Beauveria bassiana b-bb-1 and biocontrol agent and application thereof in prevention and treatment of bark beetles

Abstract

The present application relates to the field of microbial technology, in particular to a Beauveria bassiana B-BB-1, a biocontrol agent and application thereof in prevention and treatment of bark beetles, wherein the Beauveria bassiana B-BB-1 grows faster on PDA medium at 28 DEG C; the spore yield is large on SDAY medium at 25 DEG C, and the Beauveria bassiana B-BB-1 is easy to culture, and through indoor virulence determination, it is proved that the Beauveria bassiana B-BB-1 has high pathogenicity to Platypus quercivorus and Phoridus nodosus, when the spore concentration is 1x10 8 ML, the mortality of P. quercivorus adults reaches 96.15% on the 5th day, when the spore concentration is higher than 1x10 8 ML, the mortality of P. nodosus adults reaches 79.29% on the 3rd day after treatment, and reaches 100% on the 4th day, and the Beauveria bassiana B-BB-1 has the potential to be developed into a biological control agent for forest pests of bark beetles.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Beauveria bassiana B-BB-1, a biocontrol agent, and its application in the control of bark beetles. Background Technology

[0002] Lesser beetle on slope ( Euwallacea interjectus ), belonging to the genus *Bartholinae* of the subfamily Scolytinae in the family Curculionidae of the order Coleoptera. Euwallacea The slope bark beetle (Slope-shaped bark beetle) is a fungivorous bark beetle that cultivates symbiotic Fusarium fungi in its tunnels as its nutrient source. The bark beetle and its symbiotic fungi work together to damage the tree, ultimately leading to its death. It is highly invasive and causes significant damage to plants such as poplar, fig, and avocado.

[0003] Liquidambar beetle ( Acanthotomicus suncei ), belonging to the genus *Scolytinae* of the subfamily Scolytinae in the family Curculionidae of the order Coleoptera. Acanthotomicus The *Liquidambar formosana* (tree borer) is a borer that damages *Liquidambar formosana*. Its type specimen was discovered and collected from native *Liquidambar formosana* species in Nanjing as early as 1980. *Liquidambar formosana* has been widely introduced as an ornamental tree species in recent years, but the *Liquidambar formosana* borer has caused significant damage, resulting in large-scale infestations in Shanghai, Suzhou, Jiangsu, and other areas, causing substantial economic losses and the death of many *Liquidambar formosana* trees.

[0004] Bark beetles (including the slope bark beetle or the sweetgum bark beetle) live elusive lifestyles, making conventional control methods ineffective. With the gradual advancement of national ecological civilization construction, biological control has broad application prospects in the field of pest control due to its advantages such as strong environmental compatibility and low likelihood of developing drug resistance.

[0005] Because there are many types of host insects for biological control, and different strains have different host ranges and pathogenicity, there is an urgent need for a biological control strain that is highly pathogenic to both the slope bark beetle and the sweetgum bark beetle. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a strain of Beauveria bassiana B-BB-1, a biocontrol agent, and its application in controlling bark beetles. The Beauveria bassiana B-BB-1 provided by this invention exhibits high pathogenicity against both the slope bark beetle and the sweetgum bark beetle, and has the potential to be developed into a biological control agent for bark beetle forest pests.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a strain of Beauveria bassiana ( ).Beauveria bassiana The Beauveria bassiana B-BB-1 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2023270.

[0008] The present invention also provides a biocontrol agent, wherein the biocontrol agent comprises Beauveria bassiana B-BB-1 as described in the above technical solution.

[0009] Preferably, the spore concentration of Beauveria bassiana B-BB-1 in the biocontrol agent is ≥1×10⁻⁶. 7 per mL.

[0010] This invention provides a method for preparing the biocontrol agent described in the above technical solution, comprising the following steps: The Beauveria bassiana B-BB-1 described in the above technical solution was inoculated into a culture medium and cultured to obtain the biocontrol agent. The culture medium includes PDA medium, SDAY medium, or Czapek's medium.

[0011] Preferably, the culture temperature is 25~31℃.

