Metarhizium anisopliae engineering bacterium expressing spider venom peptide Hv1a and application of metarhizium anisopliae engineering bacterium in control of chilo suppressalis

Abstract

The invention belongs to the technical field of biological prevention and control of agricultural pests, and particularly relates to a metarhizium anisopliae engineering bacterium for expressing spider venom peptide Hv1a and application of the metarhizium anisopliae engineering bacterium in prevention and control of chilo suppressalis. The invention discloses the obvious insecticidal activity of Metarhizium guerizense ARSEF 977 on chilo suppressalis for the first time, on the basis, an Hv1a toxic peptide gene derived from a spider is introduced into the strain through a genetic engineering technology, an engineering bacterium for stably and efficiently expressing the spider insecticidal peptide Hv1a is successfully constructed, the engineering bacterium plays a synergistic effect, the insecticidal effect on the chilo suppressalis is further improved, and the application prospect is wide. The death rate after 7 days of treatment reaches up to 96.7%, and the method can be used for controlling chilo suppressalis. The invention provides a high-potential starting strain for developing a special fungal insecticide for chilo suppressalis, provides a new material and method for promoting the development of a green and efficient chilo suppressalis prevention and control technology, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, specifically relating to an engineered strain of Metarhizium anisopliae expressing spider venom peptide Hv1a and its application in controlling rice stem borer. Background Technology

[0002] The rice stem borer (Chilo suppressalis) is one of the most difficult pests to control on rice. A boring insect, its larvae burrow into the rice stalks to feed, causing symptoms such as dead heart and whiteheads, which can lead to significant yield reduction or even total crop failure. Currently, the control of the rice stem borer still heavily relies on chemical pesticides. Long-term and excessive use has not only led to increasing pesticide resistance in the pest but also caused problems such as pesticide residues, environmental pollution, and agricultural product quality and safety. Therefore, the development of new, efficient, and environmentally friendly control products and technologies is urgently needed.

[0003] Microbial pesticides, as an important category of biological pesticides, have become a hot topic in research and application in recent years because they can kill pests through infection, parasitism, or the production of active substances. Currently, the only single-agent microbial pesticide products used to control the rice stem borer are Bacillus thuringiensis (Bt), Beauveria bassiana, and Metarhizium anisopliae. While microbial pesticide products offer advantages in rice stem borer control, such as safety and relatively low resistance rates, they also generally suffer from slow insecticidal speed, mortality rates highly dependent on environmental conditions, and unstable efficacy, making it difficult to fully meet the demand for highly efficient and rapid control in the field.

[0004] To overcome the limitations of natural microbial pesticides, genetic engineering techniques have been explored for the genetic modification of biocontrol bacteria. Some studies have attempted to introduce exogenous toxin genes and insecticidal protein genes into fungal vectors such as *Beauveria bassiana* and *Metarhizium anisopliae* to enhance their insecticidal toxicity and speed of action. Among these, the spider venom peptide Hv1a, derived from the Australian funnel-web spider, is a neurotoxin that specifically acts on the voltage-gated calcium channels of insects. It is highly toxic to a variety of insects but relatively safe for mammals, thus possessing the potential to be developed into a novel biopesticide. However, the construction of such engineered bacteria still faces multiple challenges, including exogenous gene expression efficiency, genetic stability of the engineered bacteria, field adaptability, and safety evaluation. Currently, highly effective engineered fungal formulations targeting the rice stem borer are still relatively lacking. Summary of the Invention

[0005] This invention addresses a gap in existing technologies. Based on the discovery of a strain of Metarhizium guizhouense with high insecticidal activity against the rice stem borer, this invention utilizes genetic engineering technology to introduce the Hv1a toxic peptide gene derived from spiders into the Metarhizium guizhouense strain, successfully constructing a stable and highly efficient engineered strain expressing the spider insecticidal peptide Hv1a. Tests have shown that this engineered strain exhibits excellent insecticidal effects against the rice stem borer and can be used for its control. This provides new materials and methods for promoting the development of green pest control technologies and has broad application prospects.

