W-5 strain for improving pesticide tolerance of natural enemy insects and preparation method of W-5 strain

Abstract

The invention discloses a W-5 strain for improving pesticide tolerance of natural enemy insects and a culture method of the W-5 strain, the W-5 strain can well grow in a lowest-salt culture medium with lambda-lambda-cyhalothrin as a unique carbon source, the OD600 of a cultured bacteria solution is continuously increased, and the degradation rate of cyhalothrin pesticide reaches 98.24%. Furthermore, after the tenebrio molitor treated by the W-5 is fed to the hunting fuscipes, the activity of two types of key detoxifying enzymes, namely glutathione S-transferase and cytochrome P450, in the body of the hunting fuscipes can be remarkably improved. After the strain is continuously fed for one week, the W-5 is successfully colonized in intestinal tracts of the hpactor fuscipes, and the survival rate of the hpactor fuscipes under the stress of lambda-lambda-cyhalothrin is increased to 90.625%. Therefore, through double mechanisms of'direct microbial degradation 'and'host detoxification metabolism activation', the W-5 synergistically enhances the resistance of the Harpactor fuscipes to the lambda-lambda-cyhalothrin.

Description

Technical Field

[0001] This invention belongs to the field of strain technology, specifically relating to a W-5 strain that improves the pesticide tolerance of natural enemy insects and its preparation method. Background Technology

[0002] The painted assassin bug is an indispensable "biological guardian" in the agricultural ecosystem. Through its powerful predatory abilities, it provides sustainable and pollution-free pest control services for agricultural production. Studies have shown that a single painted assassin bug (especially older nymphs and adults) can prey on a large number of pests throughout its life cycle. A single adult can consume dozens or even hundreds of aphids per day.

[0003] However, the scarlet assassin bug is highly sensitive to broad-spectrum chemical pesticides such as organophosphates and pyrethroids. Therefore, when pesticide application is necessary, selective insecticides such as certain insect growth regulators and microbial pesticides must be prioritized, and application should be avoided during peak periods of natural enemy activity. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a W-5 strain for enhancing pesticide tolerance in natural enemy insects, along with its construction method and applications. The *Staphylococcus saprophyticus* W-5 strain described in this invention can synergistically enhance the resistance of the *Aspergillus oryzae* to λ-cyhalothrin through a dual mechanism of "direct microbial degradation" and "host detoxification metabolic activation."

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0006] A strain of strain W-5 that enhances the pesticide tolerance of natural enemy insects, classified as Stapyhlococcus sp. W-5, with accession number GDMCC No. 67176, deposited on October 29, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0007] Preferably, the 16S rDNA sequence of the W-5 strain is shown in SEQ ID No. 1.

[0008] The application of the W-5 strain, which enhances the pesticide tolerance of natural enemy insects, in improving the host's tolerance to λ-cyhalothrin.

[0009] The method for cultivating the W-5 strain to enhance pesticide tolerance in natural enemy insects includes the following steps:

[0010] (1) Grind the intestinal tissue of the natural enemy insects thoroughly to obtain a tissue homogenate. After centrifugation, collect the supernatant for later use.

[0011] (2) Take the supernatant obtained in step (1), add it to the liquid culture medium, mix thoroughly, place it in a constant temperature shaker, shake and culture to obtain bacterial solution; dilute the obtained bacterial solution to obtain bacterial solution dilution;

[0012] (3) Take the bacterial solution obtained in step (2), spread it evenly on a solid culture medium plate, and carry out plate culture; pick out single colonies with different morphologies, and purify them by streak plate method to obtain the W-5 strain.

[0013] In step (1), the natural enemy insect is the adult female assassin bug; the centrifugation treatment is centrifugation at 3000 r / min for 5 min.

[0014] In step (2), the liquid culture medium is any one of LB liquid culture medium, EB liquid culture medium or NA liquid culture medium.

[0015] In step (2), the culture solution is placed in a constant temperature shaker at 37°C and shaken at 200 r / min for 12 h to obtain the culture solution;

[0016] The dilution specifically involves adding sterile liquid culture medium or PBS to the obtained bacterial culture for dilution. This dilution process is repeated multiple times to obtain 10... -1 Up to 10 -9 Gradual dilution of bacterial culture.

