High-ultraviolet-resistant Beauveria bassiana strain, its method for targeted mutagenesis, and uses thereof

Abstract

This application relates to the field of biotechnology, specifically to a highly UV-resistant Beauveria bassiana strain, its target mutagenesis method and uses. The highly UV-resistant Beauveria bassiana strain of this application has a preservation number of CGMCC No. 22466 and a preservation date of July 5, 2021. The strain is obtained by using the Beauveria bassiana wild strain CGMCC No. 13566 as the starting strain, through multiple repeated stresses by sub-lethal dose radiation simulating solar UVB and target selection, and has the property of high UV resistance. The fungal insecticide prepared by the strain can be successfully used all-weather in the green control of pests.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a highly UV-resistant Beauveria bassiana strain, its targeted mutagenesis method, and uses thereof.

Background Art

[0002] Beauveria bassiana is a broad-spectrum entomopathogenic fungus that mainly reproduces asexually and forms conidia. Currently, Beauveria bassiana conidia are prepared as a formulated fungal insecticide and are used globally for the biological control of pests. In particular, in China, the control of Dendrolimus punctatus and Chilo suppressalis using Beauveria bassiana has become the world's largest pest control project, showing a good sustainable control effect on pests.

[0003] However, the formulated Beauveria bassiana is damaged by sunlight ultraviolet rays during field use, and the stability and sustainability of the pest control effect are often affected. Especially in summer when strong sunlight radiation is frequent and large-scale pest outbreaks occur frequently, this is particularly obvious. Therefore, the radiation of sunlight ultraviolet rays limits the all-weather use of fungal insecticides for the green control of pests. Here, sunlight ultraviolet rays contain two types of harmful radiation components, UVB (290~320nm) and UVA (320~400nm). Fortunately, the shortest and most harmful UVC rays (<290nm) are completely filtered and removed by the atmospheric ozone layer before reaching the earth's surface. Therefore, in sunlight, the damage to formulated fungal cells by UVB ultraviolet radiation is the greatest, and the influence of the longer wavelength UVA is very limited.

[0004] Botryosphaeria bassiana has 1 to 2 photolyases. Usually, the mechanism by which eukaryotic cells repair DNA damage caused by ultraviolet radiation mainly depends on the photoreactivation effect of photolyase located in the cell nucleus. This photolyase can quickly repair DNA damage caused by radiation with visible light, thereby restoring the activity of a large number of damaged cells. Therefore, the expression level of the photolyase gene mainly determines the photorepair ability of fungal cells against DNA damage.

[0005] Currently, there are two methods to improve the expression level of the photolyase gene in fungal cells. First, it is a method of selecting a highly expressed target gene using a foreign resistance molecular marker. The ultraviolet-resistant strains selected by this method belong to recombinant strains because they cannot avoid the foreign resistance gene, so there is a risk of ecological safety. Second, it is a method of mutagenesis and selection without using a foreign resistance molecular marker, which is a method of selecting highly ultraviolet-resistant strains. However, the current resistance of the selected Botryosphaeria bassiana to ultraviolet radiation is limited, and it is difficult to use it well all-weather in the green control of pests.

Summary of the Invention

Problems to be Solved by the Invention

[0006] In order to solve the problem of the low ultraviolet resistance performance of conventional fungal pesticides, this application can select Botryosphaeria bassiana that efficiently expresses the photoreactivation gene. This strain has a strong ultraviolet resistance effect, and the fungal pesticide prepared by this strain can be used well all-weather in the green control of pests.

Means for Solving the Problems

[0007] In a first aspect, this application provides a highly ultraviolet-resistant Botryosphaeria bassiana strain with a preservation number of CGMCC No. 22466 and a preservation date of July 5, 2021.

[0008] The high-UV-tolerant Beauveria bassiana strain of the present application has a 53% higher resistance to UVB radiation in its conidia than the starting strain, a 98-fold increase in the expression level of the main photolyase gene that repairs DNA damage, characteristics related to the remaining possibilities of biological control that are completely consistent with the starting strain, and does not contain any foreign resistance molecular markers with potential ecological safety risks. Therefore, it can be used as a production strain for high-UV-tolerant fungal insecticides and has important application value.

[0009] Preferably, the corrected mortality rate on the 6th day after body wall puncture infection of the high-UV-tolerant Beauveria bassiana strain is ≧50%, and the corrected mortality rate on the 7th day after body wall puncture infection is ≧60%.

