Serratia marcescens and application of serratia marcescens in improvement of host pesticide tolerance

Abstract

The invention discloses serratia marcescens and application of the serratia marcescens in improvement of host pesticide tolerance. The serratia marcescens is a serratia marcescens LB3 strain, the strain is preserved in the Guangdong Microbial Culture Collection Center on June 19, 2025, and the preservation number is GDMCC No: 66557. The serratia marcescens LB3 strain disclosed by the invention has the advantages that the preservation number is CGMCC No: 66557; the LB3 strain can effectively degrade the lambda-cyhalothrin, the activity of glutathione S-transferase, carboxylesterase and cytochrome P450 in a host can be improved by ingestion of the strain, the tolerance of the host to the lambda-cyhalothrin is improved, and the survival rate of the host under the stress of the lambda-cyhalothrin is improved. Based on the strain, the tolerance of natural enemy insects to the lambda-cyhalothrin can be improved, the influence of the lambda-cyhalothrin on the natural enemy insects is reduced, and effective prevention and control of pests are promoted.

Description

A strain of Serratia marcescens and its application in improving host pesticide tolerance Technical Field

[0001] This invention belongs to the field of microbial and plant pest control technology. More specifically, it relates to a strain of Serratia marcescens and its application in improving host pesticide tolerance. Background Technology

[0002] Pyrethroid insecticides are a class of synthetic, biomimetic insecticides with characteristics such as high efficiency, low toxicity, broad spectrum, and easy degradation, and are widely used in agriculture and public health. However, with the increasing use of pyrethroid insecticides, their potential hazards are gradually becoming apparent. Due to their broad spectrum, pyrethroid insecticides are also highly toxic to many non-target insects, including many important natural enemies. Furthermore, because the mechanism of action of pyrethroid insecticides is mainly through contact and stomach poisoning, while killing pests, non-target insects can easily come into direct contact with the pesticide while moving in the field, leading to death or abnormal behavior.

[0003] The painted assassin bug (Euagoras plagiatus) is a predatory insect belonging to the genus Euagoras in the family Asparagidae. As an important natural enemy insect in agricultural ecosystems, it plays a positive role in controlling the development and spread of pests. More than half of the painted assassin bugs exposed to pyrethroid insecticides will die. To ensure that natural enemy insects such as the painted assassin bug can continuously and effectively play their role in the biological control of pests, it is urgent to provide methods to improve the tolerance of natural enemy insects to pyrethroid insecticides.

[0004] *Serratia marcescens* is a Gram-negative bacillus belonging to the genus *Serratia* in the family Enterobacteriaceae. Its colonies often produce a red, non-water-soluble pyrethroid pigment, although some strains do not produce pigment. Technicians isolated a strain of *Serratia marcescens* WF01, capable of degrading pyrethroid insecticides, from the leaves of wheat plants in saline-alkali land that had been subjected to long-term use of highly effective cypermethrin. However, it is unclear whether it can enhance the pesticide tolerance of natural enemy insects. Furthermore, reports indicate that *Serratia marcescens* S-JS1 reduces the resistance of planthoppers to insecticides. After feeding on rice seedlings treated with *Serratia marcescens*, the activity of carboxylesterase (CarE) in planthoppers was not significantly different from the control group, while the activity of glutathione S-transferase (GST) showed a trend of first decreasing and then increasing. The activities of peroxidase (POD) and superoxide dismutase (SOD) were both lower than the control group. Summary of the Invention

[0005] This invention addresses the lack of methods to improve the tolerance of natural enemy insects to pyrethroid insecticides by providing a strain of Serratia marcescens and its application in improving host pesticide tolerance.

[0006] The first objective of this invention is to provide a strain of Serratia marcescens LB3.

[0007] A second objective of this invention is to provide the application of the LB3 strain in improving the tolerance of *Aspergillus oryzae* to pyrethroid insecticides.

[0008] A third object of the present invention is to provide the use of the LB3 strain in the preparation of products for improving the tolerance of *Aspergillus oryzae* insects to pyrethroid insecticides.