[0017] In step (3), take 100 μL of 10 -5 Up to 10 -9 Diluted bacterial solutions are evenly spread on solid culture medium plates.

[0018] In step (3), the solid culture medium plate is any one of LB solid culture medium plate, EB solid culture medium plate or NA solid culture medium plate.

[0019] In step (3), the plate culture conditions are: placed in a 37°C constant temperature incubator for 48 h;

[0020] The purification culture was carried out by passage purification every 48 hours for 5 consecutive generations.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) This invention provides a W-5 strain that enhances the pesticide tolerance of natural enemy insects. The W-5 strain can grow well in a minimum salt medium with 50 mg / L λ-cyhalothrin as the sole carbon source. After 72 h of culture, the bacterial culture OD 600The degradation rate of lambda-cyhalothrin pesticide continued to rise, reaching 98.24%. Furthermore, feeding W-5-treated mealworms to *Aspergillus simonii* significantly enhanced the activity of two key detoxification enzymes—glutathione S-transferase and cytochrome P450. After one week of continuous feeding with this strain, W-5 successfully colonized the intestines of *Aspergillus simonii*, increasing the survival rate of *Aspergillus simonii* under lambda-cyhalothrin stress to 90.625%. In summary, *Staphylococcus saprophyticus* W-5 can synergistically enhance the resistance of *Aspergillus simonii* to lambda-cyhalothrin through a dual mechanism of "direct microbial degradation" and "host detoxification metabolic activation." The W-5 strain described in this invention provides a new technical pathway for enhancing the pesticide tolerance of natural enemy insects using insect symbiotic microorganisms, and has significant value for promoting the practical application of *Aspergillus simonii* in field biological control.

[0023] (2) This invention provides a method for culturing the W-5 strain to improve the pesticide tolerance of natural enemy insects. The method involves first thoroughly grinding the intestinal tissue of the *Astrophytum asterias* to obtain a tissue homogenate, centrifuging to obtain the supernatant for liquid culture, diluting the resulting bacterial solution, and then performing plate culture and purification to finally prepare the W-5 strain. The W-5 strain can grow well in a minimum salt medium with 50 mg / L λ-cyhalothrin as the sole carbon source. After 72 h of culture, the OD of the bacterial solution... 600 The degradation rate of lambda-cyhalothrin pesticide continued to rise, reaching 98.24%. Furthermore, feeding W-5-treated mealworms to *Aspergillus simonii* significantly enhanced the activity of two key detoxification enzymes—glutathione S-transferase and cytochrome P450. After one week of continuous feeding with this strain, W-5 successfully colonized the intestines of *Aspergillus simonii*, increasing the survival rate of *Aspergillus simonii* under lambda-cyhalothrin stress to 90.625%. In summary, *Staphylococcus saprophyticus* W-5 can synergistically enhance the resistance of *Aspergillus simonii* to lambda-cyhalothrin through a dual mechanism of "direct microbial degradation" and "host detoxification metabolic activation." The W-5 strain described in this invention provides a new technical pathway for enhancing the pesticide tolerance of natural enemy insects using insect symbiotic microorganisms, and has significant value for promoting the practical application of *Aspergillus simonii* in field biological control. Attached Figure Description

[0024] 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.

[0025] Figure 1This is a photograph of the bacterial strain on a plate.

[0026] Figure 2 Figure showing the phylogenetic analysis results of strain W-5;

[0027] Figure 3 The HPLC standard curve for λ-cyhalothrin;

[0028] Figure 4 This is a growth curve of strain W-5;

[0029] Figure 5 The degradation effect of strain W-5 on λ-cyhalothrin;

[0030] Figure 6 Determination of colonization of strain W-5 in the gut of the variegated assassin bug;

[0031] Figure 7 The effect of strain W-5 on the glutathione S-transferase (GST) activity of the variegated assassin bug;

[0032] Figure 8 The effect of strain W-5 on the carboxylesterase (CarE) activity of the variegated assassin bug;

[0033] Figure 9 The effect of strain W-5 on the survival rate of the variegated assassin bug. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] The above technical solution will be described in detail below with reference to specific embodiments.