[0006] One objective of this invention is to provide an engineered strain of *Metarhizium guizhouense* expressing spider venom peptide Hv1a. The engineered strain is formed by transferring the spider venom peptide Hv1a gene into *Metarhizium guizhouense*, enabling it to stably express and secrete spider venom peptide Hv1a. The amino acid sequence of the spider venom peptide Hv1a is shown in SEQ ID NO.2, and the *Metarhizium guizhouense* strain is *Metarhizium guizhouense* ARSEF 977.

[0007] Furthermore, the nucleotide sequence of the spider venom peptide Hv1a gene is shown in SEQ ID NO.1.

[0008] Furthermore, the method for preparing the Guizhou Metarhizium anisopliae engineered strain expressing spider venom peptide Hv1a includes: ligating the Hv1a venom peptide gene into the pCAMBIA0380 expression vector, transforming the recombinant vector into Guizhou Metarhizium anisopliae using Agrobacterium-mediated genetic transformation, and screening to obtain recombinant engineered strains that fully express spider venom peptide Hv1a.

[0009] The second objective of this invention is to provide the application of *Metarhizium anisopliae* and / or engineered *Metarhizium anisopliae* expressing spider venom peptide Hv1a in any of the following: A1) Application in the control of rice stem borer; A2) Application in the preparation of products for controlling rice stem borer; The amino acid sequence of the spider venom peptide Hv1a is shown in SEQ ID NO.2; the Metarhizium guizhouense is Metarhizium guizhouense ARSEF 977.

[0010] A third objective of this invention is to provide a method for controlling the rice stem borer, the method comprising: treating the rice stem borer with *Metarhizium guizhouense* and / or engineered *Metarhizium guizhouense* strains expressing spider venom peptide Hv1a; wherein the amino acid sequence of the spider venom peptide Hv1a is shown in SEQ ID NO.2; and the *Metarhizium guizhouense* strain is *Metarhizium guizhouense* ARSEF 977.

[0011] Furthermore, the Guizhou Metarhizium anisopliae and / or Guizhou Metarhizium anisopliae engineered strains expressing spider venom peptide Hv1a are prepared into spore suspensions for inoculating rice stem borers, and / or for spraying rice stem borer-infested plants, leaves, or soil.

[0012] Furthermore, the crops damaged by the rice stem borer include, but are not limited to: rice, water chestnut, corn, sorghum, millet, and sugarcane.

[0013] Furthermore, the concentration of the spore suspension is not less than 2.00 × 10⁻⁶. 7 Spores / mL.

[0014] Furthermore, the engineered strain of *Metarhizium anisopliae* expressing spider venom peptide Hv1a is prepared by transferring the Hv1a venom peptide gene as shown in SEQ ID NO.1 into *Metarhizium anisopliae* to induce it to express spider venom peptide Hv1a.

[0015] Furthermore, the method for preparing the Guizhou Metarhizium anisopliae engineered strain expressing spider venom peptide Hv1a includes: ligating the Hv1a venom peptide gene into the pCAMBIA0380 expression vector, transforming the recombinant vector into Guizhou Metarhizium anisopliae using Agrobacterium-mediated genetic transformation, and screening to obtain recombinant engineered strains that fully express spider venom peptide Hv1a.

[0016] Furthermore, the method for preparing the engineered strain of *Metarhizium anisopliae* expressing spider venom peptide Hv1a includes: S1. Expression vector construction: The signal peptide encoding gene, Hv1a toxic peptide gene and FLAG protein encoding gene are sequentially linked to form the Hv1a-FLAG fusion expression element, which is then inserted between the Ptef1 promoter and the sur selection marker of the pCAMBIA0380 expression vector to obtain the p0380-Ptef1-Hv1a-FLAG-sur heterologous expression vector. S2. Fungal genetic transformation: The p0380-Ptef1-Hv1a-FLAG-sur heterologous expression vector was introduced into *Metarhizium anisopliae* via Agrobacterium-mediated genetic transformation. S3. Screening and identification of engineered bacteria: Using the sur selection marker on the expression vector, positive transformants were screened in M-100 medium containing chlorpyrifos antibiotic, and the integration of the Hv1a gene was verified by PCR amplification to obtain the engineered strain of Metarhizium anisopliae expressing spider venom peptide Hv1a.