[0012] This invention provides the *Beauveria bassiana* B-BB-1 described in the above-described technical solution, or the biocontrol agent described in the above-described technical solution, or the biocontrol agent prepared using the preparation method described in the above-described technical solution, for the control of weevils (…). Curculionidae Applications in pest control.

[0013] Preferably, the weevil family includes the subfamily Bark beetleinae (… Scolytinae ) pests.

[0014] Preferably, the bark beetle subfamily pests include the genus *Bark beetle* (*Bark beetle*). Euwallacea Pests and / or bark beetles ( Acanthotomicus ) pests.

[0015] Preferably, the *Bartholinae* pests include *Bartholinae slopeina* (…). Euwallacea interjectus ).

[0016] Preferably, the bark beetle pests include the sweetgum bark beetle (Symplocos spp.) Acanthotomicus suncei ).

[0017] Beneficial effects: This invention provides a strain of Beauveria bassiana ( ). Beauveria bassianaThe *Beauveria bassiana* strain B-BB-1, described in this invention, is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M2023270. This invention isolated a *Beauveria bassiana* strain, designated B-BB-1, from a diseased *Bartholinium sclerotium* species in the wild. Morphological and molecular biological identification confirmed it as a strain of *Beauveria bassiana*, and it is now deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M2023270. The *Beauveria bassiana* strain B-BB-1 described in this invention grows rapidly on PDA medium at 28°C; on SDAY medium at 25°C, it produces a large amount of spores, is easy to culture, and has been demonstrated by in vitro virulence testing to have high pathogenicity against both *Bartholinium sclerotium* and *Bartholinium styracifolium*, with a spore concentration of 1×10⁻⁶. 8 When the spore concentration was 1×10⁶ / mL, the mortality rate of adult bark beetles on the slope reached 96.15% on day 5. 8 When the concentration of insects per mL was 79.29%, the mortality rate of adult bark beetles reached 79.29% on the 3rd day after treatment and 100% on the 4th day, indicating its potential to be developed into a biological control agent for bark beetles, a type of forest pest.

[0018] Biological Preservation Instructions Beauveria bassiana strain B-BB-1, Latin name Beauveria bassiana, It was deposited on March 9, 2023, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: M2023270. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 A frontal view of Beauveria bassiana B-BB-1 colonies on PDA medium; Figure 2 This is a reverse image of a colony of Beauveria bassiana B-BB-1 on PDA medium. Figure 3 Abdominal image of a dead bark beetle on a slope infected with Beauveria bassiana B-BB-1; Figure 4 The back view of the dead state of a bark beetle on a slope infected with Beauveria bassiana B-BB-1; Figure 5 Abdominal image of a dead *Liquidambar formosana* after infection with *Beauveria bassiana* B-BB-1; Figure 6 The image shows the back of a dead *Liquidambar formosana* beetle after infection with *Beauveria bassiana* B-BB-1. Detailed Implementation

[0021] This invention provides a strain of Beauveria bassiana ( ). Beauveria bassiana The Beauveria bassiana B-BB-1 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2023270.

[0022] The *Beauveria bassiana* B-BB-1 described in this invention has white colonies on the front side on PDA medium (see...). Figure 1 The back is pale yellow (see) Figure 2 The colonies are relatively thick; initially, the colonies are fluffy and slightly raised; later, the central part of the colony is slightly concave and powdery, while the outer part is flocculent; under an optical microscope, the conidiophores are spike-shaped and attached to the vegetative hyphae, and the conidiophores are clustered on the conidiophores; the conidia are nearly spherical, smooth, transparent, and 2.0~2.8μm×1.9~2.5μm in size.

[0023] The preferred ITS sequence of Beauveria bassiana B-BB-1 described in this invention is as shown in SEQ ID NO.1, specifically: 5'--3'; The preferred EF1-α sequence of Beauveria bassiana B-BB-1 described in this invention is as shown in SEQ ID NO.2, specifically: 5'--3'.

[0024] The present invention also provides a biocontrol agent, wherein the biocontrol agent comprises Beauveria bassiana B-BB-1 as described in the above technical solution.

[0025] In this invention, the spore concentration of Beauveria bassiana B-BB-1 in the biocontrol agent is preferably ≥1×10⁻⁶. 7 Cells / mL, more preferably ≥1×10 8 per mL.

[0026] This invention also provides a method for preparing the biocontrol agent described in the above technical solution, comprising the following steps: The *Beauveria bassiana* B-BB-1 described in the above technical solution was inoculated into a culture medium and cultured to obtain the biocontrol agent.

[0027] The culture medium described in this invention preferably includes PDA medium, SDAY medium, or Czapek's medium. The *Beauveria bassiana* B-BB-1 strain described in this invention exhibits the fastest growth rate on PDA medium, reaching a colony diameter of 58.48 mm on day 16, significantly higher than that on SDAY and Czapek's media, and also shows good growth vigor. Therefore, this strain is considered suitable for growth on PDA medium. *Beauveria bassiana* B-BB-1 shows greater colony thickness and significantly higher sporulation yield on SDAY medium than the other two groups, with high spore viability. Therefore, SDAY is considered more suitable as the sporulation medium for this strain.

[0028] In this invention, the preferred culture temperature is 25-31℃. The *Beauveria bassiana* B-BB-1 strain described in this invention exhibits the fastest growth rate at 28℃, with a colony diameter reaching 58.48 mm on day 16, significantly higher than at other culture temperatures. At 25℃, the highest sporulation yield is observed on day 16, reaching 2.87 × 10⁻⁶. 6 spores / cm 2 The optimal growth temperature of Beauveria bassiana B-BB-1 described in this invention is 28°C, and the optimal spore production temperature is 25°C.

[0029] The present invention also provides the application of Beauveria bassiana B-BB-1 as described in the above technical solution, or the biocontrol agent as described in the above technical solution, or the biocontrol agent prepared by the preparation method described in the above technical solution, in the control of Curculionidae pests.

[0030] In this invention, the weevil family pests preferably include pests of the Scolytinae subfamily; the Scolytinae subfamily pests preferably include the genus *Scolytinae* (…). Euwallacea Pests and / or bark beetles ( Acanthotomicus ) pests; the preferred pests of the genus *Bartholinae* include *Bartholinae slopeina* (… Euwallacea interjectus The preferred pests of the genus *Bark beetle* include *Bark beetle styrax* (*Symplocos spp.*). Acanthotomicus suncei ).

[0031] The *Beauveria bassiana* B-BB-1 strain described in this invention exhibits high pathogenicity against both *Bark beetle* and *Liquidambar formosana*, with a spore concentration of 1×10⁻⁶. 8 When the spore concentration was 1×10⁶ / mL, the mortality rate of adult bark beetles on the slope reached 96.15% on day 5. 8 When the concentration of insects per mL was 79.29%, the mortality rate of adult bark beetles reached 79.29% on the 3rd day after treatment and 100% on the 4th day. This strain has the potential to be developed into a biological control agent for bark beetles, a forest pest. Using this strain to control bark beetles is a typical biological control method, which can avoid the problems of drug resistance and environmental pollution caused by chemical pesticides. It will provide a new product for the green control of bark beetles.

[0032] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a strain of Beauveria bassiana B-BB-1, a biocontrol agent, and its application in controlling bark beetles, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1 Isolation and identification of strains 1.1 Materials and Methods 1.1.1 Materials Adult bark beetles on diseased slopes were collected from poplar trees in Huanghai National Forest Park, Dongtai City, Jiangsu Province.

[0034] Potato glucose agar (PDA) medium: 200g potato, 20g glucose, 15g agar and 1000mL distilled water.

[0035] Aseptic operating conditions: All instruments and utensils are sterilized in a high-temperature sterilizer (121℃, 30min), and inoculation and other operations are carried out in a laminar flow hood.

[0036] Culture conditions: Incubate in a 28℃ constant temperature incubator.

[0037] 1.1.2 Isolation and purification of pathogens Isolation: The pathogen was isolated from the cadavers of infected adult *Begonia gracilis*. Infected adult *Begonia gracilis* bodies were brought back to the laboratory. The cadavers were sequentially immersed in 70% alcohol for 30 seconds, followed by immersion in 0.1% mercuric chloride solution for 3 minutes (for thorough disinfection), and finally rinsed three times with sterile water. The treated *Begonia gracilis* tissue was then inoculated onto PDA medium using an inoculation needle. The medium was cultured at 28°C. Pure strains were obtained by single-spore isolation and culture, transferred to PDA slant, and grown for 3-4 days. The strains were then stored at 4°C.

[0038] Rejuvenation: The isolated strain was cultured on PDA medium for 5 days until a large number of conidia were produced. These conidia were then collected and transferred to sterile water containing 1% Tween-80, stirred evenly with a magnetic stirrer to obtain a spore suspension. This suspension was then sprayed evenly onto the surface of adult *Bartholin's scorpion* beetles on a slope using a spray tower, and cultured under conditions of 80% or higher humidity. Dead insect bodies were collected and cultured under moist conditions. The resulting adult carcasses were then used to isolate a more virulent strain using the same method.

[0039] Purification: Pick conidial powder from the culture medium and inoculate it again onto freshly prepared PDA medium. Culture in this way for 3 generations to obtain the purified strain.

[0040] 1.1.3 Identification of Pathogens Morphological identification: Identification was performed based on the culture characteristics, hyphae, and conidia of the pathogen. The morphology of hyphae, conidia, and sporulation structures was examined under a 40×10 magnification optical microscope.

[0041] Molecular identification: DNA was extracted from this strain using a rapid fungal genomic DNA extraction kit (Sangon Biotech). PCR amplification was performed using ITS primer pairs and EF1-α primer pairs, respectively.

[0042] The ITS primer pair consists of ITS1 and ITS4, with the following specific sequences: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3, SEQ ID NO.3; ITS4: 5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO.4; The EF1-α primer pair consists of EF1-688F and EF1-1251R, with the following specific sequences: EF1-688F: 5'-CGGTCACTTGATCTACAAGTGC-3', SEQ ID NO.5; EF1-1251R: 5'-CCTCGAACTCACCAGTACCG-3', SEQ ID NO. 6.

[0043] The PCR reaction system for the PCR amplification was as follows: 10 μL of 2×EasyTaq PCR SuperMix (TransGen Biotech Co., Ltd.), 1 μL each of forward and reverse primers, 2 μL of DNA template, and 6 μL of ddH2O were added to bring the total volume of the reaction system to 20 μL. The PCR amplification conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; 72℃ extension for 10 min. After amplification, the size of the reaction products was determined by 0.5% agarose gel electrophoresis, and finally, the samples were sent to Shanghai Jieli Biotechnology Co., Ltd. for sequencing.

[0044] 1.2 Results Morphological identification: On PDA medium, the colony surface is white (see...). Figure 1 The back is pale yellow (see) Figure 2The colonies are relatively thick. Initially, the colonies are fluffy and slightly raised; later, the central part of the colony is slightly concave and powdery, while the outer part is flocculent. Under an optical microscope, the conidiophores are spike-like, attached to the vegetative hyphae, and the conidiophores are clustered on the conidiophores. The conidia are nearly spherical, smooth, and transparent, measuring 2.0–2.8 μm × 1.9–2.5 μm.

[0045] Molecular identification: The purified strain was sequenced to obtain the ITS gene sequence as shown in SEQ ID NO.1 and the EF1-α gene sequence as shown in SEQ ID NO.2. The two sequences, SEQ ID NO.1 and SEQ ID NO.2, were compared using the NCBI database. The comparison results are shown in Table 1.

[0046] Table 1. Sequence alignment results of ITS and EF1-α genes in Beauveria bassiana strain B-BB-1

[0047] Based on natural infection symptoms, indoor isolation and observation of infected insects, and according to the infection symptoms, morphological characteristics of conidia and hyphae, and molecular identification characteristics of *Beauveria bassiana*, this strain was identified as *Beauveria bassiana* and named *Beauveria bassiana*. Beauveria bassiana The strain B-BB-1 was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2023270.

[0048] Example 2: Biological characteristics of the purified strain 2.1 Materials and Methods 2.1.1 Test strains Example 1 shows the isolated and purified Beauveria bassiana strain B-BB-1.

[0049] 2.1.2 Effects of culture medium on the growth and sporulation of Beauveria bassiana strain B-BB-1 This experiment used potato dextrose agar (PDA), saxo-yeast agar (SDAY), and Czapek's medium. The SDAY medium consisted of the following components by weight percentage: 1% peptone, 4% glucose, 1% yeast extract, 2% agar, and 92% water. The Czapek's medium consisted of the following components by weight percentage: 3% sucrose, 0.3% sodium nitrate, 0.05% potassium chloride, 0.05% magnesium sulfate heptahydrate, 0.001% ferrous sulfate heptahydrate, 1.5% agar, and 95.099% water.

[0050] Each culture medium was treated as follows: A mycelial disc inoculation method was used. A 5mm hole punch was used to make a round hole in the center of the culture dish to be inoculated. Using the same punch, mycelial discs were then collected from the spore-scarce area at the edge of the colony on the culture dish of the test strain. These discs were then picked up with an inoculation needle or tweezers and inserted into the round hole to complete the inoculation. After inoculation, the culture dishes were inverted and incubated in the dark at 28℃. The cross-sectional and longitudinal diameters of the colonies were measured periodically using the cross-sectional method. After 16 days of incubation, five mycelial discs were collected at equal intervals along the diameter from the center of the colony: at the colony edge, halfway between the edge and the center, and at the center of the colony. These discs were placed in 25mL of 0.2% Tween-80 solution, shaken evenly, and a spore suspension was prepared. The spore concentration was determined using a hemocytometer, and the sporulation yield for each treatment was calculated. Each treatment was replicated at least five times. The results are shown in Table 2.

[0051] 2.1.3 Effects of temperature on the growth and sporulation of Beauveria bassiana strain B-BB-1 The test strains were inoculated using PDA medium, following the same inoculation method as in section 2.1.2, and then incubated in the dark at 25, 28, and 31°C. Growth rates were observed and measured daily starting from day 3, and sporulation was determined on day 16. Each treatment was replicated at least four times. Results are shown in Table 3.

[0052] 2.2 Results 2.2.1 Effects of culture medium on the growth and sporulation of Beauveria bassiana strain B-BB-1 Table 2 shows that the *Beauveria bassiana* strain B-BB-1 can grow on PDA, SDAY, and Czapek's agar. The strain showed the fastest growth rate on PDA, with a colony diameter of 58.48 mm on day 16, significantly higher than the SDAY and Czapek's agar groups, and exhibited better growth. Therefore, this strain is considered suitable for growth on PDA. *Beauveria bassiana* B-BB-1 showed larger colony thickness and significantly higher sporulation yield on SDAY agar compared to the other two groups, with higher spore viability. Therefore, SDAY is considered more suitable as the sporulation medium for this strain.

[0053] Table 2. Growth and sporulation of Beauveria bassiana strain B-BB-1 on different culture media.

[0054] 2.2.2 Effects of temperature on strain growth and sporulation Table 3 shows that the Beauveria bassiana strain B-BB-1 can grow at 25, 28, and 31℃. The fastest growth rate was observed at 28℃, with a colony diameter of 58.48 mm on day 16, significantly higher than other treatments. The highest sporulation yield was observed at 25℃ for 16 days, reaching 2.87 × 10⁻⁶. 6 pcs / cm 2The results were significantly higher than those from treatments at 28°C and 31°C. In summary, 28°C is the most suitable temperature for the growth of this strain, but 25°C is more suitable for sporulation.

[0055] Table 3. Growth and sporulation of Beauveria bassiana strain B-BB-1 at different culture temperatures.

[0056] Example 3: Indoor toxicity determination of purified strains against *Bartholin's slope* 3.1 Materials and Methods 3.1.1 Test insect source The slope beetle was obtained from Dongtai City, Jiangsu Province. After being brought back to the laboratory, it was raised in the Insect Physiology and Biochemistry Laboratory of Nanjing Forestry University. Adults with good activity and complete development were selected as experimental materials.

[0057] 3.1.2 Preparation of spore suspension Using the purified Beauveria bassiana culture medium from Example 1, which showed good growth and high spore production, an appropriate amount of mycelial cake was collected from the colonies using a fungal punch. The cake was then eluted with sterile water containing 0.2% Tween 80. A small amount of spore suspension was then aspirated using a sterile plastic dropper and placed onto a hemocytometer. The spores were counted and recorded under a microscope. Finally, the spore suspension was appropriately diluted to prepare a 1×10⁻⁶ solution. 8 cells / mL, 1×10 7 cells / mL, 1×10 6 A conidial suspension of 1 conidia / mL was prepared for use, and 0.2% Tween 80 sterile water was prepared as a blank control for virulence determination.

[0058] 3.1.3 Inoculation treatment The insects were treated using the immersion method. Adult *Bartholin's slope* beetles were picked up with a soft brush and transferred to the bottom of a 5 mL centrifuge tube. 1 mL of spore suspension was added using a pipette, and the tube was gently shaken to ensure the insects were fully in contact with the spore suspension. After immersion for 10 seconds, the tube was poured out, and the insects were blotted dry with filter paper. The insects were then transferred to a 9 cm diameter sterile disposable plastic petri dish lined with clean filter paper. A small amount of sterile water was added to the filter paper to keep it moist. (1×10⁻⁶) 8 cells / mL, 1×10 7 cells / mL, 1×10 6The treatment group consisted of a spore suspension at a concentration of 20 insects / mL and a control group consisted of sterile water containing 0.2% Tween 80. Each group used 20 insects, and the treatments were repeated three times. Before treatment, each group of insects was incubated at room temperature (25±3℃) for 24 hours. Individuals that died unexpectedly within a short period were removed. The surviving insects were then used for Beauveria bassiana treatment. The final number of insects treated in each group is shown in Table 4. After treatment, the insects were incubated at room temperature (25±3℃). The survival of each group of insects was observed daily, the filter paper was replenished with water, and dead insects were removed promptly. Dead insects were individually removed and kept in a warm and humid environment for observation to see if Beauveria bassiana symptoms appeared. Figure 3 and Figure 4 .

[0059] Cumulative mortality rate (%) = Total cumulative mortality at observation time / Total number of insects before treatment × 100%; Cumulative corrected mortality rate (%) = (Cumulative mortality rate in the treatment group - Cumulative mortality rate in the control group) / (1 - Cumulative mortality rate in the control group) × 100%.

[0060] 3.2 Results The results are shown in Tables 4 and 5. The results indicate that the *Beauveria bassiana* strain B-BB-1 has a significant pathogenic effect on adult *Bartholin's slope* beetles. When the treatment concentration is 1×10⁻⁶... 8 At a concentration of 1×10⁶ cells / mL, the mortality rate of adult *Bartholin's slope* beetles reached 96.15% on day 5, compared to only 3.85% in the control group; the treatment concentration was 1×10⁶ cells / mL. 7 When the number of insects / mL was 78.26%, the mortality rate of adult Bark Beetles on the slope reached 78.26% on day 6, compared with 11.54% in the control group.

[0061] Table 4. Total number of deaths of *Leymus chinensis* on slopes at different times and in different treatment groups.

[0062] Table 5. Pathogenicity of Beauveria bassiana strain B-BB-1 against Bark beetle on slope.

[0063] Example 4: Indoor toxicity determination of purified strains against Liquidambar bark beetle 4.1 Materials and Methods 4.1.1 Test insect source Liquidambar beetle Acanthotomicus suncei Originating from Jiangxi Province, the infested wood segments were sent back to the laboratory and placed in net cages, where they were regularly sprayed with water to maintain humidity. After the Liquidambar bark beetles inside the wood segments emerged, due to their tendency to ascend, the adults mostly gathered at the upper edge of the net cages. Adults with good activity, complete development, and roughly the same size were selected as experimental materials.

[0064] 4.1.2 Preparation of spore suspension Using the purified Beauveria bassiana culture medium from Example 1, which showed good growth and high spore production, an appropriate amount of mycelial cake was collected from the colonies using a fungal punch. The cake was then eluted with sterile water containing 0.2% Tween 80. A small amount of spore suspension was then aspirated using a sterile plastic dropper and placed onto a hemocytometer. The spores were counted and recorded under a microscope. Finally, the spore suspension was appropriately diluted to prepare a 1×10⁻⁶ solution. 8 cells / mL, 1×10 7 cells / mL, 1×10 6 A conidial suspension of 1 conidia / mL was prepared for use, and 0.2% Tween 80 sterile water was prepared as a blank control for virulence determination.

[0065] 4.1.3 Inoculation Treatment The insects were treated using the immersion method. Adult *Symplocos spp.* (a type of bark beetle) were picked up with a soft brush and transferred to the bottom of a 5 mL centrifuge tube. 1 mL of spore suspension was added using a pipette, and the tube was gently shaken to ensure the insects were fully in contact with the spore suspension. After immersion for 10 seconds, the tube was poured out, and the insects were blotted dry with filter paper. The insects were then transferred to a 9 cm diameter sterile disposable plastic petri dish lined with clean filter paper. A small amount of sterile water was added to the filter paper to keep it moist. (1×10⁻⁶) 8 cells / mL, 1×10 7 cells / mL, 1×10 6 The treatment group consisted of spore suspension at a concentration of 20 insects / mL and a control group consisted of sterile water containing 0.2% Tween 80. Each group used 20 insects, and the treatments were repeated three times. Before treatment, each group of insects was incubated at room temperature (25±3℃) for 24 hours. Individuals that died unexpectedly within a short period were removed. The surviving insects were then used for Beauveria bassiana treatment. The final number of insects treated in each group is shown in Table 6. After treatment, the insects were incubated at room temperature (25±3℃). The survival of each group of insects was observed daily, and dead insects were removed promptly. Dead insects were individually removed and kept in a humid environment for further observation to check for the appearance of Beauveria bassiana symptoms. Figure 5 and Figure 6 .

[0066] Cumulative mortality rate (%) = Total cumulative mortality at observation time / Total insect population before treatment × 100% Cumulative corrected mortality rate (%) = (Cumulative mortality rate of treatment group - Cumulative mortality rate of control group) / (1 - Cumulative mortality rate of control group) × 100%; when the cumulative mortality rate of treatment group is 0, its cumulative corrected mortality rate is also 0.

[0067] 4.2 Results The results are shown in Tables 6 and 7. The results indicate that *Beauveria bassiana* strain B-BB-1 has a significant pathogenic effect on adult *Bark beetle*. When the treatment concentration is higher than 1×10⁻⁶, the pathogenicity is significantly reduced. 8 At a concentration of [number] insects / mL, the mortality rate of adult Liquidambar beetles reached 79.29% on day 3 and 100% on day 4 after treatment, compared to only 6.9% in the control group; 1×10 [units / mL]7 The mortality rate of adult bark beetles in the treatment group with 1000 insects / mL reached 72.15% on day 4.

[0068] Table 6. Total number of deaths of Liquidambar bark beetles at different times and in different treatment groups

[0069] Table 7. Pathogenicity of Beauveria bassiana strain B-BB-1 against Liquidambar bark beetle

[0070] In summary, the Beauveria bassiana B-BB-1 provided by this invention has high pathogenicity against both the slope bark beetle and the sweetgum bark beetle, and has the potential to be developed into a biological control agent for bark beetle forest pests.

[0071] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Beauveria bassiana ( Beauveria bassiana B-BB-1, characterized in that, The Beauveria bassiana B-BB-1 strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2023270.

2. A biocontrol agent, characterized in that, The biocontrol agent includes Beauveria bassiana B-BB-1 as described in claim 1.

3. The biocontrol agent according to claim 2, characterized in that, The spore concentration of Beauveria bassiana B-BB-1 in the biocontrol agent is ≥1×10⁻⁶. 7 per mL.

4. The method for preparing the biocontrol agent according to claim 2 or 3, characterized in that, Includes the following steps: The Beauveria bassiana B-BB-1 of claim 1 was inoculated into a culture medium and cultured to obtain the biocontrol agent. The culture medium includes PDA medium, SDAY medium, or Czapek's medium.

5. The preparation method according to claim 4, characterized in that, The culture temperature is 25~31℃.

6. The biocontrol agent of Beauveria bassiana B-BB-1 as described in claim 1, or the biocontrol agent as described in claim 2 or 3, or the biocontrol agent prepared by the preparation method described in claim 4 or 5, is effective in controlling bark beetles on slopes ( Euwallacea interjectus ) and / or Liquidambar bark beetle ( Acanthotomicus suncei Applications in ).

Images