Uvioresistant high-toxicity beauveria bassiana mutant strain F5-Bb20240406-6 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and discloses an anti-ultraviolet high-toxicity beauveria bassiana mutant strain F5-Bb20240406-6 and an application of the mutant strain F5-Bb20240406-6. The beauveria bassiana mutant strain F5-Bb20240406-6 is obtained by carrying out ultraviolet mutagenesis screening on the beauveria bassiana Bb20240406, the lethal time of the mutant strain on tenebrio molitor, peanut aphid and other test insects is remarkably shortened, the biological characteristic is excellent, the sporulation quantity, the growth speed and the ultraviolet resistance are improved, and the activity of protease and chitinase is remarkably enhanced. The strain is applied to peanut pest control, has a good control effect on grubs and aphids, has remarkable leaf protection and yield increase effects, provides a high-quality strain resource for biological control of peanut pests, is beneficial to reducing the use of chemical pesticides, and promotes agricultural sustainable development.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a mutagenized strain F5-Bb20240406-6 of Beauveria bassiana with high virulence against ultraviolet rays and its application. Background Art

[0002] Pests such as aphids and grubs can damage a variety of crops, causing huge economic losses. At present, chemical pesticides are the main means of controlling pests and diseases. However, long-term use has led to an increase in the resistance of pests and diseases, resulting in problems of pesticide residues in agricultural products and environmental pollution, and disrupting the ecological balance of farmland.

[0003] Biological control is an ideal and environmentally friendly method for pest control. In the aspect of biological pesticide control, research shows that Beauveria bassiana can be used to control pests such as grubs and aphids. Its insecticidal principles mainly include the following aspects: (1) Attachment and invasion: The conidia of Beauveria bassiana attach to the surface of the insect body. When conditions are suitable, germ tubes germinate. The germ tubes secrete protease and chitinase, degrade the components of the insect body surface, and penetrate the cuticle into the insect body; (2) Nutrient absorption and reproduction: Inside the insect body, it multiplies massively using the insect body fluid and tissues as nutrients. The mycelium fills the body cavity, resulting in nutrient deficiency and physiological disorders in the insect; (3) Toxin secretion: When growing, it secretes toxins such as beauvericin, which invade the nervous and respiratory systems of the insect, interfere with metabolism, paralyze the insect, stop it from feeding, and accelerate its death; (4) Destruction of insect tissues: The mycelium invades tissues and organs such as muscles and fat bodies, destroys their structures and functions, forms sclerotia to consume nutrients, and causes the insect to die due to tissue damage and nutrient depletion; (5) Formation of spores: After the insect dies, the fungus produces a large number of conidia on the surface of the corpse, which are spread by wind and rain, infect new insects, and trigger an epidemic to control the number of pests.

[0004] However, the existing strains of Beauveria bassiana have insufficient virulence and unstable control effects. Among them, ultraviolet rays are one of the main reasons affecting the field control effect of Beauveria bassiana. Therefore, screening Beauveria bassiana strains with resistance to ultraviolet irradiation, high virulence, and good biological characteristics is the key to biological control. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and deficiencies in the insecticidal activity and stress resistance of the existing Beauveria bassiana strains, and provide a mutagenized strain F5-Bb20240406-6 of Beauveria bassiana with high virulence against ultraviolet rays and its application in controlling pests such as aphids and grubs. The mutagenized strain F5-Bb20240406-6 of Beauveria bassiana of the present invention is obtained by ultraviolet mutagenesis and screening, has high virulence, and has strong ultraviolet resistance.

[0006] In order to achieve the above object, the technical solution provided by the present invention is as follows: In the first aspect of the present invention, a mutagenized strain F5-Bb20240406-6 of Beauveria bassiana with high virulence and anti-ultraviolet properties is provided. The preservation number of the strain is CGMCC No. 41821, the preservation date is March 3, 2025, and the preservation classification name is Beauveria bassiana. Beauveria bassiana The preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101, and the telephone number is 8610 - 64807355.

[0007] In the second aspect of the present invention, a fermentation broth, a bacterial suspension, or a sterile supernatant thereof, or a preparation containing the mutagenized strain F5-Bb20240406-6 of Beauveria bassiana is provided.

[0008] In the third aspect of the present invention, the application of the mutagenized strain F5-Bb20240406-6 of Beauveria bassiana, or the fermentation broth, the bacterial suspension, or the sterile supernatant thereof, or the preparation in controlling white grubs and aphids is provided.

[0009] In the fourth aspect of the present invention, the application of the mutagenized strain F5-Bb20240406-6 of Beauveria bassiana, or the fermentation broth, the bacterial suspension, or the sterile supernatant thereof, or the preparation in promoting leaf retention and yield increase of peanuts is provided.

[0010] In the fifth aspect of the present invention, a bacterial agent for promoting leaf retention and yield increase of peanuts is provided. The active ingredient of the bacterial agent includes the mutagenized strain F5-Bb20240406-6 of Beauveria bassiana.

[0011] In the sixth aspect of the present invention, the application of the mutagenized strain F5-Bb20240406-6 of Beauveria bassiana, or the bacterial agent for promoting leaf retention and yield increase of peanuts in the production of bio-organic fertilizer is provided.

[0012] In the seventh aspect of the present invention, a bio-organic fertilizer is provided. The bio-organic fertilizer contains the mutagenized strain F5-Bb20240406-6 of Beauveria bassiana or the bacterial agent for promoting leaf retention and yield increase of peanuts.

[0013] Compared with the prior art, the present invention has the following advantages: (1)Improving strain performance: Through ultraviolet mutagenesis breeding, the present invention successfully obtained the Beauveria bassiana mutagenic strain F5-Bb20240406-6, whose performance was significantly improved. Compared with the parental strain, the ultraviolet resistance of F5-Bb20240406-6 was greatly enhanced. After being irradiated with a 15W ultraviolet lamp for 20 min and then cultured for 5 d, the number of single colonies of the parental strain Bb20240406 was only 45.97, while that of the mutagenic strain F5-Bb20240406-6 reached 60.23. This enables the strain to better resist ultraviolet damage in field applications, maintain a high survival rate and activity, and provides a guarantee for continuously playing the role of pest control. In terms of virulence, the median lethal time of F5-Bb20240406-6 to various pests was significantly shortened. The median lethal times for Tenebrio molitor, Aphis craccivora, Holotrichia oblita, and Spodoptera exigua were 1.48 d, 1.65 d, 1.60 d, and 3.72 d respectively, compared with 3.24 d, 2.75 d, 3.44 d, and 5.46 d of the parental strain Bb20240406, indicating a significant improvement in virulence. This means that the strain can infect pests more quickly and effectively, improve pest control efficiency, and reduce the time and degree of damage to crops by pests.