[0017] Furthermore, the signal peptide encoding gene in step S1 is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.5.

[0018] Furthermore, the complete sequence of the Hv1a-FLAG fusion expression element in step S1 is shown in SEQ ID NO.3.

[0019] Furthermore, the primers used in step S3 for PCR amplification to verify the integration of the Hv1a gene are shown in SEQ ID NO. 6-7.

[0020] Beneficial effects: 1. Novel Application of the Strains: Metarhizium guizhouense ARSEF 977 is a type strain, and no literature has yet reported its insecticidal activity against the rice stem borer. This invention reveals for the first time that Metarhizium guizhouense ARSEF 977 has a significant insecticidal effect against the rice stem borer, as shown by 1.08 × 10⁻⁶ ppm. 7 After 7 days of spore suspension immersion treatment, the mortality rate reached 70.0%, which was significantly better than control strains such as Beauveria bassiana. This discovery expands the host spectrum and application scope of this strain and provides a high-potential starting strain for the development of a dedicated fungal insecticide for rice stem borer.

[0021] 2. Significantly Enhanced Insecticidal Efficacy: Spider venom peptide Hv1a is a known insecticidal polypeptide against various pests, but there are currently no reports of its insecticidal activity against the rice stem borer. This invention, for the first time, constructed an engineered strain of *Metarhizium anisopliae* ARSEF 977 stably expressing Hv1a. This engineered strain successfully integrates the infection characteristics of fungi with the rapid action mechanism of neurotoxins, achieving an organic combination of the physical pathological process of "fungal infection" and the rapid physiological paralysis effect of "neurotoxins," producing a synergistic effect and significantly enhancing the insecticidal toxicity against the rice stem borer compared to the wild-type strain. Under the same treatment conditions, 2 × 10⁻⁶ of the engineered strain… 7 The mortality rate after 7 days of spore suspension immersion treatment reached 96.7%, far exceeding the 70.0% of the wild strain, significantly enhancing both the insecticidal speed and the final mortality rate. This synergistic mechanism overcomes the technical bottleneck of slow onset of action of single microbial pesticides, providing a new technical pathway for the development of next-generation highly efficient biological pesticides.

[0022] 3. Environmentally friendly and highly targeted: The biocontrol fungi and spider venom peptides used in this invention are all natural biological materials. The engineered bacteria act on the target pests and are highly safe for the environment and non-target organisms (including mammals). This provides effective technical support for reducing the use of chemical pesticides, reducing the risk of pesticide residues, protecting the ecological environment, and ensuring the quality and safety of agricultural products. It is in line with the direction of sustainable agricultural development and has broad application prospects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, 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 the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The insecticidal effect of the three *Eupolyphaga sinensis* strains in Example 1 of this invention on the rice stem borer is shown.

[0025] Figure 2 This is a schematic diagram of the construction of the vector expressing Hv1a protein in Example 2 of the present invention.

[0026] Figure 3 This is for the verification of the expression vector DNA level (A) and protein level (B) of the engineered strain in Example 2 of the present invention.

[0027] Figure 4 The insecticidal effect of the Mgu977 strain transformed with Hv1a in Example 2 of this invention on rice stem borer (immersion method, 7-day mortality rate).

[0028] Figure 5 The images show dead rice stem borers treated with the Mgu977 strain transformed with Hv1a in Example 2 of this invention, where A is the wild-type Mgu977 strain treatment group and B is the Mgu977 strain transformed with Hv1a treatment group. Detailed Implementation

[0029] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. Unless specifically stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless specifically stated, the reagents and materials used in the following embodiments are commercially available.