[0036] Example 1: In vitro culture and identification of intestinal strain W-5

[0037] (1) Ten healthy, similarly sized, wild-type adult female assassin bugs were randomly selected and starved for 24 hours. The bugs were surface-sterilized by immersion in 75% (v / v) ethanol for 30 seconds. They were then rinsed three times with sterile PBS buffer. The intestines were aseptically dissected and placed in a 1.5 mL centrifuge tube. The intestinal tissue was homogenized thoroughly using an electric homogenizer. The tissue homogenate was centrifuged at 3000 r / min for 5 min, and the supernatant was transferred to a new 1.5 mL centrifuge tube for later use.

[0038] (2) Bacterial culture and serial dilution: Take 5 mL of sterile LB, EB and NA liquid culture medium into sterile 15 mL centrifuge tubes respectively. Take 200 μL of the supernatant from each and add it to the above three liquid culture media, and mix thoroughly. Place in a 37°C constant temperature shaker and shake at 200 r / min for 12 h. Take 100 μL of overnight culture and add it to 900 μL of the corresponding sterile liquid culture medium or PBS (10 -1 (Dilution), mix well. Repeat the above dilution steps to obtain 10. -1 Up to 10 -9 Gradual dilution of bacterial culture.

[0039] (3) Plate coating separation and purification: Take 100 μL of 10 -5 Up to 10 -9 Diluted bacterial suspensions were evenly spread onto corresponding LB, EB, and NA solid agar plates, with three replicates for each dilution, and clearly labeled. The plates were incubated at 37°C for 48 hours. Single colonies with varying morphologies were picked and purified using the streak plate method. Subculture was performed every 48 hours for a total of five generations. The colony morphology of the final purified plate was observed and recorded (using photomicrography).

[0040] Example 2: Identification and Phylogenetic Analysis of Strain W-5

[0041] 1. Colony morphology characteristics

[0042] W-5 colony and cell morphology as follows Figure 1 As shown. Its colony morphology on LB agar plates is: white, round, raised, smooth surface, and opaque center. Figure 1 ).

[0043] 2. Molecular biological identification

[0044] (1) Genomic DNA of the preserved monoclonal strain was extracted using the TIANamp Bacteria DNA Kit from Tiangen Biotech Co., Ltd. Using the extracted DNA as a template, the 16S rDNA of the bacteria was amplified using the universal 16S rDNA primers 27F (5'-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') as upstream and downstream primers. The PCR reaction system is shown in Table 1. After preparation, the mixture was gently mixed, briefly centrifuged, and placed on a PCR instrument according to the following reaction program: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 15 s, 35 cycles; 72℃, 5 min; 4℃, end. The PCR products were detected by 1.5% agarose gel electrophoresis, purified by gel extraction, and sent to Qingke Biotechnology Co., Ltd. (Guangzhou) for sequencing.

[0045] Table 1 - Bacterial 16S rDNA PCR amplification system (20 μL)

[0046] Components Volume (μL) I5 Mix (Tsingke) 10 <![CDATA[ddH2O]]> 7 27F (10μmol) 1 1492R (10 μmol) 1 gDNA 1

[0047] (3) Phylogenetic analysis of strain W-5

[0048] Molecular biological identification: Genomic DNA of the strain was extracted and purified.

[0049] Using DNA as a template, PCR amplification of the 16S rRNA gene was performed using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'). The PCR system (20 μL) consisted of: 10 μL 2×Taq Mix polymerase, 1 μL 27F primer (10 μmol / L), 1 μL 1492R primer (10 μmol / L), 1 μL DNA template, and 7 μL ddH2O.

[0050] PCR program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 90 s, for a total of 35 cycles; 72℃ final extension for 10 min; store at 4℃. PCR products were detected by 1.0% agarose gel electrophoresis. Products containing the target band were sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing.

[0051] The 16S rDNA sequence of strain W-5 is as follows (SEQ ID No. 1):

[0052]

[0053] To accurately identify the taxonomic position of strain W-5, multiple sequence alignment and phylogenetic analysis were employed for validation. First, homologous protein sequences were obtained from the NCBI database, and multiple sequence alignment was performed using MAFFT. Based on the alignment results, a phylogenetic tree was constructed using IQ-TREE, with 1000 ultrafast guided replicates (-bb 1000) to assess branch support. Further, the obtained 16S rDNA sequences were BLASTed in NCBI, and highly similar strain sequences were screened. A phylogenetic tree was constructed using the neighbor-joining method with MEGA 7 software, and the reliability of the topology was evaluated using the bootstrap test.