[0014] (2)Optimizing biological characteristics: The mutagenic strain F5-Bb20240406-6 has obvious advantages in biological characteristics. In terms of colony culture characteristics, its morphology has changed favorably, from the villous form of the parental strain Bb20240406 to a powdery form, with an increase in concentric circles, and the color of the back changing from light yellow to yellow. This morphological change may affect the growth and metabolic patterns of the strain, and thus have a positive impact on its application. In terms of sporulation quantity and growth rate, F5-Bb20240406-6 performs more excellently. On the 3rd d, 6th d, and 9th d, the sporulation quantities of F5-Bb20240406-6 were 0.53×10 8 spores / cm², 1.14×10 8 spores / cm², and 2.28×10 8 spores / cm² respectively, all significantly higher than 0.38×10 8 spores / cm², 0.56×10 8 spores / cm², and 1.56×10 8 spores / cm² of the parental strain Bb20240406. On the 2nd d, 4th d, and 6th d, its growth diameters were 15.33 mm, 29.00 mm, and 55.00 mm respectively, also significantly higher than 11.67 mm, 24.67 mm, and 45.00 mm of the parental strain Bb20240406. The increase in sporulation quantity helps improve the transmission and infection ability of the strain in the environment, while the accelerated growth rate enables the strain to colonize and spread more quickly in the pest population, enhancing the control effect.

[0015] (3) Enhanced extracellular enzyme activity and gene expression advantages: The protease activity and chitinase activity of the mutant strain F5-Bb20240406-6 were significantly increased. The protease activity increased from 177.96 IU / mL of the parental strain Bb20240406 to 219.43 IU / mL, and the chitinase activity increased from 27.38 IU / mL to 53.99 IU / mL. Protease and chitinase play key roles in the process of Beauveria bassiana infecting pests. They can decompose the proteins and chitin in the insect cuticle, promoting the invasion and colonization of the fungus. The increase in activity means that the strain has enhanced ability to infect pests, can penetrate the pest cuticle more effectively, and improve the control effect. In addition, the expression levels of the protease gene CDEP-1 and the chitinase gene Chit1 of the mutant strain F5-Bb20240406-6 were 8.10 times and 14.27 times that of the parental strain Bb20240406, respectively, and the gene sequences were changed. This not only further confirmed the internal molecular mechanism of the increased enzyme activity, but also indicated that beneficial mutations occurred in the strain at the gene level, providing a more stable genetic basis for its excellent performance.

[0016] (4) Significantly improve the control effect of peanut pests: Applying the mutagenized strain F5-Bb20240406-6 to the control of peanut pests has achieved remarkable results. In the control of peanut underground pest Holotrichia oblita, when the application rate is 135.00 trillion spores / hm2, there are no significant differences in the damage index, control effect, yield per mu, and pod damage rate between the treatment with the mutagenized strain F5-Bb20240406-6 and the treatment with 30% phoxim microcapsule suspension, but it is significantly better than the treatment with the parental strain Bb20240406 and the water control. This indicates that the mutagenized strain F5-Bb20240406-6 has an effect equivalent to that of chemical pesticides in controlling Holotrichia oblita in peanut fields, reduces the use of chemical pesticides, and lowers the risk of environmental pollution. In the control of peanut above-ground pests, when the application rates are 18.00 trillion spores / hm2 and 36.00 trillion spores / hm2, the leaf protection effect of the mutagenized strain F5-Bb20240406-6 on peanuts has no significant difference from that of 2.5% deltamethrin emulsifiable concentrate at a dosage of 300 mL / hm2 10 - 15 days after application, and reaches relatively high values 15 days after application, which are 34.36% and 51.19% respectively; the control effect on aphids has no significant difference from that of 2.5% deltamethrin emulsifiable concentrate 7 days after application, is higher than that of 2.5% deltamethrin emulsifiable concentrate 10 - 15 days after application, and the control effect of the high application rate treatment reaches 79.36% 15 days after application; in terms of the yield increase effect, when the application rate is 36.00 trillion spores / hm2, the yield increase effect of the mutagenized strain F5-Bb20240406-6 is 18.01%, which has no significant difference from the yield increase effect of 2.5% deltamethrin emulsifiable concentrate of 14.30%, but is significantly higher than other treatments with the application of the strain and the water control. These results show that the mutagenized strain F5-Bb20240406-6 has a good control effect on peanut above-ground pests, can effectively protect peanut leaves, reduce aphid damage, and promote peanut yield increase, providing a strong guarantee for the safe production of peanuts.

[0017] (5) Promote the development of biological control: The present invention provides a new high-quality strain resource for the biological control of peanut pests, which helps to reduce the use of chemical pesticides. The extensive use of traditional chemical pesticides has led to problems such as environmental pollution and enhanced pest resistance. Beauveria bassiana, as a biological control method, has the advantages of safety, environmental protection, and low resistance generation. The successful selection of the mutagenized strain F5-Bb20240406-6 provides a more effective tool for the development of biological control technology, which helps to promote the development of agricultural production towards the green and sustainable direction. At the same time, the research methods and results of the present invention also provide a reference for the improvement and application of other entomopathogenic fungi, and have important significance for promoting the development of the entire biological control field. Description of the Drawings

[0018] Figure 1Effect of ultraviolet mutagenesis on the morphology of the strain: a1 and a2 are the front and back culture morphologies of Bb20240406; b1 and b2 are the front and back culture morphologies of F5-Bb20240406-6.

[0019] Figure 2 Effect of mutagenesis on the sporulation amount of the strain: Different lowercase letters in the figure indicate significant differences at the 0.05 level.

[0020] Figure 3 Effect of mutagenesis on the growth rate of the strain: Different lowercase letters in the figure indicate significant differences at the 0.05 level.

[0021] Figure 4 Effect of mutagenesis on the ultraviolet resistance of the strain: Different lowercase letters in the figure indicate significant differences at the 0.05 level.

[0022] Figure 5 Effect of mutagenesis on the expression level of virulence-related enzyme genes of the strain.

[0023] Figure 6 CDEP-1 gene (left) and Chit1 gene (right) before and after mutagenesis.

[0024] Figure 7 Sequence comparison of CDEP-1 gene (upper) and Chit1 gene (lower) before and after mutagenesis. Specific implementation manners

[0025] The specific implementation manners of the present invention are described below to facilitate those skilled in the art of the present technology to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation manners. For those of ordinary skill in the art of the present technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.

[0026] Example 1: Mutagenesis, isolation and identification of Beauveria bassiana Bb20240406 1. Materials The mutagenized strain of Beauveria bassiana is mutagenized on the basis of Beauveria bassiana Bb20240406. The morphological identification result of Beauveria bassiana Bb20240406 is that the middle of the colony on the front is powdery, the edge is velvety, and there are multiple radiation depressions; the middle of the colony on the back is light yellow, and the edge is white. After observation and measurement under a fluorescence microscope, the conidia are spherical or nearly spherical, and the diameter of the spores is about 2.18 μm. The preservation number of Bb20240406 is: CGMCC No. 41802, and the ITS sequence is as shown in SEQ ID No. 1.

[0027] 2. Mutagenesis of Beauveria bassiana Bb20240406 2.1 UV mutagenesis method of Beauveria bassiana Take an appropriate amount of spores of the parental strain Bb20240406, add an appropriate amount of sterile water containing 0.05% Tween-80 to make a spore suspension of 1×108 cells / mL, irradiate it under a preheated 15 W UV lamp for 20 min. Take 0.1 mL of the spore suspension and spread it evenly on a PDA medium. There are 10 plates in total, which are the mutagenized strains of the F1 generation. 3 plates without UV irradiation are used as controls. Wrap the plates with black cloth, avoid light, and place them in a constant temperature incubator at (25±1)°C for dark cultivation. After 24 h, switch to normal cultivation. Observe the colony growth after 6 d, and select the strains with good growth status for the next experiment.

[0028] 2.2 Virulence determination results of the first-generation mutagenized strains Using Bb20240406 as the parental strain for UV mutagenesis, the virulence determination results of 10 F1-generation mutagenized strains against 4 test insects are shown in Table 1. Among the 10 mutagenized strains, the LT50 of 5 mutagenized strains against Tenebrio molitor is less than that of the parental strain, the LT50 of 3 strains against Aphis craccivora is less than that of the parental strain, the LT50 of 3 strains against Holotrichia diomphalia is less than that of the parental strain, and the LT50 of 2 strains against Spodoptera exigua is less than that of the parental strain. Among them, the mutagenized strain F1-Bb20240406-3 has the smallest lethal median time LT50 and the highest virulence against Tenebrio molitor, Aphis craccivora, Holotrichia diomphalia, and Spodoptera exigua, which are 2.57 d, 2.20 d, 2.64 d, and 4.99 d respectively. The virulence is higher than that of the parental strain Bb20240406. Therefore, F1-Bb20240406-3 is selected as the parental strain for the second-generation mutagenesis.

[0029] Table 1 Determination results of the lethal median time of the first-generation mutagenized strains against 4 test insects

[0030] Note: The data in the table are mean ± standard error, and different lowercase letters indicate significant differences at the 0.05 level.

[0031] 2.3 Virulence determination results of the second-generation mutagenized strains Using the excellent mutagenized strain F1-Bb20240406-3 of the F1 generation as the parental strain, ultraviolet mutagenesis was carried out, and the virulence determination results of 10 mutagenized strains of the F2 generation against 4 test insects are shown in Table 2. Among the 10 mutagenized strains, the LT50 of 4 strains against Tenebrio molitor was less than that of the parental strain, the LT50 of 7 strains against Aphis craccivora was less than that of the parental strain, the LT50 of 6 strains against Holotrichia oblita was less than that of the parental strain, and the LT50 of 7 strains against Spodoptera exigua was less than that of the parental strain. Among them, the mutagenized strain F2-Bb20240406-8 had the smallest median lethal time LT50 and the highest virulence against Tenebrio molitor, Aphis craccivora, Holotrichia oblita, and Spodoptera exigua, which were 2.25 d, 1.94 d, 2.30 d, and 4.42 d respectively. The virulence was higher than that of the parental strains Bb20240406 and F1-Bb20240406-3. Therefore, F2-Bb20240406-8 was selected as the parental strain for the third-generation mutagenesis.

[0032] Table 2 Determination results of the median lethal time of the second-generation mutagenized strains against 4 test insects

[0033] Note: The data in the table are mean ± standard error, and different lowercase letters indicate significant differences at the 0.05 level.

[0034] 2.4 Virulence determination results of the third-generation mutagenized strains Using the excellent mutagenized strain F2-Bb20240406-8 of the F2 generation as the parental strain, ultraviolet mutagenesis was carried out, and the virulence determination results of 10 mutagenized strains of the F3 generation against 4 test insects are shown in Table 3. Among the 10 mutagenized strains, the LT50 of 7 strains against Tenebrio molitor was less than that of the parental strain, the LT50 of 8 strains against Aphis craccivora was less than that of the parental strain, the LT50 of 9 strains against Holotrichia oblita was less than that of the parental strain, and the LT50 of 9 strains against Spodoptera exigua was less than that of the parental strain. Among them, the mutagenized strain F3-Bb20240406-4 had the smallest median lethal time LT50 and the highest virulence against Tenebrio molitor, Aphis craccivora, Holotrichia oblita, and Spodoptera exigua, which were 1.75 d, 1.78 d, 2.14 d, and 4.14 d respectively. The virulence was higher than that of the parental strains Bb20240406 and F2-Bb20240406-8. Therefore, F3-Bb20240406-4 was selected as the parental strain for the fourth-generation mutagenesis.

[0035] Table 3 Determination results of the median lethal time of the third-generation mutagenized strains against 4 test insects

[0036] Note: The data in the table are mean ± standard error, and different lowercase letters indicate significant differences at the 0.05 level.

[0037] 2.5 Virulence determination results of the fourth-generation mutagenized strains Using the excellent mutagenized strain F3-Bb20240406-4 of the F3 generation as the parental strain, ultraviolet mutagenesis was carried out, and the virulence determination results of 10 mutagenized strains of the F4 generation against 4 test insects are shown in Table 4. Among the 10 mutagenized strains, the LT50 of 8 strains against Tenebrio molitor was less than that of the parental strain, the LT50 of 6 strains against Aphis craccivora was less than that of the parental strain, the LT50 of 9 strains against Holotrichia diomphalia was less than that of the parental strain, and the LT50 of 8 strains against Spodoptera exigua was less than that of the parental strain. Among them, the lethal median time LT50 of the mutagenized strain F4-Bb20240406-1 against Tenebrio molitor, Aphis craccivora, Holotrichia diomphalia, and Spodoptera exigua was the smallest and the virulence was the highest, which were 1.66 d, 1.70 d, 1.88 d, and 4.04 d respectively. The virulence was higher than that of the parental strains Bb20240406 and F3-Bb20240406-4. Therefore, F4-Bb20240406-1 was selected as the parental strain for the fifth-generation mutagenesis.

[0038] Table 4 Determination results of the lethal median time of the fourth-generation mutagenized strains against 4 test insects

[0039] Note: The data in the table are mean ± standard error, and different lowercase letters indicate significant differences at the 0.05 level.

[0040] 2.6 Virulence determination results of the fifth-generation mutagenized strains Using the excellent mutagenized strain F4-Bb20240406-1 of the F4 generation as the parental strain, ultraviolet mutagenesis was carried out, and the virulence determination results of 10 mutagenized strains of the F5 generation against 4 test insects are shown in Table 5. It can be seen from Table 14 that among the 10 mutagenized strains, the LT50 of 7 strains against Tenebrio molitor was less than that of the parental strain, the LT50 of 8 strains against Aphis craccivora was less than that of the parental strain, the LT50 of 7 strains against Holotrichia diomphalia was less than that of the parental strain, and the LT50 of 8 strains against Spodoptera exigua was less than that of the parental strain. Among them, the lethal median time LT50 of the mutagenized strain F5-Bb20240406-6 against Tenebrio molitor, Aphis craccivora, Holotrichia diomphalia, and Spodoptera exigua was the smallest and the virulence was the highest, which were 1.48 d, 1.65 d, 1.60 d, and 3.72 d respectively. The virulence was higher than that of the parental strains Bb20240406 and F4-Bb20240406-1.

[0041] Table 5 Determination results of the lethal median time of the fifth-generation mutagenized strains against 4 test insects

[0042] Note: The data in the table are mean ± standard error, and different lowercase letters indicate significant differences at the 0.05 level.

[0043] 3. Morphological description of the mutagenized strains The culture characteristics of the parental strain and the F5-generation mutagenized strains are as Figure 1As shown in the figure, the front of the colony of the parental strain Bb20240406 is white and villous, with a small number of concentric circles in the middle; the back of the colony of the parental strain Bb20240406 is light yellow. The front of the colony of F5-Bb20240406-6 is powdery white in the middle and villous at the edge, with multiple concentric circles; the back of the colony of the strain F5-Bb20240406-6 is yellow. Thus, it can be seen that the culture characteristics of the strain have changed under the condition of ultraviolet mutagenesis.

[0044] Extract DNA from the mycelium of the strain F5-Bb20240406-6, and then use the extracted genomic DNA of F5-Bb20240406-6 as a template for PCR amplification to obtain the amplified product of the fungal ITS region, that is, the ITS sequence of F5-Bb20240406-6. Sequence the ITS sequence of F5-Bb20240406-6 (see SEQ ID No. 2).

[0045] 4. Effect of Ultraviolet Mutagenesis on Sporulation of Beauveria bassiana Absorb 0.1 mL of the spore suspension with a concentration of 108 spores / mL onto the PDA medium, spread it evenly with a sterile spreader, and place it in an artificial incubator with a culture condition of temperature (25±1)°C. Incubate for 3 d, 6 d, and 9 d respectively. Scrape all the spores in the petri dish to make a bacterial suspension, and measure its concentration with a hemocytometer. Use the parental strain as a control, with 3 replicates for each treatment.

[0046] The change trend of sporulation of excellent mutagenized strains in each generation is as Figure 3 shown. The sporulation of each strain showed an upward trend from the 3rd d to the 9th d. On the 3rd d, the sporulation of the fifth-generation mutagenized strain F5-Bb20240406-6 was significantly higher than that of the parental strain Bb20240406, which were 0.38×108 spores / cm2 and 0.53×108 spores / cm2 respectively. There was no difference between the other generations of strains and the parental strain; on the 6th d, the sporulation of each generation of mutagenized strains was higher than that of the parental strain. Among them, the sporulation of the fourth-generation mutagenized strain F4-Bb20240406-1 was the highest at 1.18×108 spores / cm2, which was twice as high as the sporulation of the parental strain at 0.56×108 spores / cm2; on the 9th d, the sporulation of each generation of mutagenized strains was higher than that of the parental strain.

[0047] 5. Effect of Ultraviolet Mutagenesis on Growth Rate of Beauveria bassiana Take 0.1 mL of the spore suspension with a concentration of 108 spores / mL on the PDA medium, spread it evenly with a sterile spreader, place it in an incubator for 24 h, punch out a mycelial disc with a puncher with a diameter of 7 mm, and use sterile forceps to place the mycelial disc at the center of the PDA medium. Using the parental strain as a control, repeat each treatment 3 times, place it in an artificial incubator at a temperature of (25±1) °C for cultivation, observe regularly on the 2nd d, 4th d, and 6th d, and measure its growth diameter by the cross method.

