A strain of Beauveria bassiana highly toxic to multiple silkworm varieties and its application in silkworm production.

Abstract

This invention discloses a highly toxic Beauveria bassiana strain to multiple silkworm varieties and its application in silkworm production. The strain is Beauveria bassiana strain KW1, deposited on July 30, 2024, at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO: 64922. This invention utilizes a spore suspension of strain KW1 (spore content 1×10⁻⁶). 6 The KW1 strain was inoculated with multiple varieties of healthy fifth-instar silkworms (number of silkworms per mL), and the mortality rate was 97.1%–99.5%. Even during large-scale production of these silkworms, the mortality rate remained as high as 96%. The average body length of the resulting silkworms was 4.3±0.495 cm, the average diameter was 6.8±0.703 mm, and the maximum weight of a single silkworm reached 1.14 g, with an average weight of 0.88±0.109 g (equivalent to an average weight of 8.8 kg per 10,000 silkworms). All indicators of the silkworms, such as extractives, total ash, and moisture content, met and significantly exceeded the standards of the Pharmacopoeia of the People's Republic of China (2020 edition), indicating that the KW1 strain has promising application prospects in the large-scale standardized production of high-quality silkworms.

Description

Technical Field

[0001] This invention belongs to the technical field of preparation of the traditional Chinese medicine silkworm, specifically involving a strain of Beauveria bassiana that is highly toxic to multiple silkworm varieties and its application in the production of silkworm. Background Technology

[0002] Silkworm larvae (also known as stunted silkworms) are the dried bodies of 4th-5th instar larvae of the silkworm (Bombyx mori Linnaeus) that have died naturally (or through artificial inoculation) from the fungus Beauveria bassiana (Bals.) Vuillant. They are primarily produced in spring and autumn and are believed to have effects such as relieving wind-heat and spasms, dispelling wind and relieving pain, and resolving phlegm and nodules. Statistics show that 206 out of 10,169 existing traditional Chinese medicine preparations contain silkworm larvae, primarily used to treat symptoms such as infantile convulsions, epilepsy, and wind-heat headaches. The annual domestic demand for silkworm larvae is as high as 3,000 tons, and with its widespread application in medicine, health foods, and cosmetics, this demand is expected to increase year by year. However, the production of silkworm larvae is very limited.

[0003] Early "stiff silkworms" (or "silkworm pupae") were mainly derived from dead silkworms infected with Beauveria bassiana during the silkworm rearing period, resulting in significant instability in both the quality and yield of the medicinal material. In recent years, the production method of preparing stiff silkworms by artificially inoculating fifth-instar silkworms with Beauveria bassiana has attracted increasing attention. For example, CN110352920B provides a method for artificially culturing stiff silkworms based on Beauveria bassiana, producing silkworms with an average body length and diameter of 4.30±0.35cm and 0.5±0.1cm, respectively, and a weight of 7-8kg per 10,000 silkworms. However, currently produced stiff silkworms often fail to meet the requirements of the *Pharmacopoeia of the People's Republic of China*, as well as some local and enterprise standards, in one or more indicators such as appearance size, ash content, extractive content, and number of silk gland rings, significantly impacting the efficiency of large-scale production and clinical application. The characteristics of Beauveria bassiana strains are a key factor affecting the production efficiency and product quality of stiff silkworms. Current research also shows that different strains of Beauveria bassiana exhibit a certain selectivity for silkworm varieties, and the reported strains of Beauveria bassiana infecting relatively limited silkworm varieties.

[0004] Therefore, identifying superior Beauveria bassiana strains that are highly toxic to multiple silkworm varieties and can be used for large-scale production of high-quality silkworm pupa is of great significance to the high-quality development of the silkworm pupa industry. Summary of the Invention