[0010] Preferably, the insect attachment rate of the high-UV-tolerant Beauveria bassiana strain is 92.5 - 110.3%.

[0011] In a second aspect, the present application provides a method for targeted mutagenesis of a high-UV-tolerant Beauveria bassiana strain, and the high-UV-tolerant Beauveria bassiana strain is obtained by multiple repeated stresses with a sub-lethal dose of radiation simulating solar UVB and target screening using the wild-type Beauveria bassiana strain CGMCC No. 13566 as the starting strain.

[0012] Preferably, the method for targeted mutagenesis is (1) Using the wild-type Beauveria bassiana strain CGMCC No. 13566 as the starting strain and preparing a spore suspension of its conidia; (2) Uniformly coating the spore suspension on a culture medium plate and irradiating the plate with a sub-lethal dose of UVB in a solar radiation simulation box; (3) Culturing the irradiated surviving spores to grow colonies, selecting the colonies that grow vigorously, transferring them to a spore-forming culture plate, culturing until sufficient sporulation, using the obtained conidia for UVB tolerance measurement, selecting colonies with a further significantly increased UVB tolerance on top of the previous highest colonies, and measuring whether there are significant changes in sporulation and pathogenicity characteristics. (4) Repeating the mutagenesis and screening steps of (1), (2) and (3) until the UVB tolerance of the previous optimal target colony no longer significantly increases in the next round of iterative mutagenesis, and selecting, as the highly UV-resistant Beauveria bassiana strain, the strain showing ideal UVB tolerance in the last round of mutagenesis screening.

[0013] This application uses the wild strain of Beauveria bassiana as the starting strain, has a wide range of material sources, adopts multiple repeated stresses and target screening by sub-lethal dose radiation simulating solar UVB, is more convenient than gene manipulation and editing techniques, and the strain selected thereby is a molecular-directed strain improvement without foreign genes and should be considered as a non-genetically modified strain, and its formulated products do not require additional strict, cumbersome and costly environmental safety assessments.

[0014] Preferably, in step (1), the conidia are dispersed as a spore suspension using sterile water containing 0.01 - 0.06% polysorbate 80.

[0015] By adopting the above technical solution, polysorbate 80 is a hydrophilic surfactant, has a strong effect of disrupting cell membranes and causing irritation. When a low dose of polysorbate 80 is used in this application, it can increase the permeability of the meristematic tissue membrane, promote the occurrence of target mutagenesis in spores, and to a certain extent, improve the selection efficiency of Beauveria bassiana.

[0016] Preferably, in step (2), the medium is Sabouraud medium.

[0017] By adopting the above technical solution, the composition of the Sabouraud medium mainly includes yeast extract, glucose, peptone, and agar, which is a medium commonly used for the isolation and culture of fungi. In this application, by using the Sabouraud medium, the basic nutrients required for the growth of Beauveria bassiana can be sufficiently provided, which is an optimal option based on the purpose of this application to select strains with strong environmental tolerance. There is no need to additionally prepare a special medium, which can reduce the strain selection cost to a certain extent and enable efficient selection of mutant strains with excellent performance.

[0018] Preferably, in step (2), the sublethal dose of UVB is 0.35 - 0.40 J / cm 2 .

[0019] By adopting the above technical solution, about 95% of the conidia die due to the above sublethal dose, and the surviving small number of conidia have strong ultraviolet resistance, effectively improving the selection efficiency of Beauveria bassiana.

[0020] Preferably, the irradiated medium in step (3) is cultured under the conditions of a temperature of 22 - 28°C and a photoperiod of (10 - 14):(10 - 14).

[0021] The irradiation temperature and photoperiod are the main factors affecting the mutation of the strain. The mutagenized strains cultured under the above temperature and photoperiod contribute to selecting the high-ultraviolet-resistant Beauveria bassiana strain described in this application and adapting it to the field use environment.

