Beauveria bassiana bh002 strain as well as screening and separating method and application thereof

Abstract

The invention is applicable to the technical field of microorganisms, and provides a beauveria bassiana bh002 strain as well as a screening and separating method and application thereof. The strain is classified and named as Beauveria bassiana, and is preserved in China General Microbiological Culture Collection Center (CGMCC) on May 12, 2025, and the preservation number is CGMCC No.42004. The invention further discloses a preparation method of the Beauveria bassiana strain. The corrected death rate of the strain to larvae reaches 60.26%, larva bodies begin to rigidify within 48 hours after being inoculated, hyphae completely cover the surfaces of the larva bodies in 4-8 days, and it is proved that the strain can rapidly and efficiently infect target pests. Compared with chemical pesticides, the biological prevention and control scheme has the remarkable ecological advantages that no chemical residues exist, natural enemy organisms are not damaged, the strains can continuously play a role through natural circulation, and sustainable prevention and control support is provided for a hazel forest ecological system.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Beauveria bassiana BH002 strain and a screening and separation method and application thereof. Background Art

[0002] Entomogenous fungi, as insect pathogens, are a crucial component of microbial pesticide resources. They are not only environmentally friendly, pollution-free, and harmless to humans and animals, but they also prevent pest resurgence and are less likely to develop resistance. They possess a robust soil survival capacity and can cause epidemics in fields, playing a crucial role in controlling agricultural and forestry pest populations. Statistics show that 60% of insect deaths in nature are caused by fungal epidemics, demonstrating the crucial role of entomogenous fungi in controlling pest numbers and population density. After leaving their host, entomogenous fungi enter the soil and survive there for long periods of time. They are a specialized group of soil microorganisms, and therefore soil is considered a vast reservoir of entomogenous fungi and a crucial source for biocontrol agents for agricultural and forestry pests. The rhizosphere, the thin layer of soil directly influenced by plant roots and their secretions, is a crucial site for interactions between plants and soil microorganisms. Rhizosphere microorganisms play a vital role in nutrient transformation and absorption, plant growth promotion, pollutant degradation, organic matter decomposition, soil-borne pathogen suppression, plant health maintenance, and plant resilience to adversity. Isolating entomogenous fungi that are pathogenic to target insects from rhizosphere soil is of great significance for the development and utilization of biopesticides.

[0003] hazelnut( Corylus chinensis ), is a species of the genus Corylus in the family Betulaceae ( Corylus ) plant, a small deciduous tree or shrub. Its fruit has extremely high utilization value and is known as the "king of nuts". The occurrence of hazelnut diseases and insect pests affects the yield and quality of hazelnuts, among which the hazelnut weevil ( Curculio nucum ) is the primary pest that harms hazelnuts. In Jilin Province, the hazelnut weevil infests 20% to 30% of hazelnuts, and in severe cases, up to nine out of ten hazelnuts are infested, rendering the hazelnuts completely unusable. Control of the hazelnut weevil is urgent. The hazelnut weevil is a Coleoptera insect. The adult body surface is hard, making it difficult for chemical agents to penetrate. The larvae of the hazelnut weevil feed inside the hazelnuts and overwinter in the soil, making the use of chemical agents less effective. Furthermore, excessive use of chemical pesticides can cause drug resistance in the weevil, polluting the environment and affecting the edible safety of the fruit. Therefore, seeking a biological control method during the hazelnut weevil's overwintering stage has become an economical and effective measure to address the problem of overwintering larvae.

[0004] Soil is an essential habitat for hazelnut weevils (coral weevils) larvae to overwinter, and the entomogenic fungi found there are of great significance for controlling the weevils' damage. However, soil is rich in microbial communities (including bacteria, actinomycetes, and fungi), and the distribution of entomogenic fungi varies across regions and vegetation types. Therefore, isolating entomogenic fungi pathogenic to target insects, such as the hazelnut weevils, from rhizosphere soils of different forest types is fundamental and crucial for insect biological control. Currently, there are two main methods for obtaining entomogenic fungal strains: one involves isolating and purifying strains from naturally infected dead insects. However, this method yields highly virulent strains, making it extremely difficult to locate dead insects and requiring significant human resources. The other method involves isolation from soil: soil samples are collected from a specific area, prepared into a soil suspension, and the diluted soil suspension is applied to a selective medium for screening and isolation of fungi. While this method can yield large quantities of fungi from soil, it is labor-intensive and the isolated fungi may not be pathogenic to the target insects. To address this issue, the present invention proposes a Beauveria bassiana BH002 strain, its screening and isolation method, and its use. Summary of the Invention

[0005] The purpose of the present invention is to provide a Beauveria bassiana bh002 strain and its screening and separation method and application, aiming to solve the problems raised in the above background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions: A Beauveria bassiana bh002 strain, the strain is classified and named Beauveria bassiana ( Beauveria bassiana ), was deposited in the General Microbiology Center of China Culture Collection Administration on May 12, 2025, with the deposit number CGMCC No.42004.

[0007] A use of the Beauveria bassiana BH002 strain as described above in the preparation of a bacterial agent for controlling larvae of the hazelnut weevil.

[0008] Furthermore, the bacterial agent is a spore suspension with a spore concentration of 10 7 ~10 8 spores / ml.

[0009] A method for screening and isolating the Beauveria bassiana bh002 strain as described above comprises the following steps: Step 1: Collection of soil samples; Mixed forests, broad-leaved forests, larch forests and plantations were selected as sample plots and rhizosphere soils were collected from the sample plots; Step 2: Collection of borer-hole hazelnuts; Collecting borer-hole hazelnuts in wild hazel forests; Step 3: trapping soil entomogenous fungi; The collected soil samples were placed in open plastic bottles, and the bored hazelnuts were buried in two layers inside the bottles to simulate a natural habitat. Dead branches and leaves were also covered on the soil surface at the bottle mouths. The bottles were then placed in a biochemical incubator at 23°C, shielded from light and moisturized for 14 days. Three replicates were set up for each soil sample. After the incubation period, the soil in the bottles was poured out, and the dead insects with mycelium growing were picked out for later use. Step 4: Isolation and purification of entomogenous fungi; Soak the dead insects in 70% alcohol for 15-20 seconds, rinse them three times with sterile water, and then use the tissue separation method to cut the surface-disinfected dead insects into small pieces of tissue. Place them on inverted modified PDA plates, with four pieces of tissue placed on each plate, and invert them for culture at a constant temperature of 23°C. After hyphae grow around the tissue blocks, directly cut off the ends of some hyphae and transfer them to new PDA plates for further culture. If the hyphae are impure, perform another separation and purification operation until a purified strain is obtained. The purified strain is transferred to a modified PDA slant and stored in a culture cabinet at 4°C after the hyphae are fully grown. Step 5: Preparation of mature larvae of the hazelnut weevil; Pile the collected borer-hole hazelnuts on an open cement floor with plenty of sunlight. After the mature larvae crawl out, collect them into ziplock bags. Step 6: Preparation of spore suspension; The isolated and purified fungal strain was cultured on a modified PDA plate at 23°C for 7 days. After the colonies were full and spores were produced, the spores were washed with 0.5% Tween 80 sterile water. The washed spores were placed on a shaker and cultured at 180 rpm and 23°C for 1 day. The spore suspension was filtered with gauze and the spore concentration was diluted to 10 by microscopic counting. 7 ~10 8 spores / ml, stored in a refrigerator at 4°C for later use; Step 7: Screening of infectious strains; Mature larvae of the hazel weevil were placed in petri dishes lined with sterile filter paper, with 15 larvae placed in each dish. A prepared spore suspension was sprayed onto the dishes, with the insects being sprayed for inoculation. Sterile 0.5% Tween-80 water was used as a control. After inoculation, all treated petri dishes were sealed with plastic wrap and punctured for ventilation. The dishes were then placed in a biochemical incubator at 23°C for incubation. Observations were made daily for 8 consecutive days, and dead insects were picked out and continued to be cultured. Insects with mycelial growth were again isolated and purified for entomogenic fungi. If the strain obtained had the same characteristics as the original strain, it was considered to be an entomogenic fungus with infective activity against the hazel weevil larvae. Step 8: Pathogenicity determination; The selected strains with infective activity were inoculated into mature larvae of the hazelnut weevil, with 30 larvae placed in each dish. Each treatment was replicated three times, and sterilized 0.5% Tween 80 water was used as a control. After inoculation, all treated culture dishes were sealed with plastic wrap and punctured for ventilation, and then placed in a biochemical incubator at 23°C. After 48 hours, the plastic wrap was removed and the dish openings were covered with two layers of gauze to reduce the humidity inside the dish. The larvae were observed for mortality at regular intervals every day for 8 consecutive days. The mortality rate and the number of larvae with hyphae were counted, and the adjusted mortality rate was calculated according to the following formula: Adjusted mortality (%) = [(treatment mortality - control mortality) / 1 - control mortality)] × 100; The strain with the highest corrected mortality rate was screened out, namely Beauveria bassiana bh002.

[0010] Furthermore, the formula of the improved PDA includes: 200g potatoes, 20g glucose, 18g agar and 1000mL soil extract, which are sterilized at 121°C and 103.4kPa for 30min.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an efficient biological control application scheme for the damage caused by hazelnut weevil larvae. The strain of Beauveria bassiana BH002 isolated from the soil of natural forest area can be prepared into a spore concentration of 10 7 ~10 8 A suspension inoculant containing 10 spores / ml has a significant pathogenic effect on hazelnut weevil larvae. Experimental results showed that this strain had a corrected mortality rate of 60.26% for the larvae. The insects began to ossify within 48 hours after inoculation, and within 4-8 days, the mycelium completely covered the insect surface, demonstrating its rapid and efficient infection of the target pest. This biological control solution offers significant ecological advantages over chemical pesticides: it leaves no chemical residues, does not harm natural enemies, and the strain continues to function through natural cycles, providing sustainable control support for hazelnut forest ecosystems. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Flow chart of the screening and separation method of the present invention.

[0013] Figure 2 It is a trapping treatment for mixed forest soil samples.

[0014] Figure 3 To isolate and purify the strain.

[0015] Figure 4 The cumulative mortality rate of five strains (strain H1-2, strain H3-1, strain K1-3, strain L2-2, and strain L5-1) against hazelnut weevil larvae.

[0016] Figure 5 These are the symptoms of mature larvae of the hazelnut weevil infected by strain H1-2. DETAILED DESCRIPTION

[0017] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0018] The following describes the specific implementation of the present invention in detail with reference to specific embodiments. The experimental methods in the following embodiments, unless otherwise specified, are all conventional methods; the instruments and equipment used in the following embodiments, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following embodiments, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0019] Example 1: Entomogenous fungi trapping; 1. Soil Sample Collection: In late August, sampling plots were established at the Jiaohe State-owned Forest Protection Center in Jilin Province within four forest types: mixed forest (H), broad-leaved forest (K), larch forest (L), and plantation forest (R). Rhizosphere soil was collected from each plot using a five-point sampling method. Hazelnut weevil larvae typically overwinter in the 15-35 cm soil layer. Therefore, sampling was performed by first removing 2 cm of topsoil, then using a tube sampler to sample soil from a depth of 10-30 cm. Five different soil samples were collected from each sampling point (numbered H1-H5, K1-K5, L1-L5, and R1-R5). The samples were placed in sterile centrifuge tubes, sealed, and refrigerated before being brought back to the laboratory for future use.

[0020] 2. Collection of worm-holed hazelnuts: From late August to early September, fallen hazelnuts were collected from the wild hazelnut forest in Xinshan Village, Jiangmifeng Town, Longtan District, Jilin City. After collection, hazelnuts with worm holes were selected and placed in experimental bags and brought back to the laboratory for use.

[0021] 3. Attraction of soil entomogenous fungi: The collected soil samples were placed in open plastic bottles respectively, and 6 borer-hole hazelnuts were selected and buried in the plastic bottles in 2 layers to simulate the natural habitat, and the soil surface at the bottle mouth was covered with dead branches and leaves. The plastic bottles were then placed in a biochemical incubator at 23°C, shielded from light and moisturized for 14 days, and 3 groups of repeated experiments were set up for each soil sample. After the incubation was completed, the soil in the plastic bottle was poured out, and the dead insects with mycelium growth were picked out. The results showed that 6, 7, 9 and 6 dead insects were obtained from the soil samples of mixed forests, broad-leaved forests, larch forests and artificial forests, respectively, for a total of 28 dead insects ( Figure 2 , Table 1), and these dead insects were used for subsequent isolation of entomogenous fungi.

[0022] Table 1. Entomogenous fungi trapping

[0023] Example 2: Isolation and purification of entomogenous fungi; All experimental steps were performed in a sterile room.

[0024] 1. Separation preparation; First, scrub the workbench clean, place all items needed before work on the workbench, spray water with a sprayer to settle the dust in the air, turn on the ultraviolet lamp for sterilization for 20 minutes, and then turn it off.

[0025] Prepare the necessary equipment for separation: sterile culture dishes, modified PDA (modified potato sucrose medium) plates, modified PDA (modified potato sucrose medium) slopes, alcohol lamp, scalpel, tweezers, cotton wool, 70% alcohol, and a marker.

[0026] The formula of the improved potato sucrose culture medium includes: 200 g of potato, 20 g of glucose, 18 g of agar and 1000 mL of soil extract, and is sterilized by high pressure at 121° C. for 30 min.

[0027] 2. Separation of materials; The hazelnut weevils selected in Table 1.

[0028] 3. Surface disinfection; To kill saprophytes on the surface of the dead insects, surface disinfection is necessary. Soak the dead insects in 70% alcohol for 15-20 seconds and rinse three times with sterile water to remove any residual alcohol.

[0029] 4. Separation and purification; Using the tissue separation method, the surface-sterilized dead insects were cut into small pieces of tissue and placed on an inverted modified PDA plate. Four pieces of tissue were placed on each plate and inverted for culture at a constant temperature of 23°C. After hyphae grew around the tissue blocks, part of the hyphae ends were directly cut and transferred to a new PDA plate for further culture. If the hyphae were impure, another separation and purification operation was performed. After repeated several times, a purified strain was obtained. The purified strain was transferred to the modified PDA slope and stored in a 4°C culture cabinet after the hyphae were fully grown. The results showed that ( Figure 3 ), a total of 15 fungal strains were isolated and purified from 28 dead insects, and the dead insect numbers were H1-2, H3-1, H4-1, K1-2, K1-3, K2-2, K5-1, L1-2, L2-2, L2-3, L3-1, L5-1, L5-2, R3-1, and R5-1.

[0030] Example 3: Screening of infective strains and determination of pathogenicity; 1. Preparation of mature larvae of the hazelnut weevil; Choose an open cement ground and pile the collected borer-hole hazelnuts on the ground in sunny weather. The mature larvae will continue to crawl out of the hazelnuts and be collected in self-sealing bags.

[0031] 2. Preparation of spore suspension; The 15 isolated and purified fungal strains were cultured on modified PDA plates at 23°C for 7 days. After the colonies were full and spores were produced, the spores were washed with 0.5% Tween 80 sterile water. The washed spores were placed on a shaker and cultured at 180 rpm and 23°C for 1 day. The spore suspension was filtered with gauze to obtain the spore suspension. The spores were diluted to a concentration of 10 by microscopic counting. 7 ~10 8 spores / ml and stored in a 4°C refrigerator for later use.

[0032] Screening of infectious strains; Mature larvae of the hazel weevil were placed in petri dishes (90 mm diameter) lined with sterile filter paper, with 15 larvae placed per dish. The prepared spore suspension was sprayed onto the dishes, focusing on the insects. A sterile 0.5% Tween 80 solution was used as a control (CK). After inoculation, all treated dishes were sealed with plastic wrap and punctured for ventilation. The dishes were then placed in a biochemical incubator at 23°C and observed daily for 8 consecutive days. Any dead insects were removed and continued incubation. Any insects with mycelial growth were further isolated and purified. If the isolates obtained matched the original strain, they were considered to be infective to the hazel weevil larvae. The results are shown in Table 2. Table 2. Infectivity assay

[0033] Note: +: The re-isolated strain has the same characteristics as the original strain; -: The re-isolated strain has different characteristics from the original strain.

[0034] The experimental results in Table 2 show that no larvae died in the control group. Of the 15 fungal strains isolated, five caused larval rigor mortis after inoculation, and the properties of the re-isolated strains were consistent with those of the original strains. Five strains also caused larval rigor mortis after inoculation, but the properties of the re-isolated strains differed from those of the original strains. The remaining five strains did not cause larval rigor mortis after inoculation. Therefore, H1-2, H3-1, K1-3, L2-2, and L5-1 are strains with infectious activity.

[0035] Pathogenicity determination; The strains screened for infective activity were inoculated according to the above method (method 3, point 3 of Example 3), with 30 larvae placed per dish (using 150 mm diameter dishes). Each treatment was replicated three times, with sterile 0.5% Tween 80 water used as a control (CK). After inoculation, all treated dishes were sealed with plastic wrap and punctured for ventilation. The dishes were then incubated at 23°C in a biochemical incubator. After 48 hours, the plastic wrap was removed and replaced with two layers of gauze covering the dish openings to reduce humidity. Larvae were then regularly observed daily for 8 consecutive days for mortality, and mortality was calculated. Dead larvae were selected and further cultured to observe for mycelial growth.

[0036] After inoculation with the five strains, the mortality rates of H1-2, H3-1, and L5-1 larvae were 100.00%, 93.33%, and 100.00%, respectively, and the mortality rates of K1-3 and L2-2 larvae were 53.33% and 46.67% ( Table 3 ).

[0037] Table 3. Larval mortality after inoculation with five strains (8 days)

[0038] If the dead larvae continue to be cultured, the mycelium growth is considered to be caused by the growth of the isolated strain and has pathogenicity. The results of the pathogenicity test of the five strains on the larvae of the hazelnut weevil are shown in Table 4 and Figure 4 As shown: Table 4. Pathogenicity of five strains against larvae of the hazelnut weevil (8 days)

[0039] Note: ① The significance of differences between different treatments was calculated using the new multiple range method at the significance level of P=0.05. Treatments with the same letter had no significant differences, while treatments with different letters had significant differences.

[0040] ②The formula for calculating the adjusted mortality rate is: adjusted mortality rate (%) = [(treatment mortality rate - control mortality rate) / 1-control mortality rate)] × 100.

[0041] The results showed (Table 4 and Figure 4), all five strains are pathogenic to hazelnut weevil larvae, but there are differences in their mortality rates. H1-2, H3-1, and L5-1, larvae began to die on the 2nd day; K1-3 and L2-2 larvae began to die on the 3rd day. Judging from the cumulative mortality results, the mortality rate of strain H1-2 to mature larvae of hazelnut weevil reached 65.56%, and its pathogenic effect was significantly better than that of other strains, with significant differences. Next are H3-1 and L5-1, with mortality rates of 58.89% and 50.00% respectively. The mortality rates of the strains are greater than 50%, and they have strong pathogenicity. The mortality rates of K1-3 and L2-2 are 35.56% and 34.44% respectively. Although they are pathogenic, they are relatively weak. The corrected mortality rate of strain H1-2 is 60.26%, which is a high value and significantly higher than other strains. Symptoms of strain H1-2 infecting mature larvae of hazelnut weevil are as follows Figure 5 As shown in the figure, the inoculated larvae showed no significant changes on the first day. On the second day, the insect body darkened and showed signs of rigidity. On the third day, hyphae began to grow on the insect body surface. From the fourth to the eighth day, the hyphae gradually increased and covered the entire insect body surface. Therefore, the strain H1-2 trapped by this method is the preferred strain for controlling the damage caused by the hazelnut weevil. Strain H1-2 is the Beauveria bassiana strain bh002, which is classified as Beauveria bassiana ( Beauveria bassiana ), deposited on May 12, 2025 in the General Microbiology Center of China Culture Collection Administration, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, postal code 100101, and the deposit number is CGMCC No.42004.

[0042] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A Beauveria bassiana bh002 strain, characterized in that The strain is classified and named Beauveria bassiana ( Beauveria bassiana ), was deposited in the General Microbiology Center of China Culture Collection Administration on May 12, 2025, with the deposit number CGMCC No.42004.

2. Use of the Beauveria bassiana bh002 strain as claimed in claim 1 in the preparation of a microbial agent for controlling larvae of the hazelnut weevil.

3. The use according to claim 2, characterized in that The bacterial agent is a spore suspension with a spore concentration of 10 7 ~10 8 spores / ml.

4. A method for screening and isolating the Beauveria bassiana bh002 strain according to claim 1, characterized in that: The following steps are involved: Step 1: Collection of soil samples; Mixed forests, broad-leaved forests, larch forests and plantations were selected as sample plots and rhizosphere soils were collected from the sample plots; Step 2: Collection of borer-hole hazelnuts; Collecting borer-hole hazelnuts in wild hazel forests; Step 3: trapping soil entomogenous fungi; The collected soil samples were placed in open plastic bottles, and the bored hazelnuts were buried in two layers inside the bottles to simulate a natural habitat. Dead branches and leaves were also covered on the soil surface at the bottle mouths. The bottles were then placed in a biochemical incubator at 23°C, shielded from light and moisturized for 14 days. Three replicates were set up for each soil sample. After the incubation period, the soil in the bottles was poured out, and the dead insects with mycelium growing were picked out for later use. Step 4: Isolation and purification of entomogenous fungi; Soak the dead insects in 70% alcohol for 15-20 seconds, rinse them three times with sterile water, and then use the tissue separation method to cut the surface-disinfected dead insects into small pieces of tissue. Place them on inverted modified PDA plates, with four pieces of tissue placed on each plate, and invert them for culture at a constant temperature of 23°C. After hyphae grow around the tissue blocks, directly cut off the ends of some hyphae and transfer them to new PDA plates for further culture. If the hyphae are impure, perform another separation and purification operation until a purified strain is obtained. The purified strain is transferred to a modified PDA slant and stored in a culture cabinet at 4°C after the hyphae are fully grown. Step 5: Preparation of mature larvae of the hazelnut weevil; Pile the collected borer-hole hazelnuts on an open cement floor with plenty of sunlight. After the mature larvae crawl out, collect them into ziplock bags. Step 6: Preparation of spore suspension; The isolated and purified fungal strain was cultured on a modified PDA plate at 23°C for 7 days. After the colonies were full and spores were produced, the spores were washed with 0.5% Tween 80 sterile water. The washed spores were placed on a shaker and cultured at 180 rpm and 23°C for 1 day. The spore suspension was filtered with gauze and the spore concentration was diluted to 10 by microscopic counting. 7 ~10 8 spores / ml, stored in a refrigerator at 4°C for later use; Step 7: Screening of infectious strains; Mature larvae of the hazel weevil were placed in petri dishes lined with sterile filter paper, with 15 larvae placed in each dish. A prepared spore suspension was sprayed onto the dishes, with the insects being sprayed for inoculation. Sterile 0.5% Tween-80 water was used as a control. After inoculation, all treated petri dishes were sealed with plastic wrap and punctured for ventilation. The dishes were then placed in a biochemical incubator at 23°C for incubation. Observations were made daily for 8 consecutive days, and dead insects were picked out and continued to be cultured. Insects with mycelial growth were again isolated and purified for entomogenic fungi. If the strain obtained had the same characteristics as the original strain, it was considered to be an entomogenic fungus with infective activity against the hazel weevil larvae. Step 8: Pathogenicity determination; The selected strains with infective activity were inoculated into mature larvae of the hazelnut weevil, with 30 larvae placed in each dish. Each treatment was replicated three times, and sterilized 0.5% Tween 80 water was used as a control. After inoculation, all treated culture dishes were sealed with plastic wrap and punctured for ventilation, and then placed in a biochemical incubator at 23°C. After 48 hours, the plastic wrap was removed and the dish openings were covered with two layers of gauze to reduce the humidity inside the dish. The larvae were observed for mortality at regular intervals every day for 8 consecutive days. The mortality rate and the number of larvae with hyphae were counted, and the adjusted mortality rate was calculated according to the following formula: Adjusted mortality (%) = [(treatment mortality - control mortality) / 1 - control mortality)] × 100; The strain with the highest corrected mortality rate was screened out, namely Beauveria bassiana bh002.

5. The screening and separation method according to claim 4, characterized in that: The formula of the improved PDA includes: 200g of potato, 20g of glucose, 18g of agar and 1000mL of soil extract, and is sterilized at 121°C and 103.4kPa for 30min.

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