[0009] The fourth objective of this invention is to provide a microbial preparation.

[0010] A fifth object of the present invention is to provide the application of the aforementioned microbial preparation in improving the tolerance of *Aspergillus oryzae* to pyrethroid insecticides.

[0011] A sixth object of the present invention is to provide the use of the microbial preparation in the preparation of products for improving the tolerance of *Aspergillus oryzae* insects to pyrethroid insecticides.

[0012] A seventh objective of this invention is to provide a method for improving the tolerance of *Aspergillus oryzae* insects to pyrethroid insecticides.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution: Testing of the isolated *Serratia marcescens* strain LB3 revealed that this strain can efficiently degrade lambda-cyhalothrin, achieving a degradation rate of 92.49% within 72 hours. Furthermore, testing of the activities of three key detoxification enzymes in *Aspergillus simonii* treated with the LB3 strain showed a significant increase in the activities of glutathione S-transferase, carboxylesterase, and cytochrome P450. Moreover, one week after treatment with the LB3 strain, the survival rate of *Aspergillus simonii* under lambda-cyhalothrin stress significantly increased, reaching 87.24%. In summary, it is speculated that the LB3 strain can synergistically enhance the defense capability of *Aspergillus simonii* against lambda-cyhalothrin through a dual mechanism of "direct microbial degradation" and "host detoxification metabolic activation." Therefore, this invention seeks protection for the *Serratia marcescens* strain LB3 and its application in improving host pesticide tolerance.

[0014] This invention provides a strain of Serratia marcescens LB3, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 19, 2025, with accession number GDMCC No: 66557.

[0015] This invention seeks protection for the use of the LB3 strain in improving the tolerance of *Aspergillus oryzae* to pyrethroid insecticides.

[0016] The present invention also claims protection for the use of the LB3 strain in the preparation of products for improving the tolerance of *Aspergillus oryzae* to pyrethroid insecticides.

[0017] Specifically, the insect in the genus *Aspergillus* is *Aspergillus variegata*.

[0018] Specifically, the pyrethroid insecticide is cyhalothrin.

[0019] More specifically, the pyrethroid insecticide is lambda-cyhalothrin. Lamda-cyhalothrin is a highly effective version of lambda-cyhalothrin obtained by removing the ineffective isomer.

[0020] In a specific embodiment of the present invention, the high-efficiency cyhalothrin is λ-high-efficiency cyhalothrin.

[0021] The present invention also provides a microbial preparation containing the LB3 strain described herein.

[0022] Optionally, the microbial preparation is the bacterial powder of the LB3 strain or the bacterial liquid of the LB3 strain.

[0023] This invention seeks protection for the use of the aforementioned microbial preparation in improving the tolerance of *Aspergillus oryzae* to pyrethroid insecticides.

[0024] The present invention also claims protection for the use of the microbial preparation in the preparation of products for improving the tolerance of *Aspergillus oryzae* insects to pyrethroid insecticides.

[0025] Specifically, the insect in the genus *Aspergillus* is *Aspergillus variegata*.

[0026] Specifically, the pyrethroid insecticide is cyhalothrin.

[0027] More specifically, the pyrethroid insecticide is lambda-cyhalothrin.

[0028] In a specific embodiment of the present invention, the high-efficiency cyhalothrin is λ-high-efficiency cyhalothrin.

[0029] The present invention also provides a method for improving the tolerance of *Astrophytum* insects to pyrethroid insecticides, the method being: feeding *Astrophytum* insects with the LB3 strain or the microbial preparation.

[0030] More specifically, the LB3 strain or the microbial preparation is fed to or soaked in live prey of the genus *Astrophytum*, so that the *Astrophytum* ingests the LB3 strain or the microbial preparation by hunting the live prey.

[0031] Specifically, the insect in the genus *Aspergillus* is *Aspergillus variegata*.

[0032] Specifically, the pyrethroid insecticide is cyhalothrin.