[0022] Preferably, in the present application, based on the above-mentioned target mutagenesis method, conidia of the colonies selected in step (4) are obtained, the obtained conidia are immersed in a trehalose-ethanol aqueous solution, the spore suspension is uniformly applied to a culture medium plate, the plate is irradiated with a sublethal dose of UVB in a solar simulation radiation box, the surviving spores after irradiation are cultured to grow colonies, colonies that grow actively are selected and transferred to a sporulation culture plate, cultured until sufficient sporulation is achieved, the obtained conidia are used for UVB tolerance measurement, colonies with a further significantly increased UVB tolerance are selected on top of the previous highest colonies, and it is measured whether there are significant changes in sporulation and pathogenicity characteristics. Finally, strains showing ideal UVB tolerance in the last mutagenesis selection are selected as highly UV-resistant Beauveria bassiana strains.

[0023] By adopting the above technical solution, in the present application, the spores produced correspondingly by the strain selected in step (4) are immersed in a trehalose-ethanol aqueous solution. Here, trehalose is used extracellularly, and it can not only effectively improve the UV resistance of the spores after immersion, and promote the spores to germinate rapidly and grow stably at a sublethal dose of UVB, but also promote the grown strains to have a high expression level of the main photolyase gene for repairing DNA damage. This may be because trehalose adheres to the outside of the strain and exerts a good protective effect, leading to the stable expression of the photolyase gene inside the cell. Also, the ethanol among them improves the permeability of the cell wall and cell membrane of the spores to a certain extent, enabling the nutrients in the culture medium to quickly enter the cell and supply the nutrients necessary for the expression of the photolyase gene.

[0024] In a third aspect, the present application provides the use of a highly UV-resistant Beauveria bassiana strain in the preparation of a fungal insecticide that overcomes the common major technical bottleneck of the lack of field stability of conventional fungal insecticides and has good field stability, persistence, and pest control effect.

Brief Description of the Drawings

[0025]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0026] Hereinafter, the present application will be described in more detail with reference to the drawings, examples and comparative examples.

[0027] Raw material supply source All raw materials of this application are products sold on the market. Specifically, refer to Table 1 below.

[0028] Table 1 Supply sources of raw materials of this application

Table 1

[0029] Example 1 A method for targeted mutagenesis of Beauveria bassiana strains with high UV resistance, (1) The Beauveria bassiana wild strain CGMCC No. 13566 (abbreviated as the wild strain) is the starting strain. Conidia produced by normal culture on a Sabouraud medium (SDAY) plate are suspended in sterilized water containing 0.02% polysorbate 80 (allowed to vary in the range of 0.01 - 0.06%) to prepare a suspension of 10 7 spores / mL. (2) Under sterile conditions, 60 μL of the above spore suspension is evenly spread on an SDAY plate in the same amount. After the air is dried for several minutes (drying the applied spore suspension), the plate is placed on the sample stage (12 cm × 16 cm) of the solar ultraviolet radiation simulation box Bio - Sun ++ UV Chamber (Vilber Lourmat, Marne - la - Vallee, France) and irradiated with UVB at a sublethal dose of 0.38 J / cm 2 (allowed to vary in the range of 0.35 - 0.4 J / cm 2 ) so that about 95% of the spores die. (3) Immediately after irradiating the plate coated with spores, cover it and culture under the conditions of a temperature of 25 °C and a photoperiod of 12:12 (allowed to vary within the range of 22 - 28 °C, (10 - 14):(10 - 14)). Select colonies that grow actively until a small number of surviving spores form colonies, transfer them to a new SDAY plate, culture until sufficient sporulation occurs, use the obtained spores for UVB resistance measurement, select colonies with a further significantly increased UVB resistance on top of the previous highest colonies, and measure whether there are significant changes in sporulation and pathogenicity characteristics. (4) Repeat the mutagenesis and selection steps of (1), (2), and (3) until the UVB resistance of the previous optimal target colonies no longer significantly increases in the next round of repeated mutagenesis. Select the mutagenized strain showing ideal UVB resistance in the last round of mutagenesis selection as the highly UV-resistant Beauveria bassiana strain.

[0030] Biological identification of mutagenized strains (colony morphology and microscopic characteristics, measurement of gene sequences)

[0031] In this application, the highly UV-resistant Beauveria bassiana strain selected by the above mutagenesis was sent to the Institute of Microbiology, Chinese Academy of Sciences for biological identification and preservation. The submission date was July 5, 2021, the strain number was TICZJU618 (deposit number at the China Center for Type Culture Collection CGMCC No. 22466), and the identification result was Beauveria bassiana.

[0032] The colony morphology and microscopic characteristics (see Figure 1) are as follows.