[0005] This invention addresses the problems of low production efficiency and poor quality of silkworm molting, and the lack of superior Beauveria bassiana strains. It provides a Beauveria bassiana strain with high toxicity against multiple silkworm varieties and its application in silkworm molting production. This strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on July 30, 2024, with accession number GDMCC NO: 64922. This invention first utilizes spore suspensions of strain KW1 and four other different Beauveria bassiana strains (each containing 1×10⁻⁶ spores). 6 When fifth-instar silkworms of the Yuecan 6 strain were inoculated with spores (per mL), the rigor mortis rate of strain KW1 was found to be as high as 99.5%, far exceeding that of the other four strains. Then, using a spore suspension of strain KW1 (containing 1×10⁻⁶ spores / mL)... 6 Five-instar silkworms of four different silkworm varieties (Huakang No. 3, Jinxiu No. 2, Jingsong × Haoyue, and Liangguang No. 2) were inoculated with a spore suspension of Beauveria bassiana (KW1) at a concentration of 100 spores per mL. The mortality rate of all five varieties was higher than 97%, with the highest mortality rate (99.5%) and average weight per silkworm (0.90 ± 0.099 g) observed in the Huakang No. 3 silkworms. Finally, a healthy five-instar silkworm from the Huakang No. 3 variety was inoculated with a spore suspension of KW1. The mortality rate remained as high as 96%, and the total weight of the dried silkworms reached 26 kg. The maximum weight of a single silkworm was 1.14 g, and the average weight was 0.88 ± 0.109 g. Furthermore, the resulting silkworms were hard and brittle, easily broken, and the cross-section showed four bright brown or bright black silk gland rings. All indicators (tested by a third-party institution) of the silkworms met the standards of the Pharmacopoeia of the People's Republic of China (2020 edition), indicating excellent quality. This demonstrates that the Beauveria bassiana KW1 strain has good application prospects in the large-scale standardized production of high-quality silkworms.

[0006] The first objective of this invention is to provide a strain of Beauveria bassiana and its inoculum.

[0007] A second objective of this invention is to provide the application of the Beauveria bassiana strain and the bacterial agent in the production of silkworm pupae.

[0008] A third objective of this invention is to provide a method for producing silkworm pupa based on the Beauveria bassiana strain and the bacterial agent.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention, through the isolation and purification of strains of *Beauveria bassiana* from silkworm pupae, identified and named *Beauveria bassiana* strain KW1. This strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on July 30, 2024, with the accession number GDMCC NO: 64922. This invention first utilized spore suspensions of strain KW1 and four other different *Beauveria bassiana* strains (each containing 1×10⁻⁶ spores). 6When fifth-instar silkworms of the Yuecan 6 strain were inoculated with spores (per mL), the rigor mortis rate of strain KW1 was found to be as high as 99.5%, far exceeding that of the other four strains. Then, using a spore suspension of strain KW1 (containing 1×10⁻⁶ spores / mL)... 6 Four different silkworm varieties (Huakang No. 3, Jinxiu No. 2, Jingsong × Haoyue, and Liangguang No. 2) were inoculated with the above-mentioned spore suspension (spores / mL). The mortality rate of all five instar silkworms was higher than 97%, with Huakang No. 3 exhibiting the highest mortality rate and average weight per silkworm. Finally, this invention used the above-mentioned spore suspension to inoculate a healthy fifth-instar Huakang No. 3 silkworm. The mortality rate was still as high as 96%, and the total weight of the dried silkworms reached 26 kg. The maximum weight of a single silkworm was 1.14 g, and the average weight was 0.88 ± 0.109 g. Furthermore, the resulting silkworms were hard and brittle, easily broken, and the cross-section showed four bright brown or bright black silk gland rings. All quality indicators (tested by a third-party institution) met the standards of the Pharmacopoeia of the People's Republic of China (2020 edition). The silkworms were of excellent quality, indicating that the Beauveria bassiana KW1 strain has good application prospects in the large-scale standardized production of high-quality silkworms.

[0011] This invention seeks protection for a strain of Beauveria bassiana.

[0012] Specifically, the strain is Beauveria bassiana strain KW1, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 30, 2024, with the accession number GDMCC NO: 64922.

[0013] Specifically, the strain was isolated and purified from silkworms naturally infected with Beauveria bassiana. The colonies of the strain have a slightly raised center, are white, grow in concentric radial patterns, and have obvious annular grooves. The hyphae are colorless, slender, and smooth. The conidiophores are attached to the vegetative hyphae, and the conidia are spherical or nearly spherical, transparent, and smooth.

