Strong-stress-resistance beauveria bassiana strain CIPPBb075 with high pathogenicity to grassland caterpillars and application of beauveria bassiana strain CIPPBb075

Abstract

The invention relates to the field of insect prevention and control, in particular to a high-stress-resistance beauveria bassiana strain CIPPBb075 with high pathogenicity to grassland caterpillars and application of the high-stress-resistance beauveria bassiana strain CIPPBb075. According to the invention, diseased insects parasitized by beauveria bassiana are collected from a field, and the strain is separated and purified, so that the CIPPBb075 strain has relatively high toxicity to grassland caterpillar; the germination rate is increased along with temperature rise, and the spore germination rate is obviously higher than that of other strains; the ultraviolet resistance at 25 DEG C is high; the extracellular protease activity is high; the activity of extracellular chitinase is high. Therefore, the CIPPBb075 strain has relatively strong stress resistance, and can be used as an effective microbial agent for preventing and treating the larvae of the grassland caterpillar compared with other strains.

Description

TECHNICAL FIELD

[0001] 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 Euprisia cinerascens and application thereof. BACKGROUND

[0002] Euprisia cinerascens belongs to the genus Euprisia and is the most destructive pest in the alpine meadow of Qinghai-Tibet Plateau. At present, chemical pesticides are the main measure to deal with the outbreak of Euprisia cinerascens, and the unreasonable use of chemical pesticides leads to the increase of pesticide resistance of the pest, therefore, exploring the use of microbial resources to control pests is an important means in the management of pest resistance.

[0003] Beauveria bassiana is a common insect pathogenic fungus, which has strong pathogenicity and strong adaptability, and can reproduce and spread with the growth of crops, and gradually becomes a popular fungus for application in the prevention and control of agricultural and forest pests. Temperature and ultraviolet radiation during the growth and development of Beauveria bassiana and the process of host body infection are important environmental factors affecting the sporulation and spore germination of Beauveria bassiana, therefore, it is necessary to obtain Beauveria bassiana with strong stress resistance. SUMMARY

[0004] The purpose of the present application is to provide a strong stress-resistant Beauveria bassiana strain with high pathogenicity to Euprisia cinerascens.

[0005] The purpose of the present application is to provide a strong stress-resistant Beauveria bassiana strain with high pathogenicity to Euprisia cinerascens.

[0006] The Beauveria bassiana strain CIPPBb075 according to the present application has a preservation number of CGMCC No.41828. Beauveria bassiana The present application provides the application of the above-mentioned Beauveria bassiana strain CIPPBb075 in the prevention and control of Euprisia cinerascens.

[0007] Beauveria bassiana The present application provides the application of the above-mentioned Beauveria bassiana strain CIPPBb075 in the prevention and control of Euprisia cinerascens.

[0008] The present application provides the application of the above-mentioned Beauveria bassiana strain CIPPBb075 in the prevention and control of Euprisia cinerascens. 50 ​The spore concentration was 2.51*106 spores / mL; the germination rate increased with the increase of temperature, and the spore germination rate was significantly higher than that of other strains; the ultraviolet resistance of CIPPBb075 strain at 25℃ was significantly higher than that of other strains; enzymes are very important in the process of invasion, and the extracellular protease activity of CIPPBb075 is significantly higher than that of other strains; the extracellular chitinase activity of CIPPBb075 is the highest. The spores of CIPPBb075 have stronger stress resistance and can survive better in severe interfoliar environment. Therefore, the stress resistance of CIPPBb075 strain is stronger than that of other strains, and it can be used as an effective microbial source drug for preventing and treating grassland caterpillar larvae.

[0009] The Beauveria bassiana (Bb) strain CIPPBb075 of the application is preserved in the China General Microbiological Culture Collection Center (CGMCC) on March 12, 2025, and is named as Beauveria bassiana (Bb) and has a preservation number of CGMCC No. 41828. Beauveria bassiana The Beauveria bassiana (Bb) strain CIPPBb075 of the application is preserved in the China General Microbiological Culture Collection Center (CGMCC) on March 12, 2025, and is named as Beauveria bassiana (Bb) and has a preservation number of CGMCC No. 41828. Beauveria bassiana The Beauveria bassiana (Bb) strain CIPPBb075 of the application is preserved in the China General Microbiological Culture Collection Center (CGMCC) on March 12, 2025, and is named as Beauveria bassiana (Bb) and has a preservation number of CGMCC No. 41828. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 The figure shows the daily cumulative mortality rate of the grassland caterpillar larvae treated with fungi. DETAILED DESCRIPTION Example 1 Isolation of Beauveria bassiana CIPPBb075

[0011] The collected diseased lepidopteran and hemipteran larvae sclerotized insects were gently picked up with a inoculation needle from the surface of the sclerotized insects in a sterile ultraclean workbench and inoculated on SDAY medium. The formula of SDAY is: glucose 40 g, peptone 10 g, yeast extract powder 10 g, agar 20 g, and distilled water 1000 mL. The culture dish was placed in a constant temperature incubator with a temperature of (25±1)℃, a relative humidity of 75%, and a light cycle of 14L:10D. When single colonies grew on the plate, the uncontaminated mycelium or spores were picked and transferred to a new PDA medium for purification culture. The process was repeated until colonies with consistent morphological characteristics grew on the plate, and the strain was numbered and identified as Beauveria bassiana. After the purified strain fully produced spores, the strain was inoculated on a slant medium and stored in a 4℃ refrigerator.

[0012] 1.2 Preliminary determination of the activity of entomogenous fungi on grassland caterpillar larvae 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.

[0013] 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%.

[0014] like Figure 1 As shown, the indoor toxicity test results all showed a concentration of 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.

[0015] 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 .

[0016] Table 1. Toxicity of entomopathogenic fungi to grassland caterpillar larvae .

[0017] 1.3 Effects of temperature on the biological characteristics of Beauveria bassiana 6 strains of treatment, each treatment 3 times. In PPDA medium, inoculated with mycelial block diameter of 5 mm, respectively, in different temperature (15, 20, 25, 30, 35 ℃) constant temperature incubator (humidity 90%, dark environment) culture. After 7 days of culture, the colony diameter of each treatment was measured by cross method, and after 14 days, 0.05% Tween-80 sterile water was added to each treatment to wash the spores, which were filtered through 3 layers of gauze and measured under a biological microscope using a hemocytometer.

[0018] The spores of the test Beauveria bassiana had a wide temperature germination range, and could normally germinate at the tested temperature range (15-35℃) with a high germination rate. The germination rate of B. bassiana at 25℃ increased with temperature, and the germination rates of IPPBb075 and CIPPBb102 were significantly higher than those of other strains, with germination rates of 95.81% and 95.35%, respectively. The germination rate of B. bassiana strain CIPPBb264 was significantly affected at 35℃, with a germination rate of 78.24%.

[0019] Table 2 Spore germination rate of B. bassiana strains .

[0020] 1.4 Effect of UV irradiation on the biological characteristics of B. bassiana 6 strains of treatment, each treatment 3 times. Mycelial block was inoculated on PPDA medium and irradiated under UV lamp tube at about 25 cm for 0, 20, 40, 60, 80, 100, and 120 min, and then placed in a constant temperature incubator (temperature 25℃, humidity 90%) for culture. After 7 days of culture, the colony diameter of each treatment was measured by cross method, and after 14 days, 0.05% Tween-80 sterile water was added to each treatment to wash the spores, which were filtered through 3 layers of gauze and measured under a biological microscope using a hemocytometer.

[0021] UV irradiation had a significant inhibitory effect on the germination of B. bassiana spores, and the germination rate decreased significantly with increasing irradiation time. The UV resistance of CIPPBb075 strain 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. The second was CIPPBb102 strain, with spore germination rates of 81%, 76.4%, and 64.45% after irradiation for 20, 40, and 60 min, respectively. The UV resistance of CIPPBb075 strain was still significantly higher than that of other strains after irradiation for 80, 100, and 120 min, with spore germination rates of 56.9%, 55.9%, and 52.8%, respectively.

[0022] Table 3 Spore germination rate of B. bassiana strains under UV irradiation .