[0033] The submitted strain grows rapidly on potato dextrose medium. Under dark conditions at 25 °C for 7 days, the diameter of the colony is 30 - 35 cm, the texture is dense, fibrous, white, slightly raised, the back of the colony is light brown, and no soluble pigment is visible.

[0034] The specialization of the conidiophore stalk is not clear. The sporogenic cells are beaker-shaped, straight or curved, with a size of 6.1 - 35.8 × 1.5 - 2.5 μm. The neck is slender, the tip extends in a zigzag shape, with a width of less than 1 μm, and they exist alone or in clusters. The conidia are broadly elliptical, almost spherical, colorless, with a smooth wall, and a size of 1.5 - 3.0 μm. No sexual sporogenic structure has been confirmed.

[0035] The measurement results of the rRNA gene sequences are as follows.

[0036] It contains the complete sequences of the 18S rRNA fragment, ITS1, 5.8S rRNA, ITS2, and the 28S region sequence fragment, and the sequence is shown in SEQ ID NO: 1.

[0037] Reproducibility of the target mutagenesis method

[0038] According to the target mutagenesis method of Example 1, three experimenters were selected to conduct tests respectively and the above biological identification was carried out. As a result, the colony morphology, microscopic characteristics, and preserved gene sequences of the mutagenized strains obtained by the three experimenters were all consistent. Thus, it can be seen that the target mutagenesis method of this application has reproducibility and the mutagenized strains of this application can be stably selected.

[0039] Performance comparison test between the mutagenized strain and the wild strain

[0040] Select a mutagenized strain showing ideal UVB resistance in the last mutagenesis screening and conduct the following comparative experiment with the wild strain.

[0041] 1. UVB resistance measurement Measurement method: Apply 60 μL of the conidial suspension (10 7 conidia / mL) evenly on an SDAY plate (9 cm in diameter), place it on the sample stage of the above solar ultraviolet radiation simulation box, and expose it to a gradient dose (0.1 - 0.5 J / cm 2) Perform UVB irradiation, cover the irradiated plate after irradiation, and then culture for 24 hours under the conditions of 25 °C and a photoperiod of 12:12. Use the non-irradiated plate as a control. During the culture period, starting from the 4th hour, every 2 hours, detect the total number of spores and the number of germinated spores in three fields of view under a microscope, calculate the spore survival index (value obtained by dividing the percentage by 100), and perform model fitting analysis on the survival index at the gradient dose to obtain the median lethal dose LD of UVB 50 Each dosage experiment is independently repeated 3 times.

[0042] For the detection results, refer to Figures 2A and 2B.

[0043] 2. Measurement of the expression level of the main photolyase gene Measurement method: Apply 100 μL of the conidial suspension of the wild strain and the mutagenized strain in the same amount on the SDAY plate pasted with cellophane, culture for 3 days under the conditions of 25 °C and a photoperiod of 12:12, then harvest the culture, grind it in liquid nitrogen, and use the RNAiso Plus Kit reagent kit (TaKaRa, Dalian, China) to extract the total RNA of each strain. Further, use the PrimeScript RT reagent Kit reagent kit (TaKaRa) to reverse transcribe the RNA into cDNA. Using the obtained cDNA as a template, perform real-time quantitative PCR analysis with the action of SYBR Premix Ex Taq enzyme (TaKaRa) to measure the transcription level of the photolyase gene phr2 in the cDNA of each strain, and use the β-actin gene as an internal reference. The target gene is estimated in the mutagenized strain using the 2 -△△CT method. For each strain, three independent cDNA samples are repeatedly measured.

[0044] For the detection results, refer to Figure 2C.

[0045] 3. Measurement of pathogenicity by injection infection of the body wall and hemocoel Measurement method: Use the 5th instar larvae of Galleria mellonella, a model insect, as the test insects. As the normal body wall infection inoculation method, for every 35 insects, use 40 mL of the spore suspension (107 Soak them in it for 10 seconds at (number of spores per mL). As a method of inoculating by injecting into the hemocoel, for each larva in each group, use microinjection to inject 5 μL of the spore suspension (10 5 (number of spores per mL) into the hemocoel. Then transfer the test insects in each group into a transparent plastic box, and under the conditions of 25°C and a photoperiod of 12:12, observe and record the number of dead and living insects every day, and stop until all have died. Using the same amount of immersion or injection treatment with 0.02% polysorbate 80 solution as a control, calculate the corrected mortality rate every day. Each treatment is repeated 3 times. The obtained time-mortality curve is subjected to model fitting analysis, and the time LT 50 required for 50% death of the test insects of each strain by different inoculation methods is calculated.

