Beauveria bassiana strain CN055 and application of Beauveria bassiana strain CN055 in prevention and treatment of bean aphids

Abstract

The invention relates to the field of plant protection, in particular to a beauveria bassiana strain CN055 and application of the beauveria bassiana strain CN055 in prevention and treatment of bean aphids. Studies prove that the beauveria bassiana strain CN055 provided by the invention has bean aphid pathogenicity and excellent pathogenicity, has relatively strong activity of growing to produce spores and producing chitinase protease, has the potential of being developed into a biological control preparation, and can be applied to control of aphids.

Description

Technical Field

[0001] This invention relates to the field of plant protection, specifically to Beauveria bassiana strain CN055 and its application in the control of bean aphids. Background Technology

[0002] There are more than 20 species of fungi in the genus Beauveria bassiana, among which Beauveria bassiana is the most common. Beauveria bassiana with Beauveria bassiana B. brongniartii The pathogenicity of insects is the most studied. Beauveria bassiana mainly infects the host by conidia. The growth status of the strain and the yield of conidia are important traits directly related to the production of insecticidal fungi. Different strains have differences in colony growth rate, initial sporulation time, sporulation amount, spore germination rate and pathogenicity. When developing biological control fungi products, it is necessary to screen strains based on their growth characteristics. Among them, the sporulation amount and pathogenicity are important screening indicators for product development.

[0003] Bean aphids ( Aphis craccivora) Belongs to the order Hemiptera, family Aphididae Aphididae Entomopathogenic fungi (EMFs) are one of the global agricultural pests, with a wide host range. They are piercing-sucking insects that cause serious damage to leguminous plant production. Entomopathogenic fungi have a wide geographical distribution and host range, capable of parasitizing Hemiptera, Lepidoptera, Coleoptera, and other orders. They are relatively safe for humans, livestock, crops, natural enemies of pests, and the natural environment, making them a promising biological control resource for pest management. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Beauveria bassiana.

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

[0006] Another object of the present invention is to provide a fungal agent comprising the above-mentioned Beauveria bassiana strain.

[0007] The present invention is Beauveria bassiana ( Beauveria bassiana The strain CN055, with accession number CCTCC NO:M 20251522, is deposited at the China Center for Type Culture Collection on July 2, 2025.

[0008] Studies have confirmed that the present invention is based on Beauveria bassiana ( ). Beauveria bassiana Strain CN055 exhibits pathogenicity and excellent pathogenicity to bean aphids, and also possesses strong growth, sporulation, and chitinase / protease activity, making it a potential biocontrol agent that can be applied to the control of aphids. Attached Figure Description

[0009] Figure 1 Displaying a phylogenetic tree of 15 strains constructed based on ITS gene sequences; Figure 2 The assay shows the ability of Beauveria bassiana to produce extracellular chitinase and protease. a: extracellular chitinase, b: protease. Detailed Implementation

[0010] Test strains: 15 strains of Beauveria bassiana were tested from the longhorn beetle. Thyestilla gebleri It was isolated and purified from adult mummified worms; The test culture medium included a fungal purification medium: maltose 6.25 g / L, malt extract 6.25 g / L, yeast extract 1 g / L, peptone 6.25 g / L, KH2PO4 1.25 g / L, MgSO4·7H2O 0.6 g / L, pH 7.0.

[0011] The production of extracellular proteases and chitinases was determined using casein-agar (CaA) and chitosan (CA) medium.

[0012] Bioassays of Beauveria bassiana isolates against the Oriental bean aphid: The spores of the strain were prepared into a spore suspension using sterile water containing 0.1% Tween-80, and the suspension was adjusted to 1 × 10⁻⁶ after counting with a hemocytometer. 7 Spore concentration / mL. Aphids with uniform growth were selected and placed in 9 cm diameter 1% water agar culture dishes lined with broad bean leaves. The pre-prepared spore suspension was sprayed onto the leaves using a handheld sprayer and allowed to air dry at room temperature. A control was prepared by spraying with 0.1% Tween-80 sterile water. 25 aphids were treated per dish, and each treatment was repeated 3 times. The number of dead aphids was counted every 24 hours. The aphids were considered dead if they did not move when gently touched with a fine brush. The observation period was 5 days.

[0013] Data processing: All results are expressed as mean ± standard deviation (SD). Multiple comparisons were performed using one-way ANOVA and Tukey's honestly significant difference (HSD) test (SPSS 26.0). P <0.05 (significant difference). Example 1: Isolation and purification of strains

[0014] 15 strains of Beauveria bassiana from the longhorn beetle Thyestilla gebleri It was isolated and purified from adult worms.

[0015] Isolation and purification of strains: Selective isolation of *Beauveria bassiana* was performed by adding 70 mg / L dapoxetine, 125 mg / L cephalosporin, and 138 mg / L kanamycin sulfate to fungal purification medium cooled to 50–55°C. Under aseptic conditions, hyphae were picked from the surface of *Beauveria bassiana* using a sterile toothpick and streaked continuously, with three replicates. After incubation at 28°C for 3–5 days, single colonies were picked and transferred to purification medium. After purification three times, PPDA slants were stored at 4°C for later use.

[0016] The purified strains were inoculated onto the surface of PPDA solid medium lined with sterile cellophane and cultured at 28°C for 5-7 days. Mycelia were collected and genomic DNA was extracted from each strain.

[0017] Using fungal genomic DNA as a template, the rDNA-ITS region of the strain was amplified by PCR using the universal primer ITS1. The PCR product was sent for sequencing, and the amplified ITS sequence was preliminarily determined by BLAST similarity comparison on the NCBI website.

[0018] The sequences obtained in the experiment were compared and analyzed with the ITS sequences of Beauveria bassiana downloaded from GenBank. The sequences were saved in FAS format using software, and a phylogenetic tree was constructed using software. A single-gene phylogenetic tree was built using the neighbor-joining method, with a bootstrap value set to 1000. Species diversity analysis was performed based on the phylogenetic results.

[0019] The amplified rDNA-ITS sequences of the 15 strains were approximately 570 bp in length. Sequence alignment with nucleic acid sequences in GenBank revealed that 11 strains were similar to *Beauveria bassiana*. Beauveria bassiana The homology was higher than 99%, and four Beauveria bassiana strains (CN002, CN003, CN009 and CN043) were similar to Beauveria sulfate. B. sulfurescens The homology was higher than 99%. ITS sequences with close phylogenetic relationships were downloaded from GenBank, with Paecilomyces lilacinus as the primary sequence. Purpureocillium lilacinum NR165946.1) was an outgroup. A phylogenetic tree was constructed using MEGA X software using the neighbor-joining method (Figure 1). The results showed that all Beauveria bassiana strains clustered with Paecilomyces lilacinus in different branches, with 11 strains clustering with Beauveria bassiana coccidioides. B. bassiana Clustered into one category; CN002, CN003, CN009, and CN043 are associated with Beauveria bassiana ( ). B. sulfurescensPP384656.1) clustered into one group and formed a distinct branch with other strains of the Beauveria genus. Therefore, 11 strains in the isolates were preliminarily identified as Beauveria bassiana, and CN002, CN003, CN009 and CN043 were preliminarily identified as Beauveria sulfate. Example 2: Growth characteristics and sporulation capacity of Beauveria bassiana isolates

[0020] The purified Beauveria bassiana conidia were prepared with 0.1% Tween-80 to form a solution of 1.0 × 10⁻⁶ spores. 7 Spores / mL suspension. Sterile filter paper with a diameter of 5 mm was placed in the center of PPDA, SDAY, and MEA plates. 5 µL of spore suspension was inoculated onto the filter paper using a pipette. After drying, the plates were incubated upside down in incubators at 20℃, 25℃, and 28℃, respectively, with each incubation repeated three times. On day 7 post-inoculation, the colony diameter was measured using a digital caliper. The average value of the measurements was calculated by crosswise measurements, and the colony growth rate of each Beauveria bassiana strain was compared by calculating the growth diameter. Simultaneously, four mycelial discs were punched at half the distance from the center of each colony using a 5 mm punch. These discs were placed in 5 mL of 0.1% Tween-80 sterile water and shaken to ensure complete spore dispersion. The spore concentration was determined using a hemocytometer and converted to a unit area (mm²). 2 The sporulation yield of each strain was measured three times.

[0021] As shown in Tables 1-3, the average colony diameter was optimal on PPDA medium. Under the same medium and different temperatures, the average colony diameter of the strains showed a greater advantage at 25°C, followed by 20°C and 28°C. Therefore, PPDA medium and 25°C incubation are suitable for the growth of most Beauveria bassiana isolates.

[0022] Comparing the growth of different strains on different temperatures and culture media, the results showed that strains CN003, CN005, CN015, CN019, and CN033 exhibited superior growth compared to other strains on different culture media. At 20, 25, and 28℃, these five strains showed higher growth on PPDA medium than other strains, and also higher growth on SDAY and MEA media. Among them, strain CN003 showed the highest growth on PPDA medium at 25℃ (40.22±0.67 mm), significantly higher than the growth of this strain on PPDA at 20 and 28℃. P <0.05), but there was no significant difference in growth diameter compared with strains CN005, CN015, CN019 and CN033 (colony diameter >39 mm) on PPDA medium at 25℃.

[0023] Table 1. Growth diameter of Beauveria bassiana strains at different temperatures and PPDA (7 days)1) .

[0024] Table 2. Growth diameter of Beauveria bassiana strains at different temperatures and saturates (7 days) 1) .

[0025] Table 3. Growth diameter of Beauveria bassiana strains at different temperatures and MEA (7 days) 1) .

[0026] Results are expressed as mean ± standard deviation (SD). One-way ANOVA was performed using Tukey's honest significant difference (HSD) test. Lowercase letters in the table indicate differences at the 0.05 level among different strains in the same culture medium at the same temperature; uppercase letters indicate differences at the 0.05 level among the same strains in the same culture medium at different temperatures.

[0027] As shown in Tables 4-6, the sporulation results of the 15 Beauveria bassiana strains tested on PPDA, SDAY, and MEA media at 20, 25, and 28℃ showed that, at the same temperature, the average sporulation of the 15 Beauveria bassiana strains on different media generally showed PPDA > SDAY > MEA. On PPDA and MEA media, the sporulation of the strains at different temperatures showed 20℃ > 28℃ > 25℃; specifically, on PPDA and MEA plates at 20℃, the average sporulation of the 15 Beauveria bassiana strains was 0.88 × 10⁻⁶. 6 and 0.15×10 6 pcs / mm 2 However, on SDAY medium, the sporulation yield of the strains at different temperatures showed a pattern of 25℃ > 20℃ > 28℃, with the average sporulation yield of 15 Beauveria bassiana strains at 25℃ being 0.24 × 10⁻⁶. 6 spores / mm 2 .

[0028] The sporulation rates of strains CN004, CN011, CN012, and CN055 on PPDA medium at different temperatures were significantly higher than those of other strains. Among them, strain CN055 exhibited the highest sporulation rate on PPDA plates at 28℃, reaching (1.99±0.57)×10⁻⁶. 6 pcs / mm 2 The sporulation rate was significantly higher than that of other strains at the same temperature (28℃). P<0.05). However, strains CN004, CN011, and CN012 exhibited better sporulation at 20℃ than at 25℃ and 28℃. Therefore, overall, PPDA medium is suitable for sporulation of most Beauveria bassiana species under 20℃ culture conditions.

[0029] Table 4. Sporulation rate of Beauveria bassiana strains at different temperatures on PPDA medium (7 days) 1) .

[0030] Table 5. Sporulation yield of Beauveria bassiana strains at different temperatures on SDAY medium (7 days) 1) .

[0031] Table 6. Sporulation yield of Beauveria bassiana strains at different temperatures on MEA medium (7 days) 1) . Example 3: Virulence determination of Beauveria bassiana isolates

[0032] The spores of the strain were prepared into a spore suspension using sterile water containing 0.1% Tween-80, and the suspension was adjusted to 1 × 10⁻⁶ after counting with a hemocytometer. 7 Spore concentration / mL. Aphids with uniform growth were selected and placed in 9 cm diameter 1% water agar culture dishes lined with broad bean leaves. The pre-prepared spore suspension was sprayed onto the leaves using a handheld sprayer and allowed to air dry at room temperature. A control was prepared by spraying with 0.1% Tween-80 sterile water. 25 aphids were treated per dish, and each treatment was repeated 3 times. The number of dead aphids was counted every 24 hours. The aphids were considered dead if they did not move when gently touched with a fine brush. The observation period was 5 days.

[0033] All 15 Beauveria bassiana strains inoculated with the fungus caused aphid disease. CN019 and CN033 showed weaker pathogenicity, with cumulative corrected mortality rates of 41.3% and 55.6% respectively 5 days after inoculation. CN055, CN043, CN040, and CN002 all had a lethal time of less than 3.3 days, indicating stronger pathogenicity. The lethal time (LT) of the strains on aphids was also considered. 50 The results showed that LT of 5 strains 50 CN033 differs from other strains in that its median lethal time (LT) is [not specified]. 50 The longest lethal time was 4.635 days, LT for CN004. 50 The shortest duration was 2.911 days (Table 7). These results indicate that strains CN055, CN043, CN040, and CN002 exhibit strong toxicity against aphids.

[0034] Table 7. Mid-term lethality (LT) of different Beauveria bassiana species against aphids 50 ) strain number virulence regression equation Correlation coefficient Deadly time CN002 y = 19.048x - 8.8889 R² = 0.937 3.1077 ±0.13 de CN003 y = 18.952x - 15.222 R² = 0.9431 3.4492 ±0.23 cde CN004 y = 20.857x - 10.778 R² = 0.8964 2.911 ±0.17 e CN005 y = 19.476x - 16.778 R² = 0.9288 3.6012 ±0.24 bcd CN006 y = 18.762x - 18.444 R² = 0.9578 3.6711 ±0.34 bcd CN009 y = 21.429x - 21.667 R² = 0.9225 3.3629 ±0.08 cde CN011 y = 18.333x - 11.111 R² = 0.9341 3.384 ±0.24 cde CN012 y = 17.524x - 11.333 R² = 0.9596 3.5653±0.43 bcd CN015 y = 15.048x - 7.1111 R² = 0.9426 3.8091 ±0.02 bc CN019 y = 13x - 7.4444 R² = 0.9585 4.4133 ± 0.34a CN033 y = 10.857x + 0.3333 R² = 0.94 4.6335 ±0.32 a CN040 y = 19x - 11.778 R² = 0.9481 3.262 ±0.14 cde CN043 y = 19.238x - 12.333 R² = 0.9533 3.2439 ±0.15 cde CN054 y = 15.429x - 12.889 R² = 0.94 4.0817 ±0.54 ab CN055 y = 17.143x - 5 R² = 0.9351 3.2072 ±0.35 cde . Example 4: Determination of chitinase and protease production activities and extracellular enzyme activities of Beauveria bassiana isolates

[0035] Enzyme activity indicators are used as an auxiliary screening method to quickly identify highly virulent strains. The infection and pathogenicity of *Beauveria bassiana* in insect hosts generally involves several stages: conidial attachment, spore germination, body wall penetration, hyphal growth within the hemocoel, nutrient uptake, toxin release, and ultimately, destruction of host tissue, leading to insect death. The ability to penetrate the insect's body wall is crucial for successful infection by entomopathogenic fungi. This process involves the formation of specialized infection structures (appendage cells), the generation of mechanical forces, and the action of epidermal degrading enzymes (chitinases, proteases, lipases, etc.).

[0036] Proteins, chitin, and lipids are the main components of insect body walls. Studies have shown that chitinases and proteases in pathogenic fungi are related to the epidermis of the insects they invade, and play a role in the germination, invasion, and infection processes of fungal spores, suggesting their correlation with the virulence of the strain. In particular, the extracellular proteases secreted by the fungi degrade host epidermal components during infection, activating the overexpression of hemolymphocyte oxidase in the host, leading to host death from poisoning. This indicates a correlation between the activity of chitinases and proteases in the strain and their insecticidal potential.

[0037] Conidia from 15 Beauveria bassiana strains were prepared into 1.0 × 10⁻⁶ spores using 0.1% Tween-80. 8 A spore suspension of spores / mL was inoculated at 3 μL onto the center of CA and CaA plates using a pipette. After incubating the CA plates at 28℃ for 9 days, the surface was covered with 5% NaOH solution. For the CaA plates, after incubating at 28℃ for 5 days, the surface was covered with 0.4 mol / L trichloroacetic acid solution. A clear, transparent ring was observed around the colony. Enzyme activity was expressed as a diameter ratio (diameter ratio = diameter of the transparent ring / colony diameter). Three replicates were performed for each strain.

[0038] Extracellular chitinase induction and detection: 40 mL of sterile chitinase induction medium was placed in a 250 mL Erlenmeyer flask, and 1.0 × 10⁻⁶ cells / mL were added. 8 The conidia of the Beauveria bassiana isolate at 2% (spores / mL) V / VInoculation was performed at (25±1)℃ and 180 r / min in a constant temperature shaker, with 3 replicates per group. On the 5th day after induction, the culture medium was centrifuged at 4℃ and 6000 r / min for 10 min, and the supernatant was 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 40°C water bath for 1 h. DNS reagent was then added, and the mixture was boiled in water for 5 min. After cooling to room temperature, the volume was adjusted to 25 mL with ultrapure water, and the absorbance was measured at 540 nm. Three samples were taken from each treatment, and the measurements were performed 3 times. The average value was used to calculate the chitinase activity. The amount of enzyme required to convert 1 μg of N-acetylglucosamine per minute was defined as 1 enzyme activity unit.

[0039] Extracellular protease induction and detection: 50 mL of sterile protease induction medium was added to a 250 mL Erlenmeyer flask, and the strain spores were inoculated and induced according to 1.5.2. On the 5th day after induction, the culture medium was centrifuged at 4℃ and 6000 r / min for 10 min, and the supernatant was 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 40°C water bath for 10 min. After removal, trichloroacetic acid was added to terminate the reaction. After centrifugation at 25°C and 12,000 r / min for 5 min, the supernatant was collected. 2 mL of 0.4 mol / L Na2CO3 and 1 mL of Folin-Ciocalteu reagent were added and mixed well. The mixture was then subjected to a colorimetric reaction at 40°C for 20 min. The absorbance was measured at 680 nm. Three samples were taken from each treatment and measured three times. The absorbance was calculated as the average value. The protease activity was calculated according to the standard curve. The amount of enzyme required to convert 1 μg of tyrosine per minute was defined as 1 enzyme activity unit.

[0040] By comparing the growth, sporulation, and enzyme production capabilities of various Beauveria bassiana strains, candidate strains were selected for further testing of their extracellular chitinase (CN006, CN012, CN040, CN055) and protease (CN009, CN019, CN040, CN055) activities. Small amounts of culture medium were collected on days 1, 3, 5, 7, and 9 after induction to prepare crude enzyme solutions, and their enzyme activities were measured and compared.

[0041] 4.1 Chitinase activity of different Beauveria bassiana strains like Figure 2As shown, after culturing different strains on CA medium for 9 days, clear zones appeared in all CA plates treated with 5% NaOH solution, indicating that all Beauveria bassiana strains have the ability to produce chitinase. The top five strains in terms of clear zone diameter ratio were CN006 > CN004 > CN012 > CN055 > CN040, with strain CN006 having the highest average clear zone diameter (34.28 ± 0.3) mm (1.48 ± 0.01), significantly higher than the other strains. P <0.05 (Table 8). After detecting the extracellular chitinase activity of each strain on day 5 of induction culture, the top five strains in terms of extracellular chitinase activity were CN009 > CN006 > CN055 > CN040 ​​> CN012, with enzyme activities all greater than 18 U. Among them, strain CN009 had the highest extracellular chitinase activity, at (23.37 ± 0.66) U / mL, significantly higher than the other strains ( P <0.05). Furthermore, the diameter ratio of the clear zone and the extracellular chitinase activity of strains CN006, CN012, CN040, and CN055 were all among the top five, showing consistency.

[0042] Table 8. Chitinase Activities of Different Beauveria bassiana Strains 1) strain number Average colony diameter / mm Average transparent ring diameter / mm Diameter ratio Extracellular chitinase activity (U / mL) CN002 29.27±0.32 32.75±0.29 (1.12±0.01)e (3.8±1.01)gh CN003 28.47±0.48 32.05±0.65 (1.13±0.02)e (10.22±0.18)d CN004 26.6±0.9 35.74±0.39 (1.34±0.01)b (3.17±0.18)h CN005 29.88±0.28 31.79±0.04 (1.06±0)fg (8.15±0.31)e CN006 23.17±0.46 34.28±0.3 (1.48±0.01)a (20.63±0.36)b CN009 18.91±0.26 19.05±0.3 (1.01±0.02)h (23.37±0.66)a CN011 26.32±0.41 32.38±0.12 (1.23±0 d)d (3.06±0.32)h CN012 25.58±0.56 32.78±0.32 (1.28±0.01)c (18±0.66)c CN015 30.62±0.32 32.15±0.28 (1.05±0.01)fg (10.85±0.18)d CN019 29.9±0.2 31.41±0.59 (1.05±0.02)fg (5.38±0.48)fg CN033 30.54±0.69 31.58±0.23 (1.03±0.01)gh (5.59±0.55)f CN040 27.83±0.28 33.37±0.19 (1.2±0.01)d (20.11±0.55)b CN043 30.38±0.63 32.72±0.15 (1.08±0.01)f (7.59±0.66)e CN054 29.65±0.26 31.97±0.36 (1.08±0.01)f (5.9±0.32)f CN055 27.49±0.69 33.43±0.35 (1.22±0.01)d (21.26±0.79)b 1) The data in the table are mean ± standard deviation. Different lowercase letters in the same column indicate that the difference is significant at the 0.05 level according to the one-way ANOVA (Tukey's honest significant difference (HSD) test), and the same applies below.

[0043] 4.2 Protease activity of different Beauveria bassiana strains After culturing different strains on CaA medium for 5 days, all CaA plates treated with trichloroacetic acid solution showed obvious clear zones, and the diameter ratio of the clear zones for each strain was higher than 1.9, indicating that all 15 Beauveria bassiana strains had protease production capabilities. The strains with the highest clear zone diameter ratios were CN009 > CN040 ​​> CN055 > CN004 > CN019, with strain CN009 having the highest average clear zone diameter (33.56 ± 0.17) mm and the largest diameter ratio (2.34 ± 0.01), significantly higher than the other strains. P<0.05 (Table 9). After detecting the extracellular protease activity of each strain on day 5 after induction culture, it was found that the protease activity of each strain was higher than 10⁸ U / mL. The top five strains in terms of extracellular protease activity were CN055 > CN002 > CN009 > CN040 ​​> CN019, with strain CN055 exhibiting the highest chitinase activity at (249.41 ± 1.35) U / mL, significantly higher than the other strains ( P <0.05). Furthermore, strains CN009, CN019, CN040, and CN055 all ranked among the top five in terms of the diameter ratio of their clear zones and protease activities, demonstrating consistency.

[0044] Table 9. Protease activities of different Beauveria bassiana strains strain number Average colony diameter / mm Average transparent ring diameter / mm Diameter ratio Extracellular protease activity (U / mL) CN002 16.42±0.32 33.92±0.87 (2.07±0.05)cd (211.69±4.1)b CN003 14.21±0.26 29.39±0.74 (2.07±0.05)cd (152.57±15.3)def CN004 15.47±0.75 34.11±1.32 (2.2±0.09)abc (108.37±2.65)g CN005 14.75±0.23 29.05±0.66 (1.97±0.04)d (148.94±11.68)f CN006 13.55±0.31 28.13±0.64 (2.08±0.05)cd (147.78±11.01)f CN009 14.36±0.49 33.56±0.17 (2.34±0.01)a (201.96±14.23)b CN011 15.22±0.45 33.45±0.28 (2.2±0.02)abc (150.24±15.17)ef CN012 15.69±0.52 31.79±0.38 (2.03±0.02)d (150.76±5.84)ef CN015 14.37±0.21 28.88±0.47 (2.01±0.03)d (184.46±11.13)bcd CN019 13.78±0.43 29.1±0.54 (2.11±0.04)bcd (187.83±16.41)bc CN033 14.82±0.6 29.23±0.85 (1.97±0.06)d (183.56±10.67)bcde CN040 14.92±0.07 33.32±0.62 (2.23±0.04)ab (197.82±15.38)b CN043 14.26±0.1 29.97±0.67 (2.1±0.05)bcd (133.65±3.26)fg CN054 14.08±0.63 28.81±1.17 (2.05±0.08)d (157.63±10.53)cdef CN055 15.24±0.75 33.62±0.11 (2.21±0.01)abc (249.41±1.35)a .

[0045] 4.3 Dynamics of chitinase and extracellular protease production in candidate strains Further determination of extracellular enzyme activity was conducted on strains exhibiting consistent ratios of clear zone diameter and extracellular enzyme activity. The results showed that the production trends of chitinase and protease were generally consistent across all strains throughout the growth period. Specifically, chitinase production did not significantly increase from days 1 to 3 of growth, reaching its peak on day 5, and then declined. The highest chitinase activity was observed in strain CN055, reaching (21.26 ± 0.79) U / mL, significantly higher than that of strains CN006, CN012, and CN040. P <0.05) (Table 10). The production of protease by each strain increased slightly on day 3, entered a plateau period from day 5 to 7, and reached its highest level on day 9. Among them, strain CN055 produced the highest protease activity on day 9, which was (472.5±0.39) U / mL, significantly higher than strains CN009, CN019 and CN040. P <0.05) (Table 11).

[0046] Table 10. Significant differences in chitinase activity of Beauveria bassiana at different time points. .

[0047] Table 11. Significant differences in protease activity of Beauveria bassiana at different time points. .

[0048] 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 CN055, characterized in that, The preservation number of the Beauveria bassiana strain CN055 is CCTCCNO:M 20251522.

2. The application of the Beauveria bassiana strain CN055 according to claim 1 for the control of aphids.

3. A fungicide for controlling aphids, characterized in that, The inoculum contains spores of the Beauveria bassiana strain CN055 as described in claim 1.

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