[0014] The present invention also claims protection for a fungal agent containing the Beauveria bassiana KW1 strain.

[0015] This invention also seeks protection for the Beauveria bassiana strain KW1 and the application of the bacterial agent in the production of silkworm pupae.

[0016] The present invention also claims protection for a method for producing stiff silkworms, wherein stiff silkworms are produced by inoculating silkworms with the Beauveria bassiana strain KW1 or the bacterial agent.

[0017] The present invention is applicable to a variety of silkworm varieties, such as Yuecan No. 6, Huakang No. 3, Jinxiu No. 2, Jingsong × Haoyue, and Liangguang No. 2.

[0018] As a preferred embodiment, when using the Beauveria bassiana KW1 strain to produce silkworm pupa, the temperature of the production environment (pupa staphylococcal chamber) is maintained at 25-26°C.

[0019] As a preferred implementation, the relative humidity of the production environment (rigidification chamber) is not less than 85%.

[0020] As an optional implementation, the inoculation method is spraying with a spore suspension, wherein the spore content of the spore suspension is not less than 1×10⁻⁶. 6 per mL.

[0021] The present invention has the following beneficial effects:

[0022] This invention provides a highly toxic Beauveria bassiana strain KW1 to multiple silkworm varieties. This strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on July 30, 2024, with accession number GDMCC NO: 64922. When producing silkworm larvae using Beauveria bassiana strain KW1, a spore content of 1×10⁻⁶ is used. 6 Inoculating healthy fifth-instar silkworms of the Yuecan 6 strain with a spore suspension of 1 × 10⁶ spores / mL resulted in a mortality rate of 99.5%, significantly higher than the other four strains (all with mortality rates not exceeding 18.5%). Then, using the same KW1 strain spore suspension (spore content 1 × 10⁶ spores / mL)... 6 Five-instar silkworms of four different silkworm varieties (Huakang No. 3, Jinxiu No. 2, Jingsong × Haoyue, and Liangguang No. 2) were inoculated with a mortality rate of over 97% for all five varieties. The mortality rate of Huakang No. 3 silkworms was the highest (99.5%) and the average weight of a single silkworm (0.90 ± 0.099 g).

[0023] Furthermore, when this invention inoculated a healthy fifth-instar silkworm of variety Huakang No. 3 with the aforementioned spore suspension, the mortality rate was still as high as 96%, and the total weight of all the silkworms reached 26 kg. The average body length of a single silkworm was 4.3 ± 0.495 cm, the average diameter was 6.8 ± 0.703 mm, the maximum weight of a single silkworm was 1.14 g, and the average weight was 0.88 ± 0.109 g. Moreover, the obtained silkworms were hard and brittle, easily broken, and four bright brown or bright black silk gland rings were visible on the cross-section. Other indicators such as the content of extractives, impurities, moisture, total ash, and acid-insoluble ash (tested by a third-party institution) all met the standards of the Pharmacopoeia of the People's Republic of China (2020 edition), indicating excellent quality silkworms. The Beauveria bassiana strain KW1 provided by this invention has good application prospects in the large-scale standardized production of high-quality silkworms. Attached Figure Description

[0024] Figure 1 The images show the colony morphology from both sides and the micrographs of conidia of the isolated strain. The scale bar for the colony images is 1 cm, and the scale bar for the conidia images is 100 μm.

[0025] Figure 2A phylogenetic tree constructed based on the 18S rDNA+ITS sequence of Beauveria bassiana strain KW1.

[0026] Figure 3 Analysis of the mortality rate of fifth-instar silkworms of Yuecan No. 6 infected with Beauveria bassiana strain KW1 and four other different tested Beauveria bassiana strains (all using a spore content of 1×10⁻⁶). 6 (Spore suspension per mL).

[0027] Figure 4 Comparative analysis of the rigor mortis rate of different silkworm varieties inoculated with Beauveria bassiana strain KW1 (spore suspension content 1×10⁻⁶) 6 (units / mL).