[0046] For the detection results, refer to Figure 3A.

[0047] 4. Measuring the adhesion ability of conidia to the insect body surface to determine the success rate of normal body wall infection Measurement method: Take the hind wings of Locusta migratoria manilensis, soak them in a 37% H2O2 aqueous solution for 5 minutes for sterilization, wash them 3 times with sterile water, and then stick them on a 0.7% water agar plate. Drop 5 μL of the spore suspension (10 7 (number of spores per mL) in the same amount at the center of the surface of the hind wings, and evenly spread it with an inoculation loop. Incubate at 25°C for 8 hours, immediately remove the hind wings and place them on a glass slide, observe 3 fields of view under a microscope, and count the number of conidia in each field of view. The observed hind wings are then washed with sterile water for 30 seconds later to remove the spores adhering to the body wall of the hind wings, observe 3 fields of view on the wing surface again under a microscope, and count the remaining number of spores. Calculate the percentage of the number of washed spores to the number of spores on the wing surface before washing, and the adhesion rate of conidia to the body wall of the locust hind wings can be obtained.

[0048] For the detection results, refer to Figure 3B.

[0049] 5. Measurement of the total enzyme activity of the main insect body wall degrading enzymes Measurement method: The conidial suspension of each strain was inoculated into a basic culture medium CDB (3% sucrose, 0.3% NaNO3, 0.1% K2HPO4, 0.05% KCl, 0.05% MgSO4, and 0.001% FeSO4) with 0.3% bovine serum albumin (BSA) as the sole nitrogen source, and the final concentration was 10 4 conidia / mL. It was shaken at 25°C (150 r / min), cultured for 3 days, then filtered to collect the mycelium, dried at 75°C, and the biomass was measured. The supernatant of the culture solution was centrifuged at 13,500×g for 2 minutes at 4°C, and the supernatant was collected as the crude extract and used for the measurement of the total enzyme activities of secreted extracellular enzymes (collectively referred to as extracellular enzymes such as protease, chitinase, lipase, abbreviated as ECE) and Pr1 family protease. For the measurement of the total enzyme activity of extracellular enzymes, 100 μL of a 5 mg / mL azoprotein (dissolved in 50 mM Tris-HCl, pH 8.0) solution was taken and thoroughly mixed with 100 μL of the protein extract denatured by a 15-minute boiling water bath (control group) or the non-denatured protein crude extract (experimental group). After incubation in the dark at 37°C for 1 hour, 400 μL of 10% (w / v) trichloroacetic acid was added to stop the reaction. After centrifugation at 12,000×g for 5 minutes, the supernatant was aspirated and transferred to a new centrifuge tube, and thoroughly mixed with 700 μL of 525 mM NaOH, and the absorbance value (OD 442 ) was read at a wavelength of 442 nm. For the measurement of the total enzyme activity of Pr1 protease, 100 μL of the protein crude extract inactivated by a boiling water bath (control group) or not inactivated was taken and thoroughly mixed with 50 μL of a 1 mM reaction substrate (succinyl-(alanine)2-proline-phenylalanine-p-nitroanilide) and 850 μL of Tris-HCl buffer (15 mM, pH 8.5). It was left at 28°C for 1 hour, 250 μL of 30% (w / v) acetic acid was added to stop the reaction. The reaction system was placed in an ice bath for 15 minutes, centrifuged at 13,000×g for 5 minutes at 4°C, the supernatant was taken, and the absorbance value (OD 410 ) was read at a wavelength of 410 nm. As the enzyme activity unit, OD 442 or OD 410The increment is defined as an increase of 0.01 in the reading value, and the total enzyme activity indicates the number of extracellular enzyme activity units contained in 1 milliliter of the culture supernatant (U / mL).

[0050] Refer to Figure 3C for the detection results.

[0051] 6. Measurement of normal growth and spore formation levels Measurement method: Refer to the spore number measurement method in SB / T 10315-1999 for measurement.

[0052] Refer to Figure 3D for the detection results.

[0053] 7. Observation of spore formation level on the surface of the worm body Measurement method: Visual measurement.

