chemical pesticides in controlling agricultural pests. SUMMARY

[0006] To overcome the aforementioned deficiencies in the prior art, this application provides the application of the microbial natural product GameXPeptide-A in improving the insecticide's efficacy against pests. GameXPeptide-A is isolated and purified from the fermentation product of the symbiotic bacterium strain XBD101, which is associated with entomopathogenic nematodes. The entomopathogenic nematode symbiotic bacterium strain XBD101 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo:22056. Studies have found that GameXPeptide-A, when used in combination with insecticides such as Metarhizium anisopliae, Beauveria bassiana, nucleopolyhedrovirus, chlorantraniliprole, abamectin-acetamiprid, emamectin benzoate, and abamectin, can effectively enhance the insecticidal activity of these insecticides against agricultural pests such as beet armyworm, thrips, citrus red spider mite, 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, and biological insecticides to effectively improve the insecticidal activity of these insecticides against agricultural pests such as Lepidoptera, Diptera, Coleoptera, Orthoptera, Hemiptera, and Homoptera. It can be used in the development and application of new insect immunosuppressants or insecticide synergists.

[0007] To achieve the above-mentioned objectives, this application provides the following technical solution:

[0008] On the one hand, this application provides the application of the microbial natural product GameXPeptide-A in improving the insecticide's efficacy against pests.

[0009] Optionally, the GameXPeptide-A is isolated and purified from the fermentation product of the symbiotic bacteria strain XBD101 of entomopathogenic nematodes.

[0010] Optionally, the fermentation product of the entomopathogenic nematode symbiotic bacterium XBD101 is obtained by fermenting the entomopathogenic nematode symbiotic bacterium XBD101 in a culture medium.

[0011] Optionally, the culture medium may sequentially include an isolation culture medium, a seed culture medium, and a fermentation culture medium.

[0012] Optionally, the separation medium includes 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, GameXPeptide-A needs to be dissolved and diluted with dimethyl sulfoxide to 10-100 ppm.

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

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

[0018] Optionally, the insecticide includes one or more of the following: Metarhizium anisopliae, Beauveria bassiana, abamectin-acetamiprid, spinosad, etoxazole, bifenthrin-etoxazole, abamectin, nucleopolyhedrovirus, chlorantraniliprole, and abamectin.

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

[0020] Optionally, the pests include one or more of the following: thrips, citrus red spider mites, corn borers, beet armyworms, and diamondback moths.

[0021] Optionally, the application involves using GameXPeptide-A in conjunction with an insecticide.

[0022] Optionally, the application involves using GameXPeptide-A in combination with a diluted insecticide.

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

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

[0025] Optionally, the diluted solution of the insecticide is obtained by diluting the insecticide with water by 1,000 to 20,000 times.

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

[0027] This application discloses the application of the microbial natural product GameXPeptide-A in enhancing the insecticide's efficacy against pests. GameXPeptide-A is isolated and purified from the fermentation product of the symbiotic bacterium strain XBD101, which is *Xenorhabdus budapestensis*, a symbiotic bacterium of *Strombus budapestensis*, isolated from soil samples collected in Northeast China by the inventors' laboratory. The strain has the accession number CGMCC No:22056 (this strain has been disclosed in the inventors' previously filed patent CN113265364A). Experiments have shown that combining GameXPeptide-A with contact insecticides, stomach poison insecticides, fumigation insecticides, systemic insecticides, and biological insecticides can effectively enhance the insecticidal activity of insecticides against agricultural pests such as Lepidoptera, Diptera, Coleoptera, Orthoptera, Hemiptera, and Homoptera. This combination can be used in the development and application of new insect immunosuppressants or insecticide synergists. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The experimental results of using the compound GameXPeptide-A of this application in combination with 4% abamectin and 10% spinosad and emamectin benzoate to kill thrips are presented (Note: Values ​​represent the mean of three repeated measurements from three independent experiments ± SD; Experiment: *** indicates p < 0.001; **** indicates p < 0.0001; unmarked indicates not significant).

[0030] Figure 2 The experimental results of using the compound GameXPeptide-A of this application in combination with 30% etoxazole and 45% bifenthrin·etoxazole insecticides to kill citrus red spider mites are as follows (Note: Values ​​represent the mean of three repeated measurements from three independent experiments ± SD. Experiment: * indicates p < 0.1; ** indicates p < 0.01; **** indicates p < 0.0001; no indication indicates no significance).

[0031] Figure 3The experimental results of using the compound GameXPeptide-A in combination with 5% abamectin, nucleopolyhedrovirus, and 5% chlorantraniliprole to kill corn borers are presented (Note: Values ​​represent the mean of three repeated measurements from three independent experiments ± SD. Experiment: * indicates p < 0.1; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001; no indication indicates no significance).

[0032] Figure 4 The experimental results of using the compound GameXPeptide-A of this application in combination with the biopesticide nucleopolyhedrovirus (active ingredient content: 1 billion PIB / mL), Metarhizium anisopliae (active ingredient content: 15 billion spores / g), and Beauveria bassiana (active ingredient content: 10 billion spores / g) to kill Spodoptera litura (Note: Values ​​represent the average of three repeated measurements from three independent experiments ± SD experiment: **** indicates p < 0.0001);

[0033] Figure 5 The experimental results of using the compound GameXPeptide-A in combination with 5% abamectin insecticide to kill diamondback moth (Note: Values ​​represent the mean of three repeated measurements from three independent experiments ± SD experiment: *** indicates p < 0.001; **** indicates p < 0.0001).

[0034] Figure 6 Information regarding the compound GameXPeptide-A in this application (Note: a represents the structure of GameXPeptide-A; b represents the structure of GameXPeptide-A in DMSO-d6). 1 HNMR spectrum (500MHz); c represents the secondary mass spectrometry information of GameXPeptide-A). Detailed Implementation

[0035] The present application is further illustrated below with reference to specific embodiments. The following descriptions are merely a few embodiments of the present application and are not intended to limit the present application in any way. Although the present application discloses preferred embodiments as follows, they are not intended to limit the present application. Any modifications or variations made by those skilled in the art without departing from the scope of the technical solution of the present application using the disclosed technical content are equivalent to equivalent implementation cases and all fall within the scope of the technical solution.

[0036] Unless otherwise specified, the raw materials used in the embodiments of this application are all purchased commercially and used directly without any special treatment.

[0037] Unless otherwise specified, the analytical methods in the embodiments all adopt conventional instrument or equipment settings and conventional analytical methods.

[0038] Example 1

[0039] Purification and identification of compound GameXPeptide-A:

[0040] Pretreatment of GXP crude extract samples: Since the hfq gene is an RNA chaperone gene in nematode symbiotic bacteria that regulates the production of natural products, knocking it out silences the synthesis of most secondary metabolites, including natural products controlled by gene clusters. Therefore, we preferentially used engineering techniques to delete the hfq gene in the original XBD101 strain of this application, and constructed a mutant strain based on the Δhfq mutant strain, with the arabinose promoter controlling the synthesis of the gxpS gene cluster, named ΔP. BAD -gxpS-hfq-XBD8, and then induced fermentation of the mutant strain. First, ΔP BAD The primary seed culture of -gxpS-hfq-XBD8 was cultured. Smooth, uniformly sized single colonies were selected from NBTA agar plates using a sterile pipette tip and cultured in LB liquid medium containing 50 μg / mL ampicillin at 28℃ and 200 rpm for 18-20 h. One seed culture was then transferred to 10 L of LB medium (1 L × 10), and after fermentation for 48 hours, 5% XAD16 adsorption resin was added. Fermentation was carried out in shake flasks at 28℃ for 48 h (1 L / 2 L; 200 rpm). The XAD16 resin was separated from the supernatant by sieving, washed twice with 2 L ddH2O, and eluted with 3 L of anhydrous methanol (1 L × 4), followed by rotary evaporation to obtain 40.8 g of crude extract. Based on the differences in polarity and molecular size, ODS reversed-phase silica gel column chromatography was used for separation. Thermo Scientific TSQ Quantum Ultra triple quadrupole liquid chromatography-mass spectrometry (LC-MS) was used to analyze and track the crude extracts of each component. The target components were then separated using a gradient HPLC system, and the purified compounds were identified by secondary mass spectrometry (MS) and nuclear magnetic resonance (NMR). Results are as follows: Figure 6 As shown, the active compound GameXPeptide-A was successfully purified. LC-MS detection identified 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), 3p 1 H-NMR and 2D-NMR data (HSQC, HMBC, and TOCSY) indicate that it is composed of one valine (Val), three leucine (Leu), and one phenylalanine (Phe) amino acid residue (e.g. Figure 6a). H, COSY, HSQC and HMBC spectra in Bruker AV400 [400MHz ( 1 H) and 100MHz 13 C)] Recorded on the spectrometer (e.g. Figure 6 (b) 13 C NMR spectra at Bruker AV300 [300MHz ( 1 H) and 75MHz 13 Recorded on [C)]. Chemical shift relative to the solvent signal is 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] Experiments on the combined use of compound GameXPeptide-A and insecticides to kill pests:

[0043] For indoor experiments on thrips and citrus red spider mites: Fresh citrus and cucumber leaves were selected for use. 10 mg of pure GameX Peptide-A was dissolved in 10 mL of DMSO solution to prepare a 1 mg / mL GameX Peptide-A solution for subsequent dilution. Fresh leaves were soaked in mixtures of 10 mL of GXP solution and aqueous solutions of various insecticides at different concentrations in different proportions for 10 seconds. After drying, 20 adult mites or thrips were introduced and placed in an incubator at 27°C and 95% RH under natural light conditions. Mortality rates were observed and statistically analyzed. The results are as follows: Figure 1 As shown.

[0044] For indoor experiments on the control of Spodoptera litura, Diamondback moth, and Corn borer: 10 mg of pure GameX Peptide-A was dissolved in 10 mL of DMSO solution to prepare a 1 mg / mL GameX Peptide-A solution for subsequent dilution. 24 g of insect feed was mixed with 10 mL of GXP solution and various concentrations of insecticide aqueous solutions in different proportions. The mixture was then evenly distributed into 9 mm petri dishes and air-dried. Three replicates were set up, with 10 insects in each group. Statistical results are shown below. Figures 2-5 As shown.

[0045] Depend on Figure 1It was found that the 5-day mortality rate of thrips was significantly increased when 10 ppm (μg / mL) and 50 ppm (μg / mL) GameXPeptide-A were mixed with aqueous solutions of abamectin-acetamiprid (active ingredient content: 4%) diluted 20,000 times and spinosad-emamectin benzoate (active ingredient content: 10%) diluted 6,000 times. Specifically, the mortality rate of the 50 ppm GXP solution and the 10% spinosad-emamectin benzoate diluted 20,000 times was 41.7% higher than that of the control group treated with only 10% spinosad-emamectin benzoate.

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

[0047] Depend on Figure 3 It was found that mixing 10 ppm and 50 ppm of emamectin benzoate (effective ingredient content: 5%) diluted 20,000 times, nucleopolyhedrovirus (effective ingredient content: 1 billion PIB / mL) diluted 5,000 times, and chlorantraniliprole (effective ingredient content: 5%) diluted 10,000 times significantly increased the mortality rate of corn borers. The 50 ppm GXP treatment group showed the most significant increase in mortality, with an increase of approximately 23.4% compared to the control group.

[0048] Depend on Figure 4 It was found that when 10 ppm and 50 ppm were mixed with aqueous solutions of nucleopolyhedrovirus (active ingredient content: 1 billion PIB / mL) insecticide diluted 5000 times, Metarhizium anisopliae (active ingredient content: 15 billion spores / g) insecticide diluted 3000 times, and Beauveria bassiana (active ingredient content: 10 billion spores / g) insecticide diluted 1000 times, respectively, the mortality rate of Spodoptera litura was significantly increased. Among them, the mortality rate of the 50 ppm GXP treatment group was the most significantly increased, with a mortality rate of about 30% higher than that of the control group after 5 days.

[0049] Depend on Figure 5 It was found that when 50 ppm was mixed with abamectin (active ingredient content: 5%) diluted 20,000 times, the mortality rate of diamondback moth was significantly increased. The most significant increase was observed in the mortality rate after 3 days, which was approximately 37.7% higher than the control group.

[0050] The results above show that the GameXPeptide-A solution of different concentrations provided in this application, when used in combination with different insecticides, can effectively enhance the insecticidal activity of the insecticides, increasing the mortality rate by up to 40% or more, which is a very significant improvement.

[0051] The above experiments demonstrate that the compound GameXPeptide-A provided in this application, when used in combination with insecticides such as Metarhizium anisopliae, Beauveria bassiana, nucleopolyhedrovirus, chlorantraniliprole, abamectin, and emamectin, can effectively enhance the insecticidal activity against lepidopteran pests such as corn borer, diamondback moth, and beet armyworm. This indicates that the compound can be used in combination with contact insecticides, stomach poison insecticides, fumigation insecticides, systemic insecticides, and biological insecticides to improve the insecticidal activity of insecticides against agricultural pests such as lepidopterans, dipterans, coleopterans, orthoptera, hemiptera, and homoptera. It can be used in the development and application of new insect immunosuppressants or insecticides.

[0052] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. Application of GameX Peptide-A, a natural microbial product, in enhancing the insecticide's efficacy against pests; The GameXPeptide-A was isolated and purified from the fermentation product of the symbiotic bacteria XBD101 strain of entomopathogenic nematodes; The entomopathogenic nematode symbiotic bacteria strain XBD101 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No:22056. The insecticide is *Metarhizium anisopliae*, *Beauveria bassiana*, or abamect Acetamiprid, spinosad, etoxazole, biphenyl Etoxazole, abamectin, nucleopolyhedrovirus, chlorantraniliprole, avermectin; The pests mentioned are thrips, citrus red spider mites, corn borers, beet armyworms, and diamondback moths; The application is as follows: GameXPeptide-A was used in combination with a 3000-fold diluted aqueous solution of Metarhizium anisopliae insecticide, wherein the effective ingredient content of Metarhizium anisopliae was 15 billion spores / g; GameXPeptide-A was used in combination with a Beauveria bassiana insecticide aqueous solution diluted 1000 times, wherein the effective ingredient content of Beauveria bassiana was 10 billion spores / g; GameX Peptide-A diluted 6000 times with Avi The combined use of acetamiprid insecticide aqueous solution, and the aforementioned abamectin The active ingredient content of acetamiprid is 4%; Compare GameX Peptide-A with a multi-kill diluted 20,000 times. The combined use of abamectin insecticide aqueous solution, namely spinosad The active ingredient content of abamectin is 10%; GameXPeptide-A was used in combination with an aqueous solution of etoxazole insecticide diluted 3000 times, wherein the effective ingredient content of etoxazole was 30%. GameX Peptide-A was mixed with biphenyl diluted 4000 times. The biphenyl was used in combination with etoxazole insecticide aqueous solution. The active ingredient content of etoxazole is 45%; GameXPeptide-A was used in combination with an aqueous solution of abamectin insecticide diluted 20,000 times, wherein the active ingredient content of abamectin was 5%. GameXPeptide-A was used in combination with a nucleopolyhedrovirus insecticide aqueous solution diluted 5000 times, wherein the effective ingredient content of the nucleopolyhedrovirus was 1 billion PIB / mL; GameXPeptide-A was used in combination with a chlorantraniliprole insecticide aqueous solution diluted 10,000 times, wherein the active ingredient content of chlorantraniliprole was 5%. GameX Peptide-A was used in combination with an aqueous solution of abamectin insecticide diluted 20,000 times, wherein the active ingredient content of the abamectin was 5%. The final concentration of GameXPeptide-A when used in combination with the diluted solution of the above-mentioned insecticide is 50 μg / mL.

2. The application according to claim 1, characterized in that, The fermentation product of the entomopathogenic nematode symbiotic bacteria strain XBD101 is obtained by fermenting the entomopathogenic nematode symbiotic bacteria strain XBD101 in a culture medium. The culture medium includes, in sequence, an isolation culture medium, a seed culture medium, and a fermentation culture medium; The separation medium includes NBTA agar medium; The seed culture medium includes LB medium; The fermentation medium includes LB liquid medium.

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