[0054] like Figure 2 The figure shows the phylogenetic analysis results of strain W-5; each branch is labeled with: GenBank sequence number + strain name. The figure shows that the 16S rDNA sequence of W-5 is highly similar to that of *Staphylococcus saprophyticus*, and it is on the same branch as *Staphylococcus sp. strain KG4-1* (GenBank: PQ669068.1). Based on the above molecular phylogenetic evidence, this strain is identified as belonging to the kingdom Bacteria, phylum Firmicutes, class Bacilli, class Bacillales, family Staphylococcaceae, and genus *Stapyhlococcus*, and named *Stapyhlococcus* sp. W-5. This strain was deposited on October 29, 2025, at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No. 67176, located at No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province.

[0055] Experimental Example

[0056] 1. Analysis of the degradation ability of strain W-5 against λ-cyhalothrin

[0057] To evaluate the degradation ability of strain W-5 on λ-cyhalothrin, high-performance liquid chromatography (HPLC) was used to determine its degradation efficiency in minimum salt medium (MSM). The HPLC analytical conditions were as follows: detection wavelength 220 nm, mobile phase methanol-water (8:2 v / v), flow rate 1.0 mL / min, and injection volume 20 μL. Under these conditions, λ-cyhalothrin showed a sharp and symmetrical peak at 27 min, indicating good separation.

[0058] Calibration curves were established using a series of concentration standards (Table 2), and the regression equation was obtained as y = 39.075x + 24.595 (R²). 2 = 0.9994), such as Figure 3 As shown, the method exhibits good linearity. Based on this standard curve, the residual concentration of the target analyte in the sample is calculated according to the peak area. Degradation experimental results are as follows... Figure 5 As shown, after inoculation with strain W-5, the content of λ-cyhalothrin in MSM medium decreased significantly, with a degradation rate of 98.24%.

[0059] Table 2 - Determination of λ-cyhalothrin by W-5 strain by high performance liquid chromatography

[0060] Standard concentration / ppm 5 10 20 50 100 <![CDATA[Peak area / cm 2 > 219.10 408.20 775.80 2041.30 3907.40

[0061] 2. Assessment of the tolerance and degradation potential of strain W-5 to λ-cyhalothrin

[0062] The strains were screened and cultured using minimum salt medium (MSM). The MSM formulation (1 L) is as follows:

[0063] (NH4)2SO4 2.0g, Na2HPO4·12H2O 1.5g, KH2PO4 1.5g, MgSO4·7H2O 0.2g, CaCl2·2H2O 0.01g, FeSO4·7H2O 0.001g.

[0064] To screen for strains with λ-cyhalothrin degradation capabilities, strains isolated from the gut of the marsupial assassin bug were cultured in MSM medium with 50 mg / L λ-cyhalothrin as the sole carbon source. Two treatment groups were set up: the experimental group was inoculated with W-5, and the control group was uninoculated. OD was monitored for 7 consecutive days. 600 To assess the growth of the strain. Results showed that W-5 grew well in the experimental group, with an OD... 600 It continued to rise and eventually stabilized. Figure 4 The control group showed no growth. This result indicates that W-5 can proliferate using λ-cyhalothrin as the sole carbon source, demonstrating tolerance and degradation potential to this pesticide.

[0065] 3. Determination of the colonization ability of W-5 in the spotted assassin bug.

[0066] The assassin bug *Aegilops fasciatus* was subjected to starvation treatment. Mealworms soaked in strain W-5 were used as the treatment group, while mealworms soaked in LB pure bacterial solution served as the control group. The starved assassin bugs were then fed with this solution. After one week of feeding, the assassin bugs were dissected, and DNA was extracted from the intestines of both the control and treatment groups. Quantification was performed using W-5-specific primers, and the W-5 content in the intestines of the control and treatment groups was calculated using absolute quantification. The results showed that feeding *Aegilops fasciatus* mealworms soaked in strain W-5 significantly increased the expression level of W-5 in the intestines of the assassin bugs. Figure 6 This demonstrates that W-5 has the potential to colonize the gut of the variegated assassin bug.