[0030] Example 1: Screening of strains with high insecticidal activity against rice stem borer This embodiment screened candidate strains with high insecticidal activity against the rice stem borer from entomopathogenic fungal strains from different sources, providing excellent starting strains for subsequent genetic engineering improvement, as detailed below: 1. Preparation of test strains and spore suspension: Three entomopathogenic fungal strains from different sources were selected for activity comparison: (1) Metarhizium anisopliae strain 421 (Man421): provided by Chongqing Julixin Biotechnology Co., Ltd., prepared into a 2.88×10⁻⁶ strain using an aqueous solution containing 0.1% Triton. 7 Spores / mL spore suspension; (2) Beauveria bassiana strain (Bba-NC) from Nanchang: This strain was isolated and identified by the inventors in May 2024 from rice stem borers in Xinjian District, Nanchang City, Jiangxi Province. It was prepared using an aqueous solution containing 0.1% Triton to form a 5.55×10⁻⁶ spores. 8 Spores / mL spore suspension; (3) Metarhizium guizhouense ARSEF 977 strain (Mgu977): derived from the USDA Entomologic Fungi Collection (ARSEF). In the preliminary experiment for screening wild strains, it was prepared with an aqueous solution containing 0.1% Triton to a concentration of 1.08 × 10⁻⁶. 7 Spores / mL spore suspension; based on preliminary experimental results, in the engineered bacteria experiment, a 2.00 × 10⁻⁶ spore suspension was prepared using an aqueous solution containing 0.1% Triton. 7 Spores per milliliter of spore suspension.

[0031] Sterile water and aqueous solutions containing 0.1% (v / v) Triton were used as controls.

[0032] 2. Test insects: The rice stem borers used in the experiment were field populations collected from rice paddies in Zhijiang City, Hubei Province in 2025. They were raised indoors with artificial feed (temperature 27±1°C, relative humidity 70%±10%, photoperiod 16L:8D), and healthy, uniformly sized third-instar larvae were selected for testing.

[0033] 3. Insecticidal activity assay (immersion method): (1) Immersion treatment: The rice stem borer was treated by immersion in insects and immersion in water chestnuts. The test larvae were gently immersed in the spore suspension of the corresponding treatment or control solution, ensuring that the insect body was completely submerged. After 30 seconds, they were removed, placed on sterile filter paper to absorb excess liquid droplets, and allowed to air dry. Fresh water chestnuts were cut into thin slices of uniform thickness and immersed in the same spore suspension or control solution as the treated insects for 10 seconds. After removal, they were drained until there was no visible liquid on the surface.

[0034] (2) Feeding and observation: The treated water chestnut slices were placed in clean plastic rearing round boxes, and then 10 larvae treated by the immersion method were transferred into each box. The box opening was covered with black cloth and placed in an incubator with a temperature of 27±1°C, humidity of 80%±10% and a light cycle of 16:8 (L:D) h.

[0035] (3) Experimental design: Each strain treatment was set up in 4 replicates, with 6 larvae per replicate, for a total of 24 larvae / treatment. The control was set up in the same way. Starting from the day of treatment, the mortality of larvae was observed and recorded every 24 hours for 7 consecutive days. The larvae were considered dead if they did not react when touched with tweezers.

[0036] The mortality rate on day 7 after treatment was as follows: Figure 1 As shown, the results indicate that the Guizhou Metarhizium anisopliae strain (Mgu977) exhibited relatively low spore concentration (1.08 × 10⁻⁶). 7 At a concentration of spores / mL, it still showed the best insecticidal effect against rice stem borer. The mortality rate after 7 days of treatment reached 58.3%, which was significantly higher than the control group and far exceeded that of Man421 strain at the same concentration level. It was even better than Bba-NC strain, which had a spore concentration about 50 times higher.

[0037] This result is the first to clearly demonstrate the strong pathogenicity of *Metarhizium guizhouense* strain ARSEF 977 against the rice stem borer, showcasing its excellent application potential in rice stem borer control and expanding its application scope in biological control. Given its outstanding potential shown in the initial screening, this strain will be used as the starting and receiving strain for subsequent genetic engineering modifications, aiming to further enhance its insecticidal speed and ultimate control efficacy through the introduction of exogenous insecticidal genes and other means.