[0023] 1.5 Effect of photoperiod on mycelial growth and sporulation of strains The conidia of the tested strains were prepared into 1.0 x 10 8 The conidia of the tested strains were prepared into 1.0 x 10

[0024] The results showed that photoperiod had a significant effect on the growth of strains (p < 0.001), and all strains grew fastest under the condition of full light, followed by light and dark alternation, and slowest in total darkness, confirming the promotion of light environment on mycelial growth. There were significant differences in growth among strains: CIPPBb075 showed the best growth performance under all photoperiods, with a colony diameter significantly larger than that of other strains (p < 0.05), especially reaching 25.83 mm under full light. This stable growth advantage indicated that CIPPBb075 not only had inherent rapid growth characteristics, but also showed good adaptability to different light conditions, providing a physiological basis for its rapid colonization and ecological competitive advantage in the field, and could be used as an excellent fungal strain resource for subsequent development and application.

[0025] Table 4 Colony diameters of different strains under different photoperiods on the 7th day .

[0026] 1.6 Determination of extracellular protease activity of different Beauveria bassiana strains Referring to Bidochka's method, spore suspensions of 6 strains were inoculated onto gelatin-agar plates, and after 3 days of incubation at 26 °C, the plates were overlaid with a 15% HgCl2 solution, and clear transparent rings appeared around the colonies, with the size of enzyme production represented by the diameter of the transparent ring. Three parallel tests were set for each strain. The method was improved according to Liu Zhihui's method. The prepared spore suspension was added to the protease induction medium, and incubated at 28 °C, 150 r / min for 6 days. The enzyme solution was centrifuged at 10,000 r / min, 4 °C for 5 min, and the supernatant was taken as the crude enzyme solution. The protease activity was measured using 2% casein as the substrate, and the crude enzyme solution and 2% casein were mixed and reacted in a water bath, then 3-chloroacetic acid was added to terminate the reaction, and after centrifugation, Na2CO3 was added, followed by mixing with Folin phenol reagent for color development reaction for 20 min. The absorbance was measured at 680 nm, and the average value of the absorbance was calculated from 3 measurements for each enzyme. The protease activity was calculated according to the standard curve, and the amount of enzyme required to convert 1 μmol of tyrosine per minute was defined as one unit of enzyme activity.

[0027] 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.

[0028] Table 5. Extracellular protease activities of Beauveria bassiana from different sources. .

[0029] 1.7 Determination of extracellular chitinase activity in different strains of Beauveria bassiana 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.

[0030] 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.

[0031] Table 6 Extracellular chitinase activity of Beauveria bassiana .

[0032] 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 50The spore concentration was 2.51 × 106 spores / mL. The resistance of the six strains of Beauveria bassiana was then tested. The germination rate of B. bassiana increased with increasing temperature. The germination rates of CIPPBb075 and CIPPBb102 were significantly higher than those of the other strains, with germination rates of 95.81% and 95.35%, respectively. The UV resistance of CIPPBb075 at 25°C was significantly higher than that of the other strains, with spore germination rates of 84.9%, 77%, and 69.4% after 20, 40, and 60 min of irradiation, respectively. Enzymes are very important in the process of infection. The extracellular protease activity of CIPPBb075 was significantly higher than that of the other strains, with an enzyme activity of 10.24 U / mL. The extracellular chitinase activity of CIPPBb075 was the highest, with an enzyme activity of 369 U / mL. Based on the above experiments, CIPPBb075 has stronger resistance than the other strains and can be used as an effective microbial source of pesticide for controlling the larvae of the meadow moth.

[0033] 1.8 Field control of meadow moth A backpack sprayer was used to spray meadow grass at a constant rate. Most meadow moth individuals were in the 2-5 instar stage. The spraying concentration was 667 m 2 . Five-point sampling method was used, five 10 m × 10 m small plots were selected from each large plot, and five 1 m × 1 m plots were selected from each small plot to investigate the insect density. The same method was used to investigate the insect density before and after 3 d, 5 d, 7 d, and 10 d of spraying. The insect reduction rate = (treatment area pre-treatment insect - treatment area post-treatment insect) / treatment area pre-treatment insect × 100%; the corrected control effect = (treatment area insect reduction rate ± control area insect reduction rate) / (1 ± control area insect reduction rate) × 100%.

[0034] After 5 d of spraying, there were differences in the field control effect of the six strains of B. bassiana on meadow moth: the corrected control effect of CIPPBb074 was the highest (58.00%), which was significantly better than that of CIPPBb264 (23.21%) and CIPPBb007 (25.60%); CIPPBb075 and CIPPBb102 were at an intermediate level (42.00%, 46.60%), but there was no significant difference between them. With the passage of time, the control effect of all strains continued to rise within 7-10 d, among which CIPPBb075 and CIPPBb007 reached 85% at 10 d, which was significantly higher than that of CIPPBb104 and CIPPBb264 (52.6%, 44.6%). The results showed that CIPPBb074 had the fastest effect, while CIPPBb075 had both fast and sustained effects, making it the preferred strain for field control.

[0035] Table 7 Field control effect of B. bassiana on meadow moth .

[0036] 1.9 The ability of B. bassiana to colonize grass leaves B. bassiana CIPPBb075, CIPPBb074, CIPPBb264 and water control. Medium: selective PDA plates with chloramphenicol (0.05 g / L), carbendazim (0.01 g / L). Eluent: 1000 mL 0.85% NaCL solution, 0.5 mL Tween-80 was added, magnetic stirring to mix, 121℃ high pressure sterilization for 20 minutes. Take out 3 leaves as a biological repeat with sterile forceps. Accurately weigh the total fresh weight of the 3 leaves using an analytical balance and record it. Carefully place the leaves into a 100 mL sterile Erlenmeyer flask containing 30 mL of sterile eluent. Set the rotation speed to 180 rpm and shake at room temperature for 15 minutes. Prepare 10 -1 ,10 -2 ,10⁻³ dilutions, accurately pipette 100 μL of the diluted bacteria solution at this dilution with a sterile pipette, and drop it into the center of the plate. Use a sterile spreader to evenly spread it on the surface, so that the bacteria solution is evenly distributed. Place all the plates upright for 20-30 minutes, and then invert them in a 25℃ constant temperature incubator in the dark. Choose plates with 30-300 typical B. bassiana colonies as effective plates, and count the final number of colonies after 7 days. CFU / g FW = (average number of colonies x dilution factor x total volume of eluent) / fresh weight of leaves.

[0037] CIPPBb075 showed the best persistence. Although its initial adhesion amount was lower than that of CIPPBb074, it overtook CIPPBb074 in terms of leaf surface spore retention at 7 days and 14 days, and continued to maintain the highest level. This indicates that the spores of CIPPBb075 may have stronger stress resistance and can better survive in harsh leaf environment.

[0038] Table 8 Field control effect of B. bassiana on P. japonica (x 10 4 CFU / g FW) .

[0039] The above examples are only for understanding the technical solutions of the present application and do not limit the protection scope of the present application.

Claims

1. Beauveria bassiana ( Beauveria bassiana strain CIPPBb075, characterized in that, The Beauveria bassiana ( Beauveria bassiana The preservation number of strain CIPPBb075 is CGMCC No.41828.

2. The Beauveria bassiana as described in claim 1 ( Beauveria bassiana Application of strain CIPPBb075.

3. The application according to claim 2, characterized in that, The Beauveria bassiana ( Beauveria bassiana Strain CIPPBb075 is used to control destructive pests in grasslands.

4. The application according to claim 3, characterized in that, The Beauveria bassiana ( Beauveria bassiana Strain CIPPBb075 is used to control grassland caterpillars.

5. A method for controlling destructive pests in grasslands, characterized in that, The method includes applying the Beauveria bassiana of claim 1 (…). Beauveria bassiana Steps for obtaining spores of strain CIPPBb075.

6. The method for controlling destructive pests in grasslands according to claim 5, characterized in that, The destructive pest of the grassland is the grassland caterpillar.

7. A biological insecticide, characterized in that, The biological insecticide includes Beauveria bassiana as described in claim 1. Beauveria bassiana ) Spores of strain CIPPBb075.

8. The biological insect repellent according to claim 7, characterized in that, It also includes auxiliary materials.

9. The biological insect repellent according to claim 7, characterized in that, The biological insecticide is available in solid or liquid form.

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