[0054] Refer to Figure 3E for the detection results.

[0055] In summary, in this application, the Beauveria bassiana mutagenized strain obtained by repeated UVB sublethal dose irradiation for mutagenesis has a 53% improvement in the resistance of its conidia to UVB radiation compared to the starting strain (Figure 2B), a significant 98-fold improvement in the expression level of its photolyase gene phr2 (Figure 2C), and no exogenous resistance molecules in its cells, so there is no ecological safety risk.

[0056] Sterilization in the wild is time-consuming and laborious as it involves injecting insects one by one into the hemocoel. Therefore, it is usually carried out using the body wall puncture infection method. Moreover, since the outdoor environment is uncontrollable, the higher the efficiency of body wall puncture infection of Beauveria bassiana, the higher the insect attachment rate, and the better the insecticidal effect of Beauveria bassiana can be achieved. Referring to FIGS. 3A and 3B, the mortality rate of the Beauveria bassiana mutant strain of this example after body wall puncture infection is higher than that of the wild strain within 3 to 7 days. The corrected mortality rate on the 6th day after body wall puncture infection is 52.0% (46.2% for the wild strain), and the corrected mortality rate on the 7th day after body wall puncture infection is 61.3% (57.0% for the wild strain). As can be seen from this, the efficiency of body wall puncture infection of the mutant strain is faster. In terms of the insect attachment rate, the insect attachment rate of the mutant strain is 101.2% (ranging from 94.7 to 109.8% when adding the standard deviation), while the insect attachment rate of the wild strain is 99.0% (ranging from 90.3 to 110.0% when adding the standard deviation). That is, the conidia of the mutant strain have a higher attachment rate and attachment stability on the locust wings than the wild strain.

[0057] In addition, the growth, sporulation, and pathogenicity characteristics of the obtained Beauveria bassiana mutant strain of this example are consistent with those of the wild strain (FIGS. 3C - E).

[0058] Therefore, the mutant strain of Beauveria bassiana of this example can be used as a production strain of an ultraviolet-resistant fungal insecticide to enhance the resistance of the fungal insecticide to ultraviolet radiation and the stability of the field pest control effect. Moreover, since it has excellent body wall puncture infection efficiency and insect attachment rate, it has important application value.

[0059] Example 2 Based on the method of Example 1, this example adjusts the photoperiod from 12:12 to 10:14 and selects the corresponding mutant strain as the target.

[0060] Examples 3 - 4 Examples 3 - 4 are based on Example 1, adjusting the sublethal dose to 0.38 J / cm 2 Specifically, it is adjusted to 0.3 J / cm in Example 3 2and specifically 0.45 J / cm in Example 4 2 and corresponding mutagenized strains are selected as targets.

[0061] According to the performance comparison test process of Example 1 above, the performance of the mutagenized strains and wild strains correspondingly selected in Examples 2 to 4 was measured. As shown from the detection results, the colony morphology and microscopic characteristics of the mutagenized strains selected in Examples 2 to 4 are similar to those of the mutagenized strains selected in Example 1. The resistance of their conidia to UVB radiation is improved by 45%, 46% and 49% in turn compared with the starting strain. The expression levels of the photolyase gene phr2 that repairs DNA damage are improved by 56-fold, 67-fold and 77-fold in turn. The corrected mortality rates on the 6th day after body wall puncture infection are 50.2%, 50.5% and 51.0% in turn, and the corrected mortality rates on the 7th day after body wall puncture infection are 60.0%, 60.2% and 60.6% in turn. The insect attachment rates are 99.1% (when adding the standard deviation, all fall within the range of 92.5 - 110.3%), 100.5% (when adding the standard deviation, all fall within the range of 93.7 - 110.1%) and 100.7% (when adding the standard deviation, all fall within the range of 94.1 - 110.0%) in turn. As can be seen from this, the mutagenized strain obtained in Example 1 has better ultraviolet resistance effect, body wall puncture infection efficiency and insect attachment rate. Therefore, in this application, the target mutagenesis method of Example 1 is regarded as more preferable.