[0038] Example 2: Construction of engineered strain of *Metarhizium anisopliae* expressing Hv1a and its application in the control of rice stem borer. This embodiment uses the *Metarhizium guizhouense* ARSEF 977 strain, which has excellent control effects against the rice stem borer, as the starting recipient strain. Through genetic engineering technology, the Hv1a toxic peptide gene (Uniport ID: P56207) derived from spiders was introduced into this strain to construct an engineered strain that stably and efficiently expresses the spider insecticidal peptide Hv1a. This allows the strain to exert a synergistic effect, further enhancing the insecticidal effect against the rice stem borer and laying the core material foundation for the development of a new generation of highly efficient rice stem borer biocontrol agents.

[0039] The nucleotide sequence of the Hv1a toxic peptide is as follows: TCCCCGACTTGCATTCCATCTGGTCAACCATGTCCCTACAACGAAAATTGCTGCAGCCAATCGTGTACATTTAAGGAAAATGAAAACGGCAACACTGTTAAAAGATGCGAC (SEQ ID NO. 1) Its encoded amino acid sequence is as follows: SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD (SEQ ID NO.2) 1. Construction of expression carrier Since the signal peptide sequence of Hv1a venom peptide could not be found, the signal peptide sequence of another spider venom peptide (Uniport ID: A5A3H5) was added to the end of the Hv1a gene sequence by synthesizing a gene sequence. The gene sequence of the FLAG protein (8 amino acids: DYKDDDDK) (24 bases: GACTACAAGGACGATGACGATAAGTAA) was also added to the end of the Hv1a gene sequence, forming the Hv1a-FLAG fusion expression element. The nucleotide sequence of the signal peptide is as follows: ATGAATACCGCTACAGGTGTCATCGCTTCTTTTGGTTCTGGCGACAGTCATCGGATGCATTGAAGCAGAAGATCCAGAGCAGATCTTCAAGGAGGAGAAGCCGCCGAGAAAGTATTTCGCCGC (SEQ ID NO. 4); Its encoded amino acid sequence is: MNTATTGVIALLVLATVIGCIEAEDTRADLQGGEAAEKVFRR (SEQ ID NO.5) The complete sequence of components is as follows: ATGAATACCGCTACAGGTGTCATCGCTTCTTTTGGTTCTGGCGACAGTCATCGGATGCATTGAAGCAGAAGATACCAGAGCAGATCTTCAAGGAGGAGAAGCCGCCGAGAAAGTATTTCGCCGCTCCCCGACT TGCATTCCATCTGGTCAACCATGTCCCTACAACGAAAATTGCTGCAGCCAATCGTGTACATTTAAGGAAAATGAAAACGGCAACACTGTTAAAAGATGCGACGACTACAAGGACGATGACGATAAGTAA (SEQ ID NO.3) Then, the above sequence was given to Qingke Biotechnology Co., Ltd., where the Hv1a-FLAG fusion expression element was synthesized using gene synthesis and inserted downstream of the Ptef1 promoter in the constitutive expression vector pCAMBIA0380-Ptef1-sur, which is suitable for filamentous fungi. Figure 2 As shown, the final product is the Hv1a heterologous expression vector p0380-Ptef1-Hv1a-FLAG-sur. Here, sur encodes a resistance gene to sulfonylurea herbicides, which can be used to screen fungal transformants on media containing sulfonylurea herbicides (such as chlorimuron-methyl).

[0040] 2. Fungal genetic transformation The Hv1a heterologous expression vector constructed by the company was mixed with *Agrobacterium tumefaciens* AGL-1 competent cells, incubated on ice for 30 minutes, treated with liquid nitrogen for 5 minutes, and heat-shocked in a 37°C water bath for 5 minutes. 1 ml of YEB liquid medium was added, and the mixture was incubated at 28°C with shaking for 1 hour. The resulting culture was then spread onto YEB solid medium containing 50 μg / mL carbenicillin and 50 μg / mL kanamycin. The mixture was incubated upside down at 28°C for 2 days. Single clones were picked for PCR verification to obtain *Agrobacterium tumefaciens* containing the Hv1a heterologous expression vector.

[0041] Agrobacterium tumefaciens containing the Hv1a heterologous expression vector was inoculated into 3 mL of YEB liquid medium (containing 50 μg / mL carbenicillin and 50 μg / mL kanamycin) and cultured overnight (16–20 h) at 28 °C and 220 rpm on a shaker. The next day, the Agrobacterium cells were collected by centrifugation at 10,000 rpm for 1 min, resuspended in 1 mL of IM liquid medium, and the concentration was adjusted to OD. 660 The concentration was set at 0.15, and then cultured at 28℃ and 200 rpm for 6 hours on a shaker. The cultured Agrobacterium and wild-type Metarhizium anisopliae conidia (concentration 1×10⁻⁵) were then used to prepare a suspension of conidia. 7 Mix equal volumes of the bacterial culture (300 μg / mL cefotaxime sodium and 10 μg / mL chlorimuron-methyl) thoroughly. Spread 150 μL of the bacterial culture evenly onto IM solid medium, dry in a clean bench, and incubate at 25°C in the dark for 2 days. After 2 days, transfer the bacterial culture to M-100 selection medium (containing 300 μg / mL cefotaxime sodium and 10 μg / mL chlorimuron-methyl) for further selection. Incubate at 25°C for approximately 3-4 days until resistant single colonies appear.

[0042] 3. Screening and identification of engineered strains First, use a sterile toothpick to pick up a single colony and inoculate it onto a 48-well SDAY plate with the same resistance, and incubate at 25°C for approximately 2-3 days. Then, use a sterile toothpick to pick up colonies growing in the 48-well plate again, and use mycelial DNA as a template to perform PCR amplification to verify the correct fungal transformants. The forward primer is ID-F: TTAGTTGCCGTTCTTCCG (SEQ ID NO. 6), and the reverse primer is ID-R: CGTCATCGTCCTTGTAGTCG (SEQ ID NO. 7).

[0043] PCR amplification results as follows Figure 3 As shown in Figure A, the two fungal transformants, Mgu977-Hv1a-1 and Mgu977-Hv1a-2, have the correct DNA band at 1878 bp, indicating the correctness of recombination in the transformants.

[0044] Then, based on the correct fungal transformants mentioned above, the expression levels of Hv1a toxic peptide in fungal hyphae and secreted proteins were detected using an anti-flag antibody combined with Western blotting. The specific methods are as follows: Wild-type and mutant conidial suspensions were inoculated into SDB medium to a final concentration of 10 μL. 6 After incubating at 25°C with shaking (150 r / min) for 3 days using 1 spore / ml, the mycelia and supernatant were collected and the total protein was extracted separately. Protein samples were then separated by molecular weight using SDS-PAGE. Subsequently, the separated protein bands were transferred in situ to a PVDF membrane using electroporation to immobilize them. The membrane was then treated with blocking buffer (e.g., TBST containing 5% skim milk powder) to block non-specific binding sites. Next, the membrane was incubated sequentially with a specific primary antibody (Anti-Flag antibody) targeting the target protein and a labeled secondary antibody (e.g., horseradish peroxidase HRP), forming a complex through specific antigen-antibody binding. Finally, a chemiluminescent substrate (e.g., ECL) was added for reaction. The HRP on the secondary antibody catalyzed the substrate to generate a chemiluminescent signal, which was acquired by X-ray film exposure or an imaging system. The target protein could then be quantitatively analyzed based on band position and intensity.

[0045] The results are as follows Figure 3 As shown in Figure B, both the mycelial proteins and supernatant proteins of fungal transformants Mgu977-Hv1a-1 and Mgu977-Hv1a-2 showed the correct protein bands at 9 kDa, indicating the stability of Hv1a protein expression in the transformants. One of the fungal transformants, Mgu977-Hv1a-1, was selected for subsequent activity assays.

[0046] 4. Evaluation of the insecticidal activity of engineered strains expressing Hv1a against rice stem borer. Wild-type strain Mgu977 and Hv1a-expressing strain Mgu977-Hv1a of *Metarhizium anisopliae* were selected. Spores were collected from fungal plates and placed in centrifuge tubes containing 0.1% Triton aqueous solution, then vortexed to mix. The spore suspensions filtered into new test tubes were diluted with 0.1% (v / v) Triton aqueous solution, and counted using a hemocytometer. The spore suspensions of each strain were then adjusted to a uniform test concentration of 2 × 10⁻⁶. 7 Spores / mL suspension. A 0.1% Triton aqueous solution was used as a control (CK).

[0047] The same immersion method as in Example 1, combined with water chestnut immersion, was used to treat the rice stem borer. Third-instar larvae of the tested rice stem borer were gently immersed in the corresponding spore suspension or control solution, ensuring complete submersion. After 30 seconds, they were removed, placed on sterile filter paper to absorb excess liquid, and allowed to air dry. Fresh water chestnuts were cut into uniformly thick slices and immersed in the same spore suspension or control solution as the treated insects for 10 seconds. After removal, they were drained until no clear liquid remained on the surface. The treated water chestnut slices were placed in clean plastic rearing round boxes, and then 10 larvae treated by the immersion method were transferred into each box. The box opening was covered with a black cloth and placed in an incubator at a temperature of 27±1°C, humidity of 80%±10%, and a light cycle of 16:8 (L:D) h. Each strain treatment was performed in triplicate, with 10 larvae per replicate. The control setup was the same. From the day of treatment, larval mortality was observed and recorded every 24 hours for 7 consecutive days. The absence of any reaction when the insect is gently touched with tweezers is considered a sign of death. One-way ANOVA was performed on the mortality rates of different groups using SPSS 22.0 (IBM SPSS Statistics, USA) software, and Duncan's method was used for multiple comparisons. The significance level was set at P < 0.05.

[0048] The mortality rate of rice stem borers in each treatment group on the 7th day after treatment, along with photos of the dead insects, are as follows: Figure 4 and Figure 5As shown, the results indicated that the insecticidal activity of the engineered strain expressing Hv1a (Mgu977-Hv1a) was significantly higher than that of the wild-type strain Mgu977. Specifically, the mortality rate of the wild-type strain Mgu977 after 7 days of treatment with the rice stem borer was 70.0%, while the mortality rate of the engineered strain expressing Hv1a (Mgu977-Hv1a) reached 96.7% after 7 days. The dead insects exhibited obvious symptoms of Metarhizium anisopliae infection, including stiffness and the appearance of a distinct white and green mold layer on the surface. These results demonstrate that expressing Hv1a in the Guizhou Metarhizium anisopliae Mgu977 strain can significantly enhance its insecticidal effect against the rice stem borer, exhibiting a synergistic effect. Its insecticidal speed and final mortality rate are both superior to the wild-type strain, showing great application potential in the control of the rice stem borer.

[0049] In summary, this invention reveals for the first time the significant insecticidal activity of Metarhizium guizhouense ARSEF 977 against the rice stem borer, after 2×10⁻⁶ cycles. 7 After 7 days of spore suspension immersion treatment, the mortality rate reached 70.0%. Further, using this as the starting strain, the Hv1a toxin gene derived from spiders was introduced into the strain through genetic engineering, successfully constructing a stable and highly efficient engineered strain expressing the spider insecticidal peptide Hv1a. This strain exerted a synergistic effect, further enhancing the insecticidal efficacy against the rice stem borer. The mortality rate after 7 days of treatment with the engineered strain reached as high as 96.7%. This invention not only provides a candidate genetically engineered biocontrol strain with stronger insecticidal efficacy and faster action for the control of the rice stem borer, but also provides a successful technical example for creating high-performance microbial pesticides by fusing biocontrol factors from different sources. It lays a solid core material and technical foundation for developing a new generation of environmentally friendly and highly targeted green control products for the rice stem borer, exhibiting high environmental and non-target organism safety, aligning with the direction of sustainable agricultural development, and showing broad application prospects.

[0050] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. An engineered strain of *Metarhizium anisopliae* expressing spider venom peptide Hv1a, characterized in that, The engineered bacteria are those in which the spider venom peptide Hv1a gene is transferred into Metarhizium guizhouense to express spider venom peptide Hv1a; wherein the amino acid sequence of the spider venom peptide Hv1a is shown in SEQ ID NO.2, and the Metarhizium guizhouense is Metarhizium guizhouense ARSEF 977.

2. The engineered bacteria according to claim 1, characterized in that, The nucleotide sequence of the spider venom peptide Hv1a gene is shown in SEQ ID NO.

1.

3. The engineered bacteria according to claim 1, characterized in that, The method for preparing the Guizhou Metarhizium anisopliae engineered strain expressing spider venom peptide Hv1a includes: ligating the Hv1a venom peptide gene into the pCAMBIA0380 expression vector, transforming the recombinant vector into Guizhou Metarhizium anisopliae using Agrobacterium-mediated genetic transformation, and screening to obtain recombinant engineered strains that fully express spider venom peptide Hv1a.

4. Application of *Metarhizium anisopliae* and / or engineered *Metarhizium anisopliae* strains expressing spider venom peptide Hv1a in any of the following: A1) Application in the control of rice stem borer; A2) Application in the preparation of products for controlling rice stem borer; in, The amino acid sequence of the spider venom peptide Hv1a is shown in SEQ ID NO.2; the Metarhizium guizhouense is Metarhizium guizhouense ARSEF 977.

5. A method for controlling the rice stem borer, characterized in that, The method includes: treating *Metarhizium guizhouense* and / or engineered *Metarhizium guizhouense* expressing spider venom peptide Hv1a with *Taenia pyrenoidosa*; wherein the amino acid sequence of the spider venom peptide Hv1a is shown in SEQ ID NO.2; and the *Metarhizium guizhouense* is *Metarhizium guizhouense* ARSEF977.

6. The method according to claim 5, characterized in that, The *Metarhizium anisopliae* and / or engineered *Metarhizium anisopliae* expressing spider venom peptide Hv1a are prepared into spore suspensions for inoculation treatment of rice stem borers, and / or spraying treatment on plants, leaves, or soil of crops damaged by rice stem borers.

7. The method according to claim 6, characterized in that, The concentration of the spore suspension is not less than 2.00 × 10⁻⁶. 7 Spores / mL.

8. The method according to claim 5, characterized in that, The engineered strain of *Metarhizium anisopliae* expressing spider venom peptide Hv1a was created by transferring the Hv1a venom peptide gene, as shown in SEQ ID NO.1, into *Metarhizium anisopliae* to induce the expression of spider venom peptide Hv1a.

9. The method according to claim 8, characterized in that, The method for preparing the Guizhou Metarhizium anisopliae engineered strain expressing spider venom peptide Hv1a includes: ligating the Hv1a venom peptide gene into the pCAMBIA0380 expression vector, transforming the recombinant vector into Guizhou Metarhizium anisopliae using Agrobacterium-mediated genetic transformation, and screening to obtain recombinant engineered strains that fully express spider venom peptide Hv1a.

10. The method according to claim 9, characterized in that, The method for preparing the engineered strain of *Metarhizium anisopliae* expressing spider venom peptide Hv1a includes: S1. Expression vector construction: The signal peptide encoding gene, Hv1a toxic peptide gene and FLAG protein encoding gene are sequentially linked to form the Hv1a-FLAG fusion expression element, which is then inserted between the Ptef1 promoter and the sur selection marker of the pCAMBIA0380 expression vector to obtain the p0380-Ptef1-Hv1a-FLAG-sur heterologous expression vector. S2. Fungal genetic transformation: The p0380-Ptef1-Hv1a-FLAG-sur heterologous expression vector was introduced into *Metarhizium anisopliae* via Agrobacterium-mediated genetic transformation. S3. Screening and identification of engineered bacteria: Using the sur selection marker on the expression vector, positive transformants were screened in M-100 medium containing chlorpyrifos antibiotic, and the integration of the Hv1a gene was verified by PCR amplification to obtain the engineered strain of Metarhizium anisopliae expressing spider venom peptide Hv1a.

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