[0033] More specifically, the pyrethroid insecticide is lambda-cyhalothrin.

[0034] In a specific embodiment of the present invention, the high-efficiency cyhalothrin is λ-high-efficiency cyhalothrin.

[0035] In a specific embodiment of the present invention, the live prey is a mealworm (Tenebrio molitor).

[0036] This invention provides a *Serratia marcescens* strain LB3, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on June 19, 2025, with accession number GDMCC No: 66557. The LB3 strain described in this invention can effectively degrade lambda-cyhalothrin. Ingestion of this strain can also enhance the activity of glutathione S-transferase, carboxylesterase, and cytochrome P450 in the host, improving the host's tolerance to lambda-cyhalothrin and increasing survival rate under lambda-cyhalothrin stress. Based on the LB3 strain, it can be used to improve the tolerance of natural enemy insects to lambda-cyhalothrin, reduce the impact of lambda-cyhalothrin on natural enemy insects, and promote effective pest control. Attached Figure Description

[0037] Figure 1 shows the growth curve of strain LB3 in minimum salt medium containing 50 mg / L λ-cyhalothrin as the sole carbon source; the values ​​in the figure are mean ± SEM (n=3), and the significance of differences was analyzed using the independent samples t-test; ns represents the significance level p>0.05. This indicates a significance level of p < 0.01. This represents a significance level of p<0.001.

[0038] Figure 2 shows the colony and cell morphology of strain LB3; A in the figure is the colony morphology of strain LB3 on LB plate, and B is the cell morphology of strain LB3.

[0039] Figure 3 shows the phylogenetic analysis results of strain LB3; each branch in the figure is labeled with the species name and GenBank accession number.

[0040] Figure 4 shows the HPLC standard curve of λ-high-efficiency cyhalothrin.

[0041] Figure 5 shows the degradation rate of λ-cyhalothrin by strain LB3.

[0042] Figure 6 shows the effect of LB3 strain on glutathione S-transferase activity in *Aspergillus simonii*; ns represents the significance level, p>0.05. This indicates a significance level of p<0.05. This indicates a significance level of p<0.01.

[0043] Figure 7 shows the effect of LB3 strain on carboxylesterase activity in *Astrophytum argenteum*; ns represents the significance level, p>0.05. This indicates a significance level of p < 0.01. This indicates a significance level of p < 0.001. This represents a significance level of p<0.0001.

[0044] Figure 8 shows the effect of LB3 strain on cytochrome P450 activity in *Astrophytum argenteum*; ns represents the significance level, p>0.05. This indicates a significance level of p<0.05. This indicates a significance level of p < 0.01. This represents a significance level of p<0.0001.

[0045] Figure 9 shows the effect of LB3 strain on the survival rate of *Aspergillus simonii* under λ-cyhalothrin stress; ns represents the significance level p>0.05. This indicates a significance level of p<0.05. This represents a significance level of p<0.001. Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0047] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0048] The LB3 strain described in this invention belongs to Serratia marcescens and is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC No. 66557, deposited on June 19, 2025, at the 5th floor of the Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0049] The minimum salt medium (MSM) formulation (1 L) is as follows: (NH4)2SO4 2.0 g, Na2HPO4·12H2O 1.5 g, KH2PO4 1.5 g, MgSO4·7H2O 0.2 g, CaCl2·2H2O 0.01 g, FeSO4·7H2O 0.001 g.

[0050] Example 1 Isolation, Screening and Identification of Strains 1. Isolation of Strains Obtaining Intestinal Tissue Homogenate: Wild female adult assassin bugs were collected and starved for 24 h. The insects were then surface disinfected by immersing them in 75% (v / v) ethanol for 30 s, followed by rinsing three times with sterile PBS buffer. The intestines were dissected under aseptic conditions and placed in a 1.5 mL centrifuge tube. The intestinal tissue was thoroughly homogenized 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.