[0062] Example 5 In this example, based on the target mutagenesis method of Example 1, step (5) is further set. Specifically, The conidia of the colonies selected in step (4) are harvested, and the obtained conidia are immersed in a trehalose - ethanol aqueous solution for 10 - 15 minutes. The concentration of trehalose is 0.3 - 0.6 g / L, and the volume concentration of ethanol in the ethanol aqueous solution is 5 - 10%. Within the above range, the effect of treating the spores is similar. If it exceeds the above range, the growth of the harvested mutagenized strain is poor. In this example, specifically, it is immersed in a 0.5 g / L trehalose - 8% ethanol aqueous solution for 10 minutes to prepare a spore suspension, and Using 60 μL of the above spore suspension, uniformly coat an equal amount on an SDAY plate. After the air has dried for several minutes (drying the applied spore suspension), place the plate on the sample stage (12 cm × 16 cm) of a solar ultraviolet radiation simulation box Bio-Sun ++ UV Chamber (Vilber Lourmat, Marne-la-Vallee, France) and perform a step of irradiating with UVB at a sublethal dose of 0.38 J / cm 2 (allowed to vary in the range of 0.35 - 0.4 J / cm 2 ), and Immediately after irradiating the plate coated with spores, cover it and culture under the conditions of a temperature of 25 °C and a photoperiod of 12:12 (allowed to vary in the range of 22 - 28 °C, (10 - 14):(10 - 14)). Select actively growing colonies and transfer them to a new SDAY plate until surviving spores form colonies. Culture until sufficient sporulation occurs. Use the obtained spores for UVB tolerance measurement, select colonies with a further significantly increased UVB tolerance on top of the previous highest colonies, and measure whether there are significant changes in sporulation and pathogenicity characteristics. Selecting a mutagenized strain showing ideal UVB tolerance in the last mutagenesis screening as a highly UV-resistant Beauveria bassiana strain.

[0063] According to the performance comparison test process of Example 1 above, the performance of the mutagenized strain and the wild strain selected correspondingly in Example 5 was measured. As shown by the detection results, the colony morphology and microscopic characteristics of the mutagenized strain selected in Example 5 are similar to those of the mutagenized strain selected in Example 1. The resistance of its conidia to UVB radiation is 61% (53% in Example 1) higher than that of the starting strain. The expression level of the photolyase gene phr2 that repairs DNA damage is increased by 112 times (98 times in Example 1). The corrected mortality rate on the 6th day after body wall puncture infection is 54.3% (52.0% in Example 1), and the corrected mortality rate on the 7th day after body wall puncture infection is 62.5% (61.3% in Example 1). The insect attachment rate is 101.0% (when the standard deviation is added, all fall within the range of 96.0 - 107.2%) (101.2% in Example 1 (when the standard deviation is added, it falls within the range of 94.7 - 109.8%)).

[0064] As can be seen from this, after the mutagenized strain selected by the target mutagenesis method of Example 1 is further immersed in a trehalose - ethanol aqueous solution and the generated spores are cultured, a mutagenized strain with more excellent UV resistance effect, body wall puncture infection efficiency and insect attachment rate can be obtained. Therefore, this application further prefers the target mutagenesis method of Example 5.

[0065] This specific example is only an illustration of this application and does not limit this application. After reading this specification, those skilled in the art can make corrections to this example without creative contributions as needed, but as long as it is within the scope of the claims of this application, it will all be protected by the patent law.

Claims

1. A Beauveria bassiana strain with high UV resistance, with a deposit number of CGMCC No. 22466 and a deposit date of July 5, 2021 The Beauveria bassiana strain with high UV resistance is characterized by the above.

2. The Beauveria bassiana strain with high UV resistance has a corrected mortality rate of ≥ 50% on the 6th day after body wall puncture infection and a corrected mortality rate of ≥ 60% on the 7th day after body wall puncture infection The Beauveria bassiana strain with high UV resistance according to claim 1, characterized by the above.

3. The insect attachment rate of the Beauveria bassiana strain with high UV resistance is 92.5 - 110.3% The Beauveria bassiana strain with high UV resistance according to claim 1, characterized by the above.

4. A method for targeted mutagenesis of the Beauveria bassiana strain with high UV resistance according to claim 1, wherein the Beauveria bassiana strain with high UV resistance is obtained by using the wild Beauveria bassiana strain CGMCC No. 13566 as the starting strain and through multiple repeated stresses by sub - lethal dose radiation simulating solar UVB and target selection. The method for targeted mutagenesis is characterized by the above.

5. (1) A step of using the wild Beauveria bassiana strain CGMCC No. 13566 as the starting strain and preparing a spore suspension of its conidