Strongly stress-tolerant beauveria bassiana strain cippbb075 with high virulence against lema fuscata and its application

Abstract

The present application relates to the field of insect control, in particular to a strong stress-resistant Beauveria bassiana strain CIPPBb075 with high pathogenicity to lema frisoni and its application. The present application is collected from the field by the diseased insects parasitized by the Beauveria bassiana, and the strain is separated and purified, the CIPPBb075 strain has high virulence to lema frisoni; the germination rate increases with the increase of temperature, and the spore germination rate is significantly higher than that of other strains; the ultraviolet resistance is high at 25 DEG C; the extracellular protease activity is high; the extracellular chitinase activity is high. Therefore, the CIPPBb075 strain has strong stress resistance and other strains, and can be used as an effective microbial source drug for controlling lema frisoni larvae.

Description

Highly resistant Beauveria bassiana strain CIPPBb075 with high pathogenicity to grassland caterpillars and its application Technical Field

[0001] This invention relates to the field of insect control, specifically to the highly resistant Beauveria bassiana strain CIPPBb075, which is highly pathogenic to grassland caterpillars, and its application. Background Technology

[0002] The steppe caterpillar, belonging to the genus *Trichoderma*, is one of the most destructive pests in the alpine meadows of the Qinghai-Tibet Plateau. Currently, chemical pesticides are the main measure to deal with outbreaks of steppe caterpillars. However, the irrational use of chemical pesticides has led to increased resistance in this pest. Therefore, exploring the use of microbial resources for pest control is an important means of pest resistance management.

[0003] Beauveria bassiana is a relatively common entomopathogenic fungus, characterized by its high pathogenicity and adaptability. It reproduces and spreads rapidly with crop growth, gradually becoming a popular fungus for controlling agricultural and forestry pests. Temperature and ultraviolet radiation are important environmental factors affecting sporulation and spore germination in Beauveria bassiana during its growth, development, and infection of hosts. Therefore, obtaining Beauveria bassiana with strong resistance is essential. Summary of the Invention

[0004] The purpose of this invention is to provide a highly resistant Beauveria bassiana strain with high pathogenicity against grassland caterpillars.

[0005] Another object of the present invention is to provide the application of the above-mentioned Beauveria bassiana strain.

[0006] The Beauveria bassiana strain CIPPBb075 of the present invention has the accession number CGMCC No.41828.

[0007] This invention provides the application of the above-mentioned Beauveria bassiana strain CIPPBb075 for the control of grassland caterpillars.

[0008] This invention utilizes field-collected insects parasitized by *Beauveria bassiana*, through the isolation and purification of the strain CIPPBb075, which exhibits high virulence against grassland caterpillars. LC 50The spore density was 2.51 × 10⁶ spores / mL; the germination rate increased with increasing temperature, and the spore germination rate was significantly higher than that of other strains; the UV resistance of strain CIPPBb075 at 25℃ was significantly higher than that of other strains; enzymes are very important in the infection process, and the extracellular protease activity of CIPPBb075 was significantly higher than that of other strains; the extracellular chitinase activity of CIPPBb075 was the highest. The spores of CIPPBb075 exhibit stronger stress resistance and can survive better in harsh phyllodes environments. Therefore, strain CIPPBb075 has stronger stress resistance than other strains and can be used as an effective microbial agent for controlling grassland caterpillar larvae.

[0009] The Beauveria bassiana strain CIPPBb075 of this invention was deposited on March 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and was classified as Beauveria bassiana, with accession number CGMCC No. 41828. Attached Figure Description

[0010] Figure 1 shows the daily cumulative mortality rate of grassland caterpillar larvae under fungal treatment. Detailed Implementation

[0011] Example 1: Isolation of Beauveria bassiana CIPPBb075

[0012] Collected diseased Lepidoptera and Hemiptera larvae were collected and, using an inoculation needle, fungal spores were gently picked from the surface of the larvae in a sterilized laminar flow hood and inoculated onto SDAY agar. The SDAY formulation consisted of 40 g glucose, 10 g peptone, 10 g yeast extract powder, 20 g agar, and 1000 mL distilled water. The culture dishes were placed in a constant temperature incubator at (25±1)℃, 75% relative humidity, and a photoperiod of 14L:10D. When single colonies appeared on the plates, uncontaminated hyphae or spores were transferred to fresh PDA medium for purification. This process was repeated until colonies with consistent morphological characteristics appeared on the plates. The strains were then numbered and identified as *Beauveria bassiana*. After the purified strains had fully sporulated, they were inoculated onto slant agar and stored at 4℃.

[0013] 1.2 Preliminary determination of the activity of entomopathogenic fungi against grassland caterpillar larvae

[0014] Beauveria bassiana CIPPBb104, CIPPBb074, CIPPBb075, CIPPBb102, CIPPBb007, and CIPPBb264 were inoculated onto PDAY medium, sealed with sealing film, and placed in an incubator at 26°C. The conidia were gently scraped onto a medium containing 0.05% Tween 80 using an inoculation loop, stirred thoroughly with a magnetic stirrer, and a small amount of the spore suspension was pipetted onto a hemocytometer. After covering with a coverslip and allowing to stand for 1 minute, the spores were observed and counted under a microscope. The final concentration was 1×10⁻⁶. 6 3×10 6 9×10 6 2.7×10 7 8.1×10 7 Spore suspension at spores / mL.

[0015] Fresh leaves were harvested in the netted room before the experiment, washed with clean water, and dried. The prepared leaves were immersed in the suspension for 10 seconds, removed, and dried again for later use. Thirty larvae were fed with leaves of each concentration and placed in plastic boxes. They were then moved indoors for rearing at room temperature. Each treatment was repeated three times. The number of dead and live larvae was counted 12 days after treatment, and the corrected mortality rate was calculated. Corrected mortality rate (%) = [(treatment mortality rate - control mortality rate) / (1 - control mortality rate)] × 100%.

[0016] As shown in Figure 1, the indoor toxicity test results showed that the concentration was 8.1 × 10⁻⁶. 7 The spore counts / mL of Beauveria bassiana CIPPBb074, CIPPBb075, CIPPBb102, and CIPPBb007 showed the highest cumulative corrected mortality rate for third-instar larvae of the grassland caterpillar, with a cumulative corrected mortality rate of 100% over 14 days. The following were Beauveria bassiana CIPPBb264 and CIPPBb104, which showed cumulative corrected mortality rates of 90% and 80% for third-instar larvae of the grassland caterpillar, respectively.

[0017] Strain CIPPBb075 showed the best indoor toxicity against 3rd instar larvae of the grassland caterpillar, with an LC50 value after 14 days. 50 They are 2.51×10 6 Spores / mL, followed by strain CIPPBb102, LC after 14 days 50 3.38×10 6 .

[0018] Table 1. Toxicity of entomopathogenic fungi to grassland caterpillar larvae

[0019] .

[0020] 1.3 Effects of temperature on the biological characteristics of Beauveria bassiana

[0021] Six bacterial strains were used for treatment, with each treatment replicated three times. Mycelial blocks with a diameter of 5 mm were inoculated onto PPDA medium and incubated in constant temperature incubators (90% humidity, dark environment) at different temperatures (15, 20, 25, 30, 35℃). After 7 days of incubation, the colony diameter of each treatment was measured using the cross-hatching method. After 14 days, spores were washed off with 0.05% Tween-80 sterile water, filtered through three layers of gauze, and the spore count was determined under a biological microscope using a hemocytometer.

[0022] The tested Beauveria bassiana spores exhibit a wide temperature germination range, germinating normally within the tested temperature range (15–35℃) with a high germination rate. At 25℃, the germination rate of Beauveria bassiana increases with increasing temperature. The spore germination rates of IPPBb075 and CIPPBb102 are significantly higher than other strains, with germination rates of 95.81% and 95.35%, respectively. At 35℃, the germination rate of Beauveria bassiana strain CIPPBb264 is significantly affected, with a germination rate of 78.24%.

[0023] Table 2. Spore germination rate of Beauveria bassiana strains

[0024] .

[0025] 1.4 Effects of ultraviolet radiation on the biological characteristics of Beauveria bassiana

[0026] Six strains were used for treatment, with each treatment replicated three times. Mycelial blocks were inoculated onto PPDA medium and irradiated under a UV lamp at approximately 25 cm intervals for 0, 20, 40, 60, 80, 100, and 120 min, then incubated in a constant temperature incubator (25℃, 90% humidity). After 7 days of incubation, the colony diameter for each treatment was measured using the cross-crossing method. After 14 days, spores were washed off with 0.05% Tween-80 sterile water, filtered through three layers of gauze, and the spore count was determined under a biological microscope using a hemocytometer.

[0027] Ultraviolet (UV) irradiation significantly inhibited the germination of Beauveria bassiana spores. With increasing irradiation time, the spore germination rate decreased significantly. The CIPPBb075 strain exhibited significantly higher UV resistance than other strains, with spore germination rates of 84.9%, 77%, and 69.4% after 20, 40, and 60 minutes of irradiation, respectively. The CIPPBb102 strain was the second most effective, with spore germination rates of 81%, 76.4%, and 64.45% after 20, 40, and 60 minutes of irradiation, respectively. Even after 80, 100, and 120 minutes of irradiation, the CIPPBb075 strain still showed significantly higher UV resistance than other strains, with spore germination rates of 56.9%, 55.9%, and 52.8%, respectively.

[0028] Table 3. Spore germination rate of Beauveria bassiana strains under ultraviolet irradiation.

[0029] .

[0030] 1.5 Effects of photoperiod on strain growth and sporulation

[0031] The conidia of the tested bacterial strain were prepared using the bacterial suspension drop method with 0.05% Tween-80 to form a solution of 1.0 × 10⁻⁶. 8 A spore suspension of 1 spore / mL was pipetted into the center of a PDA agar plate at a density of 2 μL. The plates were then incubated at the optimal temperature with photoperiods of L∥D=12h∥12h, L∥D=24h∥0h, and L∥D=0h∥24h, with each treatment repeated three times. Colony diameter was measured on day 7.

[0032] The results showed that photoperiod had a significant impact on the growth of the strains (p<0.001). All strains showed the fastest growth under full light, followed by alternating light and dark, and the slowest growth in complete darkness, confirming the promoting effect of light environment on mycelial growth. Significant growth differences existed among the strains: CIPPBb075 exhibited the best growth performance under all photoperiods, with a colony diameter significantly larger than other strains (p<0.05), especially reaching 25.83 mm under full light. This stable growth advantage indicates that CIPPBb075 not only possesses inherent rapid growth characteristics but also demonstrates good adaptability to different light conditions, providing a physiological basis for its rapid colonization and ecological competitive advantage in the field, and making it a valuable strain resource for future development and application.

[0033] Table 4. Colony diameter of each strain on day 7 under different photoperiods.

[0034] .

[0035] 1.6 Determination of extracellular protease activity in different Beauveria bassiana strains -

[0036] Following Bidochka's method, spore suspensions of six bacterial strains were inoculated onto gelatin-agar plates and incubated at 26 °C for 3 days. The plates were then covered with 15% HgCl2 solution, and colonies showed a clear, transparent ring around the periphery. Enzyme production was expressed as the diameter of this transparent ring. Three replicates were performed for each strain. A modified method based on Liu Zhihui's method was also used. The prepared spore suspension was added to protease induction medium and incubated at 28 °C and 150 rpm for 6 days. The incubated enzyme solution was then centrifuged at 10,000 rpm and 4 °C for 5 minutes, and the supernatant was used as the crude enzyme solution. Protease activity was measured using 2% casein as a substrate. The crude enzyme solution and 2% casein were mixed and reacted in a water bath. After the reaction was stopped, trichloroacetic acid was added to terminate the reaction. After centrifugation, the supernatant was collected, and Na2CO3 and Folin-Ciocalteu reagent were added. After mixing, the color reaction was carried out for 20 min, and the absorbance was measured at 680 nm. For each enzyme, the absorbance was measured three times and the average value was taken. The protease activity was calculated according to the standard curve. The amount of enzyme required to convert 1 μmol of tyrosine per minute was defined as one unit of enzyme activity.

[0037] Enzymes play a crucial role in the infection process. The extracellular protease activities of six Beauveria bassiana strains were detected. The extracellular protease activities of CIPPBb075 and CIPPBb102 were significantly higher than those of other strains, with enzyme activities of 10.24 U / mL and 9.24 U / mL, respectively. CIPPBb074 and CIPPBb104 had the second highest extracellular protease activities, at 8.94 U / mL and 7.63 U / mL, respectively.

[0038] Table 5. Extracellular protease activities of Beauveria bassiana from different sources.

[0039] .

[0040] 1.7 Determination of extracellular chitinase activity in different strains of Beauveria bassiana

[0041] Configuration 1×10 7 A spore suspension at a concentration of spores / mL was added to chitinase induction medium and cultured at 26°C with shaking at 100 rpm for 7 days. The mixture was then centrifuged at 10000 rpm at 4°C for 5 minutes, and the supernatant was used as the crude enzyme solution. Chitinase activity was measured using 1% colloidal chitin as a substrate. The crude enzyme solution and 1% colloidal chitin were mixed and reacted in a water bath. DNS reagent was added, and the mixture was boiled for 5 minutes. After cooling to room temperature, distilled water was added, and the absorbance was measured at 540 nm. For each enzyme, samples were taken three times, and the average absorbance was used. Chitinase activity was calculated based on the standard curve. One unit of enzyme activity was defined as the amount of enzyme required to convert 1 μmol of N-acetylglucosamine per minute.

[0042] The extracellular chitinase activity of six Beauveria bassiana strains was tested. CIPPBb075 showed the highest extracellular chitinase activity at 369 U / mL, followed by CIPPBb102, whose extracellular chitinase activity was significantly higher than that of the other strains at 321 U / mL. The enzyme activities of CIPPBb007 and CIPPBb264 were significantly lower than those of the other strains.

[0043] Table 6 Extracellular chitinase activity of Beauveria bassiana

[0044] .

[0045] This invention utilizes field-collected insects parasitized by *Beauveria bassiana* to isolate and purify strains. Among six *Beauveria bassiana* strains, strain CIPPBb075 exhibits high virulence against grassland caterpillars. LC 50 The spore count was 2.51 × 10⁶ spores / mL. Subsequently, stress resistance tests were conducted on six strains of *Beauveria bassiana*. The germination rate of *Beauveria bassiana* increased with increasing temperature. The spore germination rates of CIPPBb075 and CIPPBb102 were significantly higher than those of other strains, with germination rates of 95.81% and 95.35%, respectively. The UV resistance of strain CIPPBb075 at 25℃ was significantly higher than that of other strains, with spore germination rates of 84.9%, 77%, and 69.4% after irradiation for 20, 40, and 60 min, respectively. Enzymes are crucial in the infection process. The extracellular protease activity of CIPPBb075 was significantly higher than that of other strains, with an activity of 10.24 U / mL. The extracellular chitinase activity was highest in CIPPBb075, at 369 U / mL. Based on the above experiments, strain CIPPBb075 exhibits strong resistance compared to other strains and can be used as an effective microbial agent for controlling grassland caterpillar larvae.

[0046] 1.8 Field control of grassland caterpillars

[0047] A backpack sprayer was used to spray the grassland forage at a constant rate, targeting the 2nd to 5th instar caterpillars. The spray concentration was [missing information] for a coverage area of ​​667 m². 2 A five-point sampling method was used, selecting five 10m × 10m plots from each large plot, and then selecting five 1m × 1m plots from each small plot to investigate insect population density. The same method was used to investigate insect population density before application and at 3, 5, 7, and 10 days after application. Insect population reduction rate = (Insect population before application in the treated area - Insect population after application in the treated area) / Insect population before application in the treated area × 100%; Corrected control efficacy = (Insect population reduction rate in the treated area ± Insect population reduction rate in the control area) / (1 ± Insect population reduction rate in the control area) × 100%.

[0048] Five days after application, the field control efficacy of the six Beauveria bassiana strains against grassland caterpillars varied: CIPPBb074 showed the highest corrected efficacy (58.00%), significantly better than CIPPBb264 (23.21%) and CIPPBb007 (25.60%); CIPPBb075 and CIPPBb102 were at intermediate levels (42.00% and 46.60%, respectively), but not significantly different from the former. Over time, the efficacy of all strains continuously increased within 7–10 days, with CIPPBb075 and CIPPBb007 reaching 85% simultaneously at 10 days, significantly higher than CIPPBb104 and CIPPBb264 (52.6% and 44.6%, respectively). The results indicate that CIPPBb074 has the fastest onset of action, while CIPPBb075 combines rapid and sustained efficacy, making it the preferred strain for field control.

[0049] Table 7. Field control efficacy of Beauveria bassiana cells against grassland caterpillars.

[0050] .

[0051] 1.9 Colonization ability of Beauveria bassiana on forage leaves

[0052] Beauveria bassiana CIPPBb075, CIPPBb074, CIPPBb264, and a water control were used. Culture medium: Selective PDA plates were prepared with chloramphenicol (0.05 g / L) and carbendazim (0.01 g / L). Eluent: 0.5 mL of Tween-80 was added to 1000 mL of 0.85% NaCl solution, and the mixture was magnetically stirred and autoclaved at 121°C for 20 minutes. Three leaves were removed as a biological replicate using sterile forceps. The total fresh weight of the three leaves was accurately weighed using an analytical balance and recorded. The leaves were carefully placed into a 100 mL sterile Erlenmeyer flask containing 30 mL of sterile eluent. The flask was shaken at 180 rpm for 15 minutes at room temperature. The eluent was diluted to prepare 10... -1 10 -2 10⁻³ dilution: Using a sterile pipette, accurately pipette 100 μL of this dilution and add it to the center of the plate. Spread the solution evenly on the surface using a sterile spreader to ensure uniform distribution. Incubate all plates upright for 20-30 minutes until the bacterial solution is absorbed by the culture medium. Then, invert the plates and incubate them in the dark at 25°C. Select plates that show 30-300 typical Beauveria bassiana colonies as valid plates. Perform a final count after 7 days. CFU / gFW = (average colony count × dilution factor × total volume of eluent) / leaf fresh weight.

[0053] CIPPBb075 exhibited the best persistence. Although its initial attachment amount was lower than that of CIPPBb074, its foliar spore retention surpassed that of CIPPBb074 on days 7 and 14, and remained at the highest level. This suggests that the spores of CIPPBb075 may have stronger stress resistance and be able to survive better in harsh phyllodes environments.

[0054] Table 8. Field control efficacy of Beauveria bassiana cells against grassland caterpillars (×10) 4 CFU / g FW)

[0055] .

[0056] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. Beauveria bassiana strain CIPPBb075, characterized in that, The Beauveria bassiana strain CIPPBb075 has the accession number CGMCC No.41828.

2. The application of the Beauveria bassiana strain CIPPBb075 according to claim 1, characterized in that, The Beauveria bassiana strain CIPPBb075 is used to control grassland caterpillars.

3. A method for controlling destructive pests in grasslands, characterized in that, The method includes the step of applying spores of the Beauveria bassiana strain CIPPBb075 as described in claim 1, wherein the grassland destructive pest is a grassland caterpillar.

4. A biological insecticide, characterized in that, The biological insecticide includes spores of the Beauveria bassiana strain CIPPBb075 as described in claim 1.

5. The biological insect repellent according to claim 4, characterized in that, It also includes auxiliary materials.

6. The biological insect repellent according to claim 4, characterized in that, The biological insecticide is available in solid or liquid form.

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