[0028] Figure 5 The images show silkworm products obtained by infecting silkworms with Beauveria bassiana strain KW1. In the image, A is the dried silkworm product (the surface is covered with white powdery aerial mycelia and conidia); B is a cross-section of the silkworm product (with a shiny black silk gland ring), and the scale bar is 1 mm. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0030] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0031] In this article, "mutilation rate" refers to the ratio of healthy fifth-instar silkworms that die from Beauveria bassiana infection before spinning their cocoons to the total number of inoculated silkworms. It is an important indicator reflecting the success rate of silkworm rearing. When different strains infect the same type of silkworm, if the same concentration of spore suspension is used for infection, the strain with a higher mutilation rate is more virulent to that silkworm. If different concentrations of spore suspension are required to achieve the same mutilation rate, then the strain with a lower concentration is more virulent to that silkworm. In addition, when the same spore suspension (with the same strain and spore content) is used to infect different varieties of silkworms, the variety with a higher mutilation rate is more susceptible to Beauveria bassiana, which also means that Beauveria bassiana is more virulent to that silkworm variety.

[0032] SAY medium: Weigh 40g glucose, 10g peptone, 10g yeast extract, and 15g agar powder, add distilled water to completely dissolve and bring the volume to 1000mL. After autoclaving for 30 minutes, spread the mixture into plates for later use.

[0033] Example 1: Isolation and Identification of Beauveria bassiana

[0034] 1. Isolation and purification of strains

[0035] In a clean bench, silkworm samples (provided by a silkworm farmer in Guangzhou, the silkworm variety being Yuecan No. 6) obtained from natural infection with Beauveria bassiana during silkworm rearing were soaked in 75% alcohol for 30 seconds, rinsed three times with sterile water, and placed on sterile absorbent paper. After the surface moisture of the silkworms was removed, they were placed on Sawyer's medium plates with the plates facing up and incubated in a constant temperature and humidity incubator at 25°C and 90% relative humidity (RH). Once pure white colonies have grown, promptly pick them with an inoculation needle and purify them on new SDAY medium plates using a multi-round streak plating method. Then, select single colonies for microscopic examination. Select colonies that show no other contaminating bacteria under microscopic examination for identification, including morphological identification and 18S rDNA+ITS sequencing analysis. Select a pure colony identified as Beauveria bassiana as the target strain and record it as Beauveria bassiana KW1 strain.

[0036] The specific methods for morphological identification and 18S rDNA+ITS sequencing analysis are as follows: "2. Morphological Identification of Strains" and "3. Molecular Biological Identification of Strains" are described below:

[0037] 2. Morphological identification of strains

[0038] The isolated and purified strains were inoculated onto SDAY medium plates and cultured at 25°C for 7 days. The shape and color of the colonies were observed and recorded, and the type, shape, and size of the conidia were observed and recorded under a microscope.

[0039] The morphological analysis of colonies and conidia of the isolated and purified strains on SDAY agar plates yielded the following results: Figure 1 As shown. By Figure 1 It can be seen that the colony has a slightly raised center, is white in color, grows in a concentric radial pattern, and has obvious annular grooves; the hyphae are colorless, slender, and smooth, with conidiophores attached to the vegetative hyphae, and the conidia are spherical or nearly spherical, transparent, and smooth. Based on the above characteristics, this strain is preliminarily identified as *Beauveria bassiana*.

[0040] 3. Molecular biological identification of the strain

[0041] The isolated and purified strain was inoculated onto SDAY medium plates and cultured at 25°C for 7 days. Spores and hyphae were scraped off, and genomic DNA was extracted using a fungal genomic DNA extraction kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.). The gene fragment containing the 18S rDNA+ITS sequence of this strain was amplified using primers 18S-F and ITS4-R. The nucleotide sequences of the primers are shown below:

[0042] Primer 18S-F: 5'-TATCTGGTTGATTCTGCCAG-3'

[0043] Primer ITS4-R: 5'-TCCTCCGCTTATTGATATGC-3'

[0044] Reaction system: 25 μL PrimeSTAR Max Premix (2X), 1 μL each of primers 18S-F and ITS4-R (concentration 10 μM), 1 μL of genomic DNA of the strain, and add ultrapure water to 50 μL.

[0045] Reaction conditions: 98℃ pre-denaturation for 5 min; 98℃ for 10 sec, 55℃ for 30 sec, 72℃ for 1 min, 30 cycles; 72℃ extension for 5 min.

[0046] After the PCR products were detected by agarose gel electrophoresis, the target fragment was recovered and purified using a standard agarose gel DNA recovery kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.), and then ligated into pMD. TM The transformants were loaded onto the 19-T vector and transformed into E. coli TOP10 competent cells. Colony PCR was then used for preliminary detection of the E. coli transformants, and positive clones were selected and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing analysis.

[0047] Homology analysis of the 18S rDNA+ITS sequence of this strain using NCBI / BLASTn revealed that the most similar strain was *Beauveria bassiana*, indicating that this bacterium belongs to the *Beauveria bassiana* family. Specifically, the 18S rDNA+ITS sequence of *Beauveria bassiana* HN6 was completely identical to that of this strain. Comparative analysis of the genomes of this strain and *Beauveria bassiana* HN6 showed that their genome sizes were 38.3 Mb and 37.1 Mb, respectively, and they encoded 10,488 and 10,000 genes, respectively, indicating significant genomic differences and suggesting they are two distinct strains.

[0048] A phylogenetic tree was constructed using MEGA 11.0 software, and the results are as follows: Figure 2 As shown, this strain aggregates with other Beauveria bassiana strains.

[0049] In summary, the strain isolated and purified from silkworm pupae was identified as belonging to the genus Beauveria and the species Beauveria bassiana, and named strain KW1. It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 30, 2024, with the accession number GDMCC NO: 64922.

[0050] Example 2: Determination of the mortality rate of silkworms infected with different strains of Beauveria bassiana

[0051] 1. Experimental Methods

[0052] The experiment was divided into 5 experimental groups, with 210 healthy Yuecan 6 silkworms (fourth instar molting silkworms) in each group, and 3 replicates in each group. Healthy fifth instar silkworms of Yuecan 6 were inoculated with *Beauveria bassiana* KW1, *Beauveria bassiana* ARSEF 2860, *Beauveria bassiana* HN6, *Beauveria bassiana* ACCC 30812, and *Beauveria bassiana* ACCC30886 to study the mortality rate of silkworms infected with different *Beauveria bassiana* strains, thereby assessing the virulence of strain KW1. *Beauveria bassiana* ACCC 30812 and *Beauveria bassiana* ACCC 30886 were from the China Agricultural Microbial Culture Collection Center, *Beauveria bassiana* HN6 was donated by Professor Guo Xijie of Jiangsu University of Science and Technology, and *Beauveria bassiana* ARSEF 2860 was donated by Professor Wang Chengshu of the Shanghai Institute of Plant Physiology and Ecology. Specific methods are as follows:

[0053] (1) Selection of fourth instar silkworms: Select healthy fourth instar silkworms that are eating normally from the silkworm bed and transfer them to the stiffening room for rearing. After they wake up from their molting, they are inoculated with Beauveria bassiana.

[0054] (2) Preparation of the inoculation environment: Use constant temperature and humidity equipment to maintain the temperature of the sterilization room at 25-26℃ and the humidity at no less than 85%; use a mechanical ventilation system to supply and exhaust air at regular intervals every day, and connect the exhaust vent to a spray sterilization device to prevent the leakage of Beauveria bassiana and its spores.

[0055] (3) Preparation of spore suspension: Beauveria bassiana, grown on SDAY medium plates for 15–20 days, was used to prepare a spore suspension with a concentration of 1 × 10⁻⁶ spores using sterile water containing 0.1% Tween-80. 6 Spore suspension of 1 spore / mL;

[0056] (4) Spraying inoculation of fifth-instar silkworms: Selected fourth-instar silkworms were transferred to a sterilization room. Two to three days after they molted, the spore suspension was added to an agricultural sprayer and sprayed onto the fifth-instar silkworms. The first spraying was applied to the surface of the silkworms, just enough to moisten the surface. The second spraying was done 15 minutes later. The mulberry leaves were then fed 15 minutes later, and the spore suspension was sprayed onto the surface of the mulberry leaves for the third inoculation. The fourth inoculation was done 15 minutes later.

[0057] (5) Collection of stiff silkworms: Regularly observe the fifth instar silkworms after inoculation. When the silkworms become stiff and diseased, pick them out in time and spread them out in a clean silkworm tray. When the stiffness rate reaches 20%, lay out the fifth instar silkworm net, give a small amount of mulberry leaves and then stop giving mulberry leaves (the small amount of mulberry leaves is a layer of mulberry leaves covering the silkworm net, so as to completely cover the silkworm net). After 48 hours, take away the silkworm net and keep the ambient temperature at 25-26℃ and the relative humidity at no less than 85%.

[0058] (6) Drying and preservation of stiffened silkworms: After stiffening is completed (until the surface of the stiffened silkworms is covered with Beauveria bassiana mycelium and the surface color of the silkworms cannot be distinguished), the wet stiffened silkworms are placed in an oven and heat-treated at 90℃ for 50 minutes to kill Beauveria bassiana and its spores; then continue drying at 60℃ until the moisture content of the stiffened silkworms is less than 13%; finally, the dried stiffened silkworms are collected and sorted. They are packed into airtight packaging bags at a specification of 1kg / bag, the air is removed and they are stored at 20℃.

[0059] 2. Experimental Results

[0060] Analysis of the mortality rate of silkworms infected with different tested Beauveria bassiana strains, such as... Figure 3 As shown. By Figure 3 It can be seen that the mortality rate of silkworm infected by Beauveria bassiana KW1 reached 99.5%, while the mortality rates of the other four Beauveria bassiana strains infected silkworms were all below 20%, specifically: the mortality rates of Beauveria bassiana ARSEF 2860, Beauveria bassiana HN6, Beauveria bassiana ACCC 30812 and Beauveria bassiana ACCC 30886 were 18.5%, 3.9%, 1.95% and 17.07%, respectively.

[0061] Example 3: Analysis of the mortality rate and weight of sterilized silkworms obtained by inoculating different varieties of silkworms with Beauveria bassiana strain KW1.

[0062] 1. Experimental materials

[0063] Beauveria bassiana KW1 strain, and four different silkworm species of healthy fifth instar silkworms (Huakang No. 3, Jinxiu No. 2, Jingsong × Haoyue, and Liangguang No. 2).

[0064] 2. Experimental Methods

[0065] The experiment was divided into four experimental groups (each silkworm species was one group), with 210 healthy silkworms (fourth instar molting silkworms) in each group, and three replicates in each group. The four silkworm species were inoculated with Beauveria bassiana strain KW1, and the mortality rate and average weight of a single molting silkworm were recorded. The specific method for infecting silkworms with Beauveria bassiana strain KW1 is described in Example 2.

[0066] 3. Experimental Results

[0067] A comparative analysis of the mushy mortality rate of different silkworm varieties inoculated with Beauveria bassiana strain KW1 was conducted, and the results are as follows: Figure 4 As shown. By Figure 4 It can be seen that the silkworm rigor mortis process was completed on the 9th day after inoculation with Beauveria bassiana strain KW1. The rigor mortis rate of different silkworm species all exceeded 97%, specifically: Huakang No. 3, 99.5%; Jinxiu No. 2, 97.1%; Jingsong × Haoyue, 97.7%; Liangguang No. 2, 99.1%.

[0068] Furthermore, the average weight of individual silkworms varied slightly among the four types of sterile silkworms: Huakang No. 3 was 0.90±0.099g, Jinxiu No. 2 was 0.79±0.102g, Jingsong×Haoyue was 0.88±0.105g, and Liangguang No. 2 was 0.73±0.102g. Among these, 80% of the sterile silkworms produced using Huakang No. 3 and Jingsong×Haoyue had an individual weight exceeding 0.8g, while only 50% of the sterile silkworms from Liangguang No. 2 and Jinxiu No. 2 reached 0.8g. Therefore, based on the sterility rate and the average weight of individual sterile silkworms, Huakang No. 3 is the most suitable silkworm variety for producing sterile silkworms, and thus it was chosen for large-scale sterile silkworm production.

[0069] Example 4: Method for large-scale production of silkworm pupae using Beauveria bassiana strain KW1

[0070] 1. Experimental materials

[0071] Beauveria bassiana strain KW1 and a healthy silkworm of Zhang Huakang No. 3

[0072] 2. Experimental Methods

[0073] A healthy fifth-instar silkworm of variety Huakang 3 was inoculated with Beauveria bassiana strain KW1. The effects of Beauveria bassiana strain on the mortality rate and quality of the silkworms were analyzed.

[0074] The specific method for producing stiff silkworms by infecting silkworms with Beauveria bassiana strain KW1 is described in Example 2.

[0075] Sampling inspection of silkworm pupae quality: Silkworm pupae samples were selected according to the sampling principles for traditional Chinese medicine stipulated in the Pharmacopoeia of the People's Republic of China (2020 edition). The body length, diameter, and weight of each silkworm pupae in the samples were measured and statistically analyzed. Then, the samples were sent to a third-party institution for testing. The test indicators included impurities, moisture, total ash, acid-insoluble ash, extractives, aflatoxin, heavy metals and harmful elements, 33 prohibited pesticides, and the content of adenine, adenosine, and uracil.

[0076] 3. Experimental Results

[0077] Inoculating a healthy silkworm larvae of the Huakang No. 3 variety with Beauveria bassiana strain KW1 resulted in a 96% mortality rate, with a total weight of 26 kg of sterile silkworms. Samples of the sterile silkworms were randomly selected for testing. Figure 5 As shown in Figure A, the measurement results indicate that the average body length of a single silkworm was 4.3 ± 0.495 cm, the average diameter was 6.8 ± 0.703 mm, and the average weight was 0.88 ± 0.109 g (the maximum weight reached 1.14 g). The obtained silkworms were hard and brittle, easily broken, and the cross-section resembled... Figure 5 As shown in Figure B, four bright brown or bright black filamentous gland rings are visible in the cross-section.

[0078] Some samples were randomly selected and sent to a third-party institution for testing. The test results are shown in Table 1. As can be seen from Table 1, all indicators of *Bombyx mori* meet the standards of the *Pharmacopoeia of the People's Republic of China* (2020 edition).

[0079] Table 1. Comparative Analysis of Detection Indicators of Self-Prepared Silkworm Sterculiae with those in the Pharmacopoeia of the People's Republic of China

[0080]

[0081]

[0082] In addition, according to the requirements of Guangdong Province's "Silkworm Formula Granules", the total content of adenine and adenosine in silkworms should be 0.04% to 0.24%. The total content of adenine and adenosine in the silkworms prepared in this embodiment is 0.10% (of which adenine is 0.01% and adenosine is 0.09%), which meets the requirements.

[0083] Some pharmaceutical companies in the industry require that the uracil content of silkworm pupae be above 0.05%. The uracil content of the silkworm pupae prepared in this example is 0.11%, which meets and is significantly higher than the minimum requirement.

[0084] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A strain of Beauveria bassiana, KW1, characterized in that, The Beauveria bassiana strain KW1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 30, 2024, with accession number GDMCC NO: 64922.

2. A microbial agent, characterized in that, Contains the Beauveria bassiana strain KW1 as described in claim 1.

3. The application of the Beauveria bassiana strain KW1 as described in claim 1 in the production of silkworm pupae.

4. The application of the microbial agent according to claim 2 in the production of silkworm pupae.

5. A method for producing silkworm pupae, characterized in that, Silkworms are produced by inoculating silkworms with the Beauveria bassiana strain KW1 as described in claim 1 or the inoculum as described in claim 2.

6. The method according to claim 5, characterized in that, The silkworm varieties mentioned are selected from any one, two, or several of the following: Yuecan No. 6, Huakang No. 3, Jinxiu No. 2, Jingsong×Haoyue, and Liangguang No.

2.

7. The method according to claim 5, characterized in that, The temperature of the production environment is maintained at 25-26℃.

8. The method according to claim 5, characterized in that, The relative humidity of the production environment shall not be lower than 85%.

9. The method according to claim 5, characterized in that, The inoculation method is spore liquid spraying.

10. The method according to claim 9, characterized in that, The spore content of the spore solution is not less than 1×10⁻⁶. 6 per mL.

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