[0067] 4. Effects of W-5 on glutathione S-transferase (GST) activity in *Astrophytum asterias*

[0068] (1) Bacterial culture treatment and sample preparation

[0069] W-5 was inoculated into LB liquid medium and cultured until OD... 600 = 0.1 (approximately 8 × 10) 7 The bacterial cells (CFU / mL) were collected by centrifugation at 1500 rpm and resuspended in ultrapure water. Mealworms were treated with an immersion method to ensure uniform adhesion of the bacterial solution to their bodies. After being blotted dry with filter paper, they were fed to third-instar nymphs of the *Astragalus membranaceus* (day 1) for one week to ensure normal feeding. After feeding, lambda-cyhalothrin (LC50) was used. 50 Nymphs were immersed in a solution of 39.230 mg / L for 5 seconds. Samples were collected at 24 h, 48 h and 72 h after treatment. 3 mg of tissue was weighed, an appropriate amount of ultrapure water was added, the mixture was homogenized and centrifuged (2000-3000 rpm, 20 min), and the supernatant was used for enzyme activity determination.

[0070] (2) GST activity assay

[0071] Referring to the kit instructions, measure the absorbance values ​​(A1 and A2) at 340 nm for 10 s and 310 s, respectively, and calculate the GST activity using the following formula:

[0072]

[0073] Where Vtotal is the total volume of the reaction system (mL), W is the fresh weight of the sample (g), and Δt is the reaction time (min).

[0074] (3) Results Analysis

[0075] like Figure 7The figure shows the effect of W-5 on the activity of glutathione S-transferase (GST) in the variegated killer bug. The values ​​in the figure are mean ± SEM (n=3). The significance of the differences was analyzed using the independent samples t-test. ns represents the significance level p > 0.05, * represents the significance level p < 0.05, and ** represents the significance level p < 0.01.

[0076] As shown in the figure, within 24-72 h, the GST enzyme activity of *Aspergillus oryzae* treated with both λ-cyhalothrin and W-5 was significantly increased at 24, 48, and 72 h, with highly significant differences compared to the control group (p < 0.05). Treatment with λ-cyhalothrin alone did not significantly increase GST activity. These results indicate that W-5 can significantly enhance the activity of glutathione S-transferase in *Aspergillus oryzae* at specific time points (24, 48, and 72 h).

[0077] 5. Effects of W-5 on cytochrome P450 activity in *Apis glazedilum*

[0078] (1) Sample preparation

[0079] Sample preparation was performed using the same method as described in Section 4, “Sample Preparation”. Samples of *Astrophytum asterias* were collected at 24 h, 48 h, and 72 h after treatment. 3 mg of tissue was weighed, homogenized, centrifuged, and the supernatant was used for cytochrome P450 activity assay.

[0080] (2) Determination of samples

[0081] Cytochrome P450 activity was determined using enzyme-linked immunosorbent assay (ELISA). The specific steps were as follows: Samples and HRP-labeled antibodies were added sequentially to the wells of an ELISA plate, and the plate was incubated at 37°C for 60 min. After washing the plate five times, substrates A and B were added sequentially, and the plate was incubated at 37°C in the dark for 15 min. Stop solution was then added, and the absorbance of each well was immediately measured at 450 nm. A standard curve was plotted based on the concentrations and OD values ​​of a series of standards, and the cytochrome P450 activity in the samples was calculated using a regression equation.

[0082] (3) Results Analysis

[0083] like Figure 8 The figure shows the effect of strain W-5 on the carboxylesterase (CarE) activity of the variegated assassin bug. The values ​​in the figure are mean ± SEM (n=3). An independent samples t-test was used to analyze the significance of the differences. ns represents a significance level of p > 0.05, ** represents a significance level of p < 0.01, *** represents a significance level of p < 0.001, and **** represents a significance level of p < 0.0001.

[0084] As shown in the figure, within 24-72 h, the cytochrome P450 enzyme activity of *Aspergillus simonii* treated with both λ-cyhalothrin and W-5 was significantly increased, with a highly significant difference compared to the blank control group (p < 0.0001). While λ-cyhalothrin alone also significantly increased enzyme activity, the increase was lower than that in the combined treatment group. These results indicate that W-5 can significantly enhance the cytochrome P450 enzyme activity in *Aspergillus simonii*.

[0085] 6. Effect of W-5 on the survival rate of *Aspergillus simonii* under λ-cyhalothrin stress.

[0086] (1) Bacterial solution treatment

[0087] Healthy, uniformly developed nymphs of the third instar on day 1 were selected as experimental subjects. Mealworms were treated using an immersion method to ensure even coating of their bodies with W-5 bacterial solution. After being blotted dry with filter paper, the nymphs were fed continuously for one week. After feeding, lambda-cyhalothrin (LC50) was used. 50 The nymphs were immersed in a solution of 39.230 mg / L for 5 seconds. Survival rates were recorded at 12 h, 24 h, 36 h, 48 h, 60 h, 72 h and 84 h after treatment.

[0088] 7. Results and Analysis

[0089] like Figure 9 The figure shows the effect of W-5 on the survival rate of the painted assassin bug. The values ​​in the figure are mean ± SEM (n=3); the independent samples t-test was used to analyze the significance of the difference. ns represents the significance level p>0.05, * represents the significance level p<0.05, and *** represents the significance level p<0.001.

[0090] As shown in the figure, there were significant differences in the survival rate of *Aspergillus simonii* among the different treatment groups. The nymph survival rate was highest (90.625%) in the "W-5 + λ-cyhalothrin" combined treatment group, while the survival rate was extremely low in the λ-cyhalothrin-only treatment group. These results indicate that W-5 can significantly improve the survival ability of *Aspergillus simonii* under λ-cyhalothrin stress, demonstrating its good application potential in protecting natural enemy insects.

[0091] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A W-5 strain that enhances the pesticide tolerance of natural enemy insects, characterized in that, The specimen is classified and named Stapyhlococcus sp., with accession number GDMCC No. 67176, deposit date October 29, 2025, and deposited at Guangdong Provincial Center for Microbial Culture Collection, located at No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

2. The W-5 strain for enhancing pesticide tolerance in natural enemy insects according to claim 1, characterized in that, The 16S rDNA sequence of the W-5 strain is shown in SEQ ID No.

1.

3. The application of the W-5 strain according to any one of claims 1-2, which enhances the pesticide tolerance of natural enemy insects, in improving the host's tolerance to λ-cyhalothrin.

4. The method for cultivating the W-5 strain for improving pesticide tolerance in natural enemy insects according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Grind the intestinal tissue of the natural enemy insects thoroughly to obtain a tissue homogenate. After centrifugation, collect the supernatant for later use. (2) Take the supernatant obtained in step (1), add it to the liquid culture medium, mix thoroughly, place it in a constant temperature shaker, shake and culture to obtain bacterial solution; dilute the obtained bacterial solution to obtain bacterial solution dilution; (3) Take the diluted bacterial solution obtained in step (2), spread it evenly on a solid culture medium plate, and after culturing, pick a single colony for purification culture to obtain the W-5 strain.

5. The cultivation method according to claim 4, characterized in that, In step (1), the natural enemy insect is the adult female assassin bug; the centrifugation treatment is: centrifugation at 3000 r / min for 5 min.

6. The cultivation method according to claim 4, characterized in that, In step (2), the liquid culture medium is any one of LB liquid culture medium, EB liquid culture medium or NA liquid culture medium.

7. The cultivation method according to claim 4, characterized in that, In step (2), the culture solution is placed in a constant temperature shaker at 37°C and shaken at 200 r / min for 12 h to obtain the culture solution; The dilution was performed by adding sterile liquid culture medium or PBS to the obtained bacterial culture. This dilution process was repeated multiple times to obtain 10... -1 Up to 10 -9 Diluted bacterial solution.

8. The cultivation method according to claim 4, characterized in that, In step (3), take 100 μL of 10 -5 Up to 10 -9 Diluted bacterial solutions are evenly spread on solid culture medium plates.

9. The cultivation method according to claim 4, characterized in that, In step (3), the solid culture medium plate is any one of LB solid culture medium plate, EB solid culture medium plate or NA solid culture medium plate.

10. The cultivation method according to claim 4, characterized in that, In step (3), the conditions for plate culture are: incubation in a 37°C constant temperature incubator for 48 h; The purification culture was carried out by passage purification every 48 hours for 5 consecutive generations.

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