[0051] Bacterial culture and serial dilution: Transfer 5 mL of sterile LB, EB, and NA liquid culture medium to sterile 15 mL centrifuge tubes, respectively; add 200 μL of the supernatant from each of these three liquid cultures, mix thoroughly, and incubate at 30℃ and 37℃ respectively in a shaker at 200 r / min for 12 h; take 100 μL of the 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.

[0052] Plate coating separation and purification: Take 100 μL of each of the following: -5 Up to 10 -9 Diluted bacterial suspensions were evenly spread onto corresponding LB, EB, and NA solid culture plates, with three replicates for each dilution, and clearly labeled. The plates were incubated at 37°C for 48 h. Single colonies with different morphologies were picked and purified using the streak plate method. The plates were passaged every 48 h for five consecutive generations. The morphology of the single colonies on the last purified plate was observed and recorded, and the purified single colonies were preserved.

[0053] 2. Strain Screening: To obtain strains with highly efficient cyhalothrin degradation function, this invention used minimum salt medium (MSM) containing 50 mg / L λ-cyhalothrin as the sole carbon source to screen strains isolated from the intestines of the variegated assassin bug. During screening, each strain was divided into two treatment groups: an inoculated experimental group and an uninoculated control group. OD was monitored for 7 consecutive days. 600 To evaluate the growth of the strain. Through screening, this invention obtained a strain capable of growing in the screening medium, namely the LB3 strain described in this invention. The growth curve of the LB3 strain in a minimum salt medium containing 50 mg / L λ-cyhalothrin as the sole carbon source is shown in Figure 1. As can be seen from Figure 1, the LB3 strain grows well in the medium, with an OD... 600The growth rate continued to rise and eventually stabilized, while no growth was observed in the blank control group. These results indicate that the LB3 strain can proliferate using λ-cyhalothrin as the sole carbon source, demonstrating tolerance and degradation potential to cyhalothrin.

[0054] 3. Morphological observation of LB3 strain: The colony morphology of LB3 strain on the last purification plate has been observed and recorded. A small number of single colonies of LB3 strain were selected for Gram staining and their cell morphology was observed under a 40x optical microscope.

[0055] The colony and cell morphology of strain LB3 are shown in Figure 2, where A represents the colony morphology of strain LB3 on LB plates, and B represents the cell morphology of strain LB3. As shown in Figure 2, A indicates that the colonies of strain LB3 are round, raised, with a smooth surface, irregular edges, and an opaque center. As shown in Figure 2, B indicates that the cells of strain LB3 are short rod-shaped, often slightly curved and approaching a coccobacillus shape, and arranged in short chains.

[0056] 4. Molecular biological identification of LB3 strain: Gene DNA of LB3 strain was extracted using the TIANamp Bacteria DNA Kit from Tiangen Biotech Co., Ltd. Using this as a template, the 16S rRNA encoding gene (16S rDNA) of LB3 strain was amplified using the universal 16S rRNA primer 27F / 1492R.

[0057] The nucleotide sequences of the universal 16S rRNA primers 27F / 1492R are shown below: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'.

[0058] The reaction system used for PCR amplification is shown in Table 1. After preparing the reaction system, gently mix and briefly centrifuge, then place it on a PCR instrument and perform PCR amplification according to the reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 90 s, 35 cycles; 72℃ for 10 min; store at 4℃. The PCR amplification products were detected by 1% agarose gel extraction, purified by gel excision, and sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0059] Table 1. Bacterial 16S rDNA PCR amplification reaction system (20 μL)

[0060] The sequencing results show that the 16S rRNA gene sequence of the LB3 strain described in this invention is shown in SEQ ID NO.1.

[0061] To accurately identify the taxonomic position of LB3 strains, this invention employs a combination of multiple sequence alignment and phylogenetic analysis. 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. Next, the obtained 16S rDNA sequences were BLASTed in NCBI to screen for highly similar strain sequences. A phylogenetic tree was then constructed using the neighbor-joining method with MEGA 7 software, and the reliability of the topology was assessed using the bootstrap test.

[0062] The phylogenetic analysis results of strain LB3 are shown in Figure 3, with each branch labeled with the species name and GenBank accession number. Figure 3 shows that the 16S rDNA sequence of strain LB3 has a 99.86% similarity to *Serratia marcescens* (accession number D0501957.1). Based on the above molecular systematic evidence, strain LB3 is identified as belonging to the kingdom Bacteria, phylum Proteobacteria, class Gammaproteobacteria, order Enterobacterales, family Enterobacteriaceae, genus *Serratia*, and *Serratia marcescens*, and named *Serratia marcescens* LB3.

[0063] The LB3 strain described in this invention has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 66557, deposit date of June 19, 2025, and address of the depository at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0064] Example 2: Degradation Ability of LB3 Strain for λ-Cyfluthrin To evaluate the degradation ability of LB3 strain for λ-cyfluthrin, this invention utilized high-performance liquid chromatography (HPLC) to determine the degradation efficiency of LB3 strain for λ-cyfluthrin in minimum salt medium (MSM). The HPLC analysis conditions were as follows: detection wavelength 220 nm, mobile phase methanol-water (v / v 8:2), flow rate 1.0 mL / min, and injection volume 20 μL. Under these conditions, λ-cyfluthrin showed a sharp and symmetrical chromatographic peak at 27 min, indicating good separation.

[0065] A calibration curve table was established with reference to the series of concentration standards shown in Table 2, and the HPLC standard curve of λ-cyhalothrin was plotted as shown in Figure 4. The regression equation was obtained as y = 39.075x + 24.595 (R² = 0.9994), indicating that the linear relationship was good.

[0066] Table 2. Establishment of Calibration Curves for a Series of Concentration Standards

[0067] The degradation ability of LB3 strain against λ-cyhalothrin is shown in Table 3.

[0068] Table 3. Degradation ability of LB3 strain against λ-cyhalothrin

[0069] Where C0 is the initial concentration of the pesticide in the liquid culture medium, and C... t The concentrations are at different times.

[0070] Based on the standard curve shown in Figure 4, the residual concentration of λ-cyhalothrin was calculated according to the peak area of ​​the inoculated MSM medium, and the degradation rate of λ-cyhalothrin by strain LB3 was obtained, as shown in Figure 5. Table 3 and Figure 5 show that after inoculation with strain LB3, the content of λ-cyhalothrin in the MSM medium decreased significantly, and the degradation rate reached 92.49% within 72 h, indicating that strain LB3 has a high degradation capacity for λ-cyhalothrin.

[0071] Example 3: Effect of LB3 strain on pesticide tolerance of *Aspergillus simonii*. This invention used *Aspergillus simonii* as the test host and λ-cyhalothrin as the test pesticide to test the effect of LB3 strain on the pesticide tolerance of *Aspergillus simonii* to λ-cyhalothrin.

[0072] 1. Toxicity determination of λ-cyhalothrin against third-instar mollusks: The technical grade λ-cyhalothrin was dissolved in 10% acetone solution to prepare a stock solution. A serial dilution method was used to dilute the stock solution to five concentration gradients: 128, 64, 32, 16, and 8 mg / L, with 10% acetone solution serving as a blank control. Third-instar nymphs (day 1) were selected for toxicity determination using the immersion method. Twenty insects were used in each treatment group. They were placed in disposable plastic petri dishes, immersed in the corresponding concentration of the pesticide solution for 5 seconds, and then removed. Excess pesticide solution was absorbed using a soft brush and transferred to clean qualitative filter paper. Individual insects were then individually packaged and reared normally. The number of dead individuals was counted 24 hours after treatment. The mortality criterion was the absence of reaction when the insect's legs were lightly touched. SPSS 26.0 was used for statistical analysis of the toxicity data. The results of the toxicity determination of λ-cyhalothrin against third-instar mollusks are shown in Table 4. Table 4 shows the 24-hour median lethal concentration (LC50) of λ-cyhalothrin against the three-instar scarlet assassin bug. 50 The concentration was 39.230 mg / L.

[0073] Table 4. Results of toxicity determination of λ-cyhalothrin against the three-instar scarlet assassin bug.

[0074] 2. Effects of LB3 strain on glutathione S-transferase (GST) activity in *Astrophytum argenteum* (1) Bacterial treatment and sample preparation: LB3 strain was inoculated into LB liquid medium and cultured until OD500. 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 treated with λ-cyhalothrin (39.230 mg / L) for 5 seconds, denoted as λ-cyhalothrin+LB3. Simultaneously, mealworms not fed with λ-cyhalothrin (not λ-cyhalothrin) and a blank control (ck) were also included. Samples were collected at 24 h, 48 h, and 72 h after treatment. 3 mg of tissue was weighed, added to an appropriate amount of ultrapure water, homogenized, and centrifuged (2000–3000 rpm, 20 min). The supernatant was used for enzyme activity assay.

[0075] (2) The GST activity assay was performed according to the instructions of the GST kit from Suzhou Keming Biotechnology Co., Ltd. The operation steps are as follows: Preheat the spectrophotometer for 30 min, set the wavelength to 340 nm, and zero it with distilled water; keep reagent 3 at 25℃ (for general species) or 37℃ (for mammals); test tube operation: take 1 mL of quartz cuvette, add 100 μL of supernatant, 900 μL of reagent 2 and 100 μL of reagent 3 in sequence, mix quickly and immediately detect the absorbance change at a wavelength of 340 nm; record the absorbance values ​​at 10 s and 310 s respectively, and denot them as A1 and A2.

[0076] GST activity is calculated using the following formula:

[0077] Where W is the fresh weight of the sample (g), and A1 and A2 are the absorbance at 10 s and 310 s, respectively.

[0078] The effect of strain LB3 on glutathione S-transferase activity in *Aspergillus simonii* is shown in Figure 6. As shown in Figure 6, within the range of 24–72 h, the GST activity in *Aspergillus simonii* treated with both λ-cyhalothrin and strain LB3 was significantly increased at 48 h, showing a highly significant difference compared to the control group (p<0.05), while no significant changes in enzyme activity were observed at 24 h and 72 h. Treatment with λ-cyhalothrin alone did not significantly increase GST activity. These results indicate that strain LB3 can significantly enhance glutathione S-transferase activity in *Aspergillus simonii* at a specific time point (48 h).

[0079] 3. Effect of LB3 strain on carboxylesterase (CarE) activity in *Astrophytum argenteum*. The bacterial culture treatment and sample preparation were the same as above. *Astrophytum argenteum* samples 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 CarE activity determination. Blank and test tubes were used for CarE activity determination. The absorbance change was monitored at 450 nm for 190 s, and recorded as ΔA (blank tube) and ΔA (test tube), respectively. Enzyme activity was calculated using the following formula: CarE activity = 67 × (ΔA (test tube)). ΔA (blank tube) ÷ W, where W is the fresh weight of the sample (g), and the CarE activity unit is U / g fresh weight, defined as the amount of enzyme required to increase the catalytic absorbance by 1 per gram of tissue per minute.

[0080] The effect of strain LB3 on carboxylesterase activity in *Aspergillus simonii* is shown in Figure 7. As shown in Figure 7, within 24–72 h, the CarE activity in the group co-treated with λ-cyhalothrin and strain LB3 was significantly increased at 48 h, showing a highly significant difference compared to the blank control group (p<0.001) and also reaching a highly significant level compared to the group treated with λ-cyhalothrin alone (p<0.0001). Although treatment with λ-cyhalothrin alone also caused a certain increase in enzyme activity, the increase was much lower than that in the combined treatment group. These results indicate that strain LB3 can significantly enhance carboxylesterase activity in *Aspergillus simonii*.

[0081] 4. Effect of LB3 strain on cytochrome P450 activity in *Astrophytum argus*. The bacterial culture treatment and sample preparation were the same as above. *Astrophytum argus* samples 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 determination. 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 the ELISA plate, and incubated at 37℃ for 60 min; after washing the plate 5 times, substrates A and B were added sequentially, and the reaction was carried out at 37℃ in the dark for 15 min; then stop solution was added, and the absorbance of each well was immediately measured at 450 nm. A standard curve was plotted based on the concentration and OD value of a series of standards, and the cytochrome P450 activity in the samples was calculated based on the regression equation.

[0082] The effect of strain LB3 on cytochrome P450 activity in *Aspergillus simonii* is shown in Figure 8. As shown in Figure 8, within 24–72 h, the cytochrome P450 enzyme activity in *Aspergillus simonii* treated with both λ-cyhalothrin and strain LB3 was significantly increased, with a highly significant difference compared to the control group (p<0.0001). While treatment with λ-cyhalothrin alone also significantly increased enzyme activity, the increase was much lower than that in the combined treatment group. These results indicate that strain LB3 can significantly enhance the cytochrome P450 enzyme activity in *Aspergillus simonii*.

[0083] 5. Effect of LB3 strain on the survival rate of *Astragalus membranaceus* under λ-cyhalothrin stress: Healthy, uniformly developed 3rd instar nymphs on day 1 were selected as experimental subjects. Mealworms were treated with LB3 bacterial solution by immersion, ensuring even coating on their body surface. After drying with filter paper, the nymphs were fed continuously for one week. After feeding, λ-cyhalothrin (LC-C) was applied. 50 The nymphs were immersed in a solution of 39.230 mg / L for 5 seconds, and the survival rate was calculated at 12 h, 24 h, 36 h, 48 h, 60 h, 72 h and 84 h after treatment.

[0084] The effect of strain LB3 on the survival rate of *Aspergillus simonii* under λ-cyhalothrin stress is shown in Figure 9. As can be seen from Figure 9, there were significant differences in the survival rate of *Aspergillus simonii* among the different treatment groups. In the combined treatment group of "LB3 + λ-cyhalothrin", the nymph survival rate was the highest, reaching 87.24%; while the survival rate of the group treated with λ-cyhalothrin alone was only 43.07%. In the solvent control group, the survival rate of nymphs fed with LB3-containing feed was 90.625%, while the survival rate of the blank control group was 100%.

[0085] The results indicate that strain LB3 can significantly improve the survival rate of the variegated assassin bug under λ-cyhalothrin stress, demonstrating its good application potential in protecting natural enemy insects.

[0086] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Serratia marcescens LB3, characterized in that, The strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 19, 2025, with accession number GDMCC No: 66557.

2. The application of the LB3 strain of claim 1 in improving the tolerance of *Aspergillus oryzae* to pyrethroid insecticides.

3. The use of the LB3 strain of claim 1 in the preparation of products for improving the tolerance of *Amanita muscaria* insects to pyrethroid insecticides.

4. A microbial preparation, characterized in that, Contains the LB3 strain as described in claim 1.

5. The application of the microbial preparation of claim 4 in improving the tolerance of *Amanita muscaria* insects to pyrethroid insecticides.

6. The use of the microbial preparation of claim 4 in the preparation of products for improving the tolerance of *Amanita muscaria* insects to pyrethroid insecticides.

7. The application according to claim 2, 3, 5 or 6, characterized in that, The insect in question is *Aspergillus oryzae*.

8. The application according to claim 2, 3, 5 or 6, characterized in that, The pyrethroid insecticide mentioned is cyhalothrin.

9. A method for improving the tolerance of *Aspergillus spp.* to pyrethroid insecticides, characterized in that, The LB3 strain of claim 1 or the microbial preparation of claim 4 is fed to insects of the genus *Aspergillus*.

10. The method according to claim 9, characterized in that, Feeding or soaking live prey of *Aspergillus oryzae* with the LB3 strain of claim 1 or the microbial preparation of claim 4, so that the *Aspergillus oryzae* ingests the LB3 strain or the microbial preparation by hunting the live prey.

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