[0048] The change trend of the growth diameter of excellent mutagenic strains in each generation is as Figure 4 shown. The growth diameters of all strains showed an upward trend from the 3rd d to the 9th d. On the 3rd d, the growth diameter of the fifth-generation mutagenic strain F5-Bb20240406-6 was significantly higher than that of the parental strain Bb20240406, which were 15.33 mm and 11.67 mm respectively. There was no difference in the growth diameter of the other generations of strains compared with the parental strain; on the 4th d, the growth diameter of the fifth-generation mutagenic strain F5-Bb20240406-6 was significantly higher than that of the parental strain Bb20240406, which were 29.00 mm and 24.67 mm respectively. There was no difference in the growth diameter of the other generations of strains compared with the parental strain; on the 9th d, the growth diameters of all generations of mutagenic strains were higher than those of the parental strain. Among them, the fourth-generation mutagenic strain F4-Bb20240406-1 and the fifth-generation mutagenic strain F5-Bb20240406-6 had the highest growth diameters, which were 54.67 mm and 55.00 mm respectively.

[0049] 6. Effect of ultraviolet mutagenesis on the ultraviolet resistance of Beauveria bassiana Take 0.1 mL of the spore suspension with a concentration of 108 spores / mL on the PDA medium, spread it evenly with a sterile spreader. After turning on a 15 W ultraviolet lamp and preheating it for 5 min, remove the lid of the petri dish coated with the spore suspension, place it 50 cm away from the ultraviolet lamp, irradiate for 20 min, then turn off the ultraviolet lamp, cover the petri dish lid, and place it in an artificial incubator at a temperature of (25±1) °C for cultivation for 5 d. Using the parental strain as a control, repeat each treatment 3 times, observe the growth of the colonies, and record the survival of the spores.

[0050] The number of single colonies of each generation of mutagenic strains after being irradiated with ultraviolet light for 20 min and then cultured for 5 d is as Figure 5As shown, the number of single colonies from the parent strain to the fifth generation of mutagenic bacteria ranged from 45.97 to 60.23. The number of single colonies of each generation of mutagenic strains was significantly higher than that of the parent strain Bb20240406, which was 45.97. It showed an increasing trend from the first generation to the fifth generation, indicating that the strain's UV resistance gradually increased with the increase of UV mutagenic generations. The fifth generation mutagenic strain F5-Bb20240406-6 had the largest number of single colonies, which was 60.23, and had the strongest UV resistance.

[0051] 7. Effect of mutagenesis on extracellular enzyme activity of strains 7.1 Preparation of enzyme solution to be tested Take 100 mL of sterilized liquid culture medium in a 250 mL Erlenmeyer flask, add 1 mL of bacterial suspension with a concentration of 108 spores / mL of the mutagenic bacteria at a ratio of 1%, use the parent strain and 0.05% Tween-80 in sterile water as controls, repeat 3 times for each treatment, culture on a rotary shaker at (25±1)℃ and 180 rpm, take the culture solution regularly, filter it with filter paper, and the filtrate is the enzyme solution to be tested.

[0052] 7.2 Extracellular protease activity assay (1) Take casein as the substrate and prepare a 1% (W / V) casein solution with 0.05 mol / L Tris-HCl buffer at a pH of 8.5. Take 1 mL of this solution in a 10-mL centrifuge tube and add 1 mL of the appropriately diluted enzyme solution to be tested. Use the parent strain and distilled water as controls. Repeat each treatment three times. After reacting in a constant temperature water bath at 37°C for 30 min, add 0.4 mol / L trichloroacetic acid to each tube. Filter and keep the filtrate for later use.

[0053] (2) Use Folin-phenol reagent for detection. Mix FolinAB solution in a ratio of 1:50, take 400 μL of the filtrate into a centrifuge tube, add 2 mL of the mixture to each tube, let it stand for 10 min, then add 200 μL of phenol reagent and let it stand for 30 min.

[0054] (3) Measure the absorbance at a wavelength of 650 nm. Draw a standard curve using the tyrosine standard solution and calculate the enzyme activity by comparing it with the standard curve. The amount of enzyme that catalyzes the production of 1 μg of tyrosine in 1 mL of the test enzyme solution is defined as one enzyme activity unit (IU / mL).

[0055] 7.3 Determination of extracellular chitinase activity (1) Take 1 mL of 1% colloidal chitin in a 10 mL centrifuge tube, add 0.5 mL of the enzyme solution to be tested. Use the parental strain and distilled water as controls. Repeat each treatment 3 times. After reacting in a constant temperature water bath at 50 °C for 1 h, take it out, appropriately dilute the reaction solution, and adjust the reducing sugar concentration to 0.1 - 1.0 mg / mL.

[0056] (2) Take 1 mL of the diluted solution to be tested in a 15 mL centrifuge tube, and use the DNS method to measure the concentration of reducing sugar. Add 2 mL of DNS reagent to each tube, boil in a water bath for 2 min, and add distilled water to 15 mL after cooling. (3) Measure the absorbance value at a wavelength of 540 nm. Draw a standard curve with a glucose standard solution, and calculate the enzyme activity by comparing with the standard curve. The amount of enzyme that catalyzes the formation of 1 μmol of reducing sugar per minute is defined as one enzyme activity unit (IU / mL).

[0057] 7.4 Determination of extracellular lipase activity (1) Take 4 mL of PVA olive oil emulsifier in a 100 mL Erlenmeyer flask, add 5 mL of phosphate buffer with a concentration of 0.025 mol / L and a pH value of 7.5, and preheat in a constant temperature water bath at 40 °C for 5 - 10 min.

[0058] (2) Add 1 mL of the enzyme solution to be tested to each bottle. Use the addition of the parental strain and distilled water as controls. Repeat each treatment 3 times. After reacting for 16 min, add 15 mL of 95% ethanol to each bottle to terminate the reaction.

[0059] (3) Use titration to measure the fatty acid concentration in the reaction solution. Add 2 drops of phenolphthalein solution to each bottle, and titrate with a 0.1 mol / L sodium hydroxide solution until the solution in the bottle turns pink and does not fade after shaking. Record the volume of the sodium hydroxide solution used for each bottle. The amount of enzyme that catalyzes the formation of 1 μmol of fatty acid per minute is defined as one enzyme activity unit (IU / mL). The determination results of the activities of three extracellular enzymes of the mutagenized strains are shown in Table 6. The activities of extracellular protease, extracellular chitinase, and extracellular lipase of the parental strain are 177.96 IU / mL, 27.38 IU / mL, and 7.95 IU / mL, respectively. The activities of extracellular protease of each strain from the first to the fifth generation of mutagenized bacteria are 223.79 IU / mL, 216.15 IU / mL, 218.96 IU / mL, 217.74 IU / mL, and 219.43 IU / mL, respectively. The activities of extracellular protease of mutagenized strains in each generation have all increased, and there are no significant differences among generations, but they are significantly different from the parental strain. The activities of extracellular chitinase of each strain from the first to the fifth generation of mutagenized bacteria are 51.00 IU / mL, 53.87 IU / mL, 52.88 IU / mL, 53.38 IU / mL, and 53.99 IU / mL, respectively. The activities of extracellular chitinase of mutagenized strains in each generation have all increased, and there are no significant differences among generations, but they are significantly different from the parental strain. The activities of extracellular lipase of each strain from the first to the fifth generation of mutagenized bacteria are 8.33 IU / mL, 8.10 IU / mL, 7.63 IU / mL, 8.27 IU / mL, and 8.44 IU / mL, respectively, and there is no significant difference from the lipase activity of 7.95 IU / mL of the parental strain.

[0060] Table 6 Effects of mutagenesis on the activities of extracellular enzymes of strains

[0061] Note: The data in the table are mean ± standard error, and different lowercase letters indicate significant differences at the 0.05 level.

[0062] 8. Effects of mutagenesis on the genes of virulence-related enzymes of strains 8.1 Effects of mutagenesis on the expression levels of virulence-related enzyme genes of strains Take the excellent strains obtained by mutagenesis, culture them in an artificial incubator at a temperature of (25 ± 1) °C for 6 days, scrape the culture, extract total RNA using a column-type fungal total RNA extraction and purification kit, reverse transcribe it into cDNA using the HiScript II Q Select RT SuperMix for qPCR (+ gDNA wiper) reverse transcription kit, adjust the cDNA concentration to 300 ng / μL with RNA-free H2O, and verify the gene expression levels using the Trans Start Green qPCR Super Mix kit. Select 18sRNA as the internal reference gene and design qRT-PCR primers (Table 7).

[0063] Table 7 qRT-PCR primers

[0064] Take a 0.2 ml centrifuge tube and add the following components: 3.0 μL of cDNA, 0.5 μL of upstream primer, 0.5 μL of downstream primer, 10 μL of 2×TransStarTip Green qPCR SuperMix, and 6 μL of ddH2O. Gently flick to mix evenly, and centrifuge to collect the droplets on the tube wall to the bottom of the tube. The Qrt-PCR reaction program is pre-denaturation at 95°C for 30 s, pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, for 42 cycles.

[0065] The expression levels of the protease gene CDEP-1 (accession number AY040532.1) and chitinase gene Chit1 (accession number GU073382.1) of the mutagenized strain F5-Bb20240406-6 relative to the parental strain Bb20240406 are as Figure 6 shown. The relative expression level of the protease gene CDEP-1 of the mutagenized strain F5-Bb20240406-6 is 8.10, and the relative expression level of the chitinase gene Chit1 of the mutagenized strain F5-Bb20240406-6 is 14.27, which is significantly higher than that of the parental strain, consistent with the results of extracellular enzyme activity determination.

[0066] 8.2 Effects of mutagenesis on the sequences of virulence-related enzyme genes of the strain Take the excellent mutagenized strain, culture it in an artificial incubator at a temperature of (25±1)°C for 6 d, scrape off the culture, extract the genomic DNA of the strain, with the parental strain Bb20240406 as a control. Design primers with DNAMAN.

[0067] Table 8 PCR primers

[0068] Take a 0.2 ml centrifuge tube and add the following components: 1.5 μL of genomic DNA, 0.5 μL of upstream primer, 0.5 μL of downstream primer, 2.5 μL of 10×Buffer, 1.0 μL of dNTP, 0.5 μL of Hif polymerase, and 18.0 μL of ddH2O. After gently flicking to mix evenly, centrifuge to collect the droplets on the tube wall to the bottom of the tube, and perform PCR reaction on a PCR amplifier. The PCR amplification reaction program: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 1 min, for 35 cycles, final extension at 72°C for 7 min. After the reaction is completed, take 4 μl of the PCR product for 0.8% agarose gel electrophoresis to detect the PCR amplification product. Recover the PCR product with a universal DNA purification and recovery kit, take the purified PCR product, and sequence it by Shanghai Sunny Biotechnology Co., Ltd.

[0069] Using the genomic DNA of the fifth-generation mutagenized strain F5-Bb20240406-6 and the parental strain Bb20240406 as templates, the protease CDEP-1 gene and chitinase Chit1 gene of this strain were obtained by PCR. The results of 0.8% agarose gel electrophoresis are as follows Figure 7 shown. The size of the CDEP-1 gene band is approximately 1500 bp, and the size of the Chit-1 gene band is approximately 1100 bp. After purification and recovery, sequencing analysis was performed. As Figure 7 shown, the sequence alignment results showed that the sequences of the protease CDEP-1 gene and chitinase Chit1 gene of the mutagenized strain F5-Bb20240406-6 both changed.

[0070] In summary, the research results show that the mutagenized strain of the present invention significantly shortens the median lethal time for test insects such as Tenebrio molitor and Aphis craccivora, has excellent biological characteristics, improved sporulation amount, growth rate and ultraviolet resistance, and significantly enhanced protease and chitinase activities. It was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on March 3, 2025, and the deposit number of the strain is CGMCC No. 41821.

[0071] Example 2 Field control effect of the mutagenized strain F5-Bb20240406-6 on peanut pests 1. Control effect of the mutagenized strain F5-Bb20240406-6 on white grubs 1.1 Field control effect of the mutagenized strain on white grubs in the peanut field in the net room Under the condition of artificially and evenly releasing 200 Holotrichia parallela in the net room, the solid fermentation material of the Bb20240406-F5 strain was obtained by the solid-liquid two-phase fermentation method, and the content of conidia was measured. Before sowing, the solid fermentation material of the mutagenized strain Bb20240406-F5 was weighed at different concentrations, supplemented with wheat bran to 30 g and mixed evenly, and then evenly spread into the sowing ditch. 30% phoxim microcapsule suspension was used as the conventional control, and the equal-concentration parental strain Bb20240406 and the sterile water fermentation material with an equal amount of 0.05% Tween-80 were used as controls. Each treatment was repeated 3 times, and the plot area was 10 m2. The investigation was carried out one day before peanut harvest. Five points were randomly selected in each plot to investigate the damage situation of peanut pods. The grading standard for peanut pod damage is shown in Table 9.

[0072] Table 9 Grading standard for peanut pod damage

[0073] Under the condition of artificially releasing Holotrichia parallela in the net room, the application effect of the mutagenic strain F5-Bb20240406-6 on peanut field white grubs is shown in Table 10. At high doses, the damage index of the treatment with the mutagenic strain F5-Bb20240406-6 was significantly lower than that of other treatments; the control effect of the low-dose treatment of Bb20240406 was significantly lower than that of other treatments, and there was no significant difference among other treatments; except for the low-dose treatment of Bb20240406, the pod damage rate of each treatment was significantly less than that of the clear water control, and there was no significant difference among all treatments; the yields of the high-dose treatment of the mutagenic strain F5-Bb20240406-6 and phoxim treatment were significantly higher than those of the low-dose treatment of the parental strain Bb20240406 treatment and the clear water control, and there was no significant difference between the two. The results show that when the high-dose treatment of the mutagenic strain F5-Bb20240406-6 is used to control white grubs in peanut fields, all evaluation indicators are similar to or better than the recommended dosage of phoxim, indicating the potential of the mutagenic strain F5-Bb20240406-6 to reduce the application of chemical pesticides in the practice of controlling white grubs in peanut fields.

[0074] Table 10 Application effect of mutagenic strains on peanut white grub pests in the net room

[0075] Note: The data in the table are mean ± standard error, and different lowercase letters indicate significant differences at the 0.05 level.

[0076] 1.2 Field control effect of mutagenic strains on peanut field white grubs The solid fermentation material of the Bb20240406-F5 strain was obtained by the solid-liquid two-phase fermentation method, and the content of conidia was determined. Before sowing, the mutagenic strain Bb20240406-F5 solid fermentation material was weighed according to different concentrations, supplemented with wheat bran to 30 g and mixed evenly, and then evenly spread into the sowing furrows. The 30% phoxim microcapsule suspension was used as the conventional control, and the fermentation materials of the parental strain Bb20240406 at the same concentration and the sterile water with the same amount of 0.05% Tween-80 were used as controls. Each treatment was repeated 4 times, and the plot area was 30 m2. The dosages of each treatment are shown in Table 11.

[0077] Table 11 Dosages of pesticides for field control effect test

[0078] The control effect of the fifth-generation mutagenized strain of Beauveria bassiana on white grubs in peanut fields is shown in Table 12. When the application concentration was 1.35 trillion spores / hm2, there were no significant differences in the damage index, control effect, yield per mu, and pod damage rate between the treatment with mutagenized strain F5-Bb20240406-6 and the treatment with 30% phoxim suspension concentrate. The damage indices of the treatment with mutagenized strain F5-Bb20240406-6 and the treatment with 30% phoxim suspension concentrate were significantly lower than 3.19% of the treatment with strain Bb20240406 and 4.92% of the water control, being 2.15% and 2.19% respectively; the control effects of the treatment with mutagenized strain F5-Bb20240406-6 and the treatment with 30% phoxim suspension concentrate were 57.08% and 55.49% respectively, which were significantly higher than 35.33% of the treatment with strain Bb20240406; the yields per mu were 2639.74 kg / hm2 and 2607.64 kg / hm2 respectively, which were significantly higher than 2233.58 kg / hm2 of the treatment with strain Bb20240406 and 2003.25 kg / hm2 of the water control; the pod damage rates of the treatment with mutagenized strain F5-Bb20240406-6 and the treatment with 30% phoxim suspension concentrate were significantly lower than 6.98% of the water control, being 3.75% and 4.10% respectively, while there was no significant difference in the pod damage rate between the treatment with strain Bb20240406 and the water control.

[0079] Table 12 Control effect of mutagenized strains on white grubs in peanut fields

[0080] Note: The data in the table are mean ± standard error, and different lowercase letters indicate significant differences at the 0.05 level.

[0081] 2. Field control effect of mutagenized strain F5-Bb20240406-6 on above-ground pests of peanuts The experiment was conducted at the experimental base of Hebei Agricultural University in Baoding, Hebei Province from August 2, 2018 to September 26, 2018 (continuous rainy days). Before spraying, the damage situation of peanut leaves and the population density of aphids were investigated. The solid fermentation material of strain Bb20240406-F5 was obtained by the solid-liquid two-phase fermentation method

[114] , and the content of conidia was determined. The solid fermentation material of the mutagenized strain was weighed according to the low application rate of 1.8 trillion spores / hm2 and the high application rate of 3.6 trillion spores / hm2, and made into a suspension with water containing 0.05% Tween-80. The parental strain Bb20240406 was used as the control, 25 g / L deltamethrin emulsifiable concentrate at 300 mL / hm2 was used as the conventional control, and water containing 0.05% Tween-80 was used as the water control. The water consumption was 600 L / hm2. Each treatment was repeated 3 times, and the area of each plot was 30 m2. It was sprayed on peanut leaves using a sprayer (CD-16B).

[0082] (1)Investigation on the damage situation of peanut leaves The investigation was carried out before the application of pesticides for the base number survey and on the 7th d, 10th d, and 15th d after the application of pesticides. Five points were randomly selected in each plot, five hills of peanuts were selected at each point, and the damage situation of the top five newly compound leaves of each hill of peanuts was investigated. The number of pest-damaged leaves and the damage level were observed and recorded. The grading standard for peanut leaf damage is shown in Table 13.

[0083] Table 13 Grading standard for peanut leaf damage

[0084] The protection effect of the mutagenic strain F5-Bb20240406-6 on peanut leaves is shown in Table 14. The corrected leaf protection effect of the strain application treatment showed an overall upward trend; while the treatment with 2.5% deltamethrin emulsifiable concentrate showed an initial increase and then a decrease, and the maximum corrected leaf protection effect appeared on the 10th d, which was 52.00%. At 7 d after the application of pesticides, with the same dosage, there was no significant difference in the corrected leaf protection effect between the treatment with F5-Bb20240406-6 and Bb20240406, and the highest corrected leaf protection effect of the 2.5% deltamethrin emulsifiable concentrate treatment was 28.32%; at 10 d after the application of pesticides, the corrected leaf protection effects of all treatments showed an upward trend. The corrected leaf protection effect of the 2.5% deltamethrin emulsifiable concentrate treatment was higher than that of the low-dose treatment of Bb20240406, and there was no significant difference from other treatments; at 15 d after the application of pesticides, the corrected leaf protection effects of the strain treatments showed an upward trend. The low-dose treatment of Bb20240406 was lower than that of the 2.5% deltamethrin emulsifiable concentrate, and there was no significant difference between other strain treatments and the 2.5% deltamethrin emulsifiable concentrate treatment.

[0085] Table 14 Leaf protection effect of mutagenic strains on peanuts

[0086] Note: The data in the table are mean ± standard error, and different lowercase letters indicate significant differences at the 0.05 level.

[0087] (2)Investigation on the number of aphids The investigation was carried out before the application of pesticides for the pest population base number survey and on the 1st d, 3rd d, 7th d, 10th d, and 15th d after the application of pesticides. Five points were randomly selected in each plot, ten hills of peanuts were selected at each point, the number of aphids on the top five newly compound leaves was investigated, and hanging tags were marked to observe and record their damage symptoms.

[0088] The control effects of different treatments on peanut aphids are shown in Table 15. Before applying pesticides, there were no significant differences among the various treatments. One day after applying pesticides, the control effect of the 2.5% deltamethrin emulsifiable concentrate treatment was higher than that of the treatment with bacteria application, at 75.91%. Three days after applying pesticides, the control effect of the treatment with bacteria application showed an upward trend, but the control effect of the 2.5% deltamethrin emulsifiable concentrate treatment was still higher than that of the treatment with bacteria application; at high dosages, the control effect of strain Bb20240406-F5 was 56.14%, which was higher than the control effect of strain Bb20240406 at 34.70%. Seven days after applying pesticides, the control effect of the treatment with bacteria application increased. At low dosages of Bb20240406, the control effect was significantly lower than that of other treatments. At high dosages of Bb20240406 and in the Bb20240406-F5 treatment, there were no significant differences in the control effect compared with the deltamethrin emulsifiable concentrate treatment. Ten days after applying pesticides, at both dosages, the control effect of Bb20240406-F5 was significantly higher than that of Bb20240406; both dosages of the Bb20240406-F5 treatment were significantly higher than the deltamethrin emulsifiable concentrate, and the control effect of the high-dosage treatment was the highest, reaching 87.82%. Fifteen days after applying pesticides, at high dosages, the control effect of Bb20240406-F5 was higher than that of the parental strain Bb20240406, and the control effects of the 4 treatments with bacteria application were all higher than that of the 2.5% deltamethrin emulsifiable concentrate treatment.

[0089] Table 15 Reduction rate of peanut aphid population by mutagenized strains

[0090] Note: The data in the table are mean ± standard error, and different lowercase letters indicate significant differences at the 0.05 level.

[0091] (3)Investigation of yield increase effect The investigation was carried out 1 day before peanut harvest. Five random points were taken in each plot, with an area of 1 m2 at each point, and the pod weight was weighed and recorded.

[0092] The yield increase effect of mutagenized strain F5-Bb20240406-6 on peanuts is shown in Table 16. The yield of the high-dosage treatment of strain F5-Bb20240406-6 was 5402.25 kg / hm2, which was higher than that of other treatments with bacteria application and the water control, and there was no significant difference compared with the 2.5% deltamethrin emulsifiable concentrate treatment. The yield increase effect of the 2.5% deltamethrin emulsifiable concentrate treatment was 14.30%, and the yield increase effect of the high-dosage treatment of strain F5-Bb20240406-6 was 18.01%. There was no significant difference between the two, and both were significantly higher than other treatments.

[0093] Table 16 Determination results of peanut yield per mu under different treatments

[0094] Note: The data in the table are mean ± standard error, and different lowercase letters indicate significant differences at the 0.05 level.

Claims

1. A UV-resistant and highly toxic Beauveria bassiana ( Beauveria bassiana ) mutagenic strain F5-Bb20240406-6, characterized in that, The mutagenic strain F5-Bb20240406-6 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 3, 2025, with the deposit number: CGMCC No.41821.

2. Containing the Beauveria bassiana according to claim 1 ( Beauveria bassiana ) The fermentation broth, bacterial suspension or sterile supernatant or preparation thereof of the mutagenized strain F5-Bb20240406-6.

3. The Beauveria bassiana according to claim 1 ( Beauveria bassiana ) Use of the mutagenized strain F5-Bb20240406-6 or the fermentation broth, bacterial suspension or sterile supernatant or preparation thereof as described in claim 2 in preventing and controlling white grubs and aphids.

4. The Beauveria bassiana according to claim 1 ( Beauveria bassiana ) Use of the mutagenic strain F5-Bb20240406-6 or the fermentation broth, bacterial suspension or sterile supernatant or preparation thereof as described in claim 2 in promoting leaf preservation and increased yield of peanut.

5. A bacterial agent for promoting peanut leaf preservation and yield increase, characterized in that: The active ingredient of the bacterial agent includes the Beauveria bassiana according to claim 1 ( Beauveria bassiana ) mutagenized strain F5-Bb20240406-6.

6. Beauveria bassiana as claimed in claim 1 ( Beauveria bassiana ) The use of the mutated strain F5-Bb20240406-6 or the bacterial agent for promoting peanut leaf preservation and increased yield as described in claim 5 in the production of biological organic fertilizer.

7. A biological organic fertilizer, characterized in that: The biological organic fertilizer contains the Beauveria bassiana described in claim 1 ( Beauveria bassiana ) The mutagenic strain F5-Bb20240406-6 or the bacterial agent for promoting leaf preservation and increased yield of peanut as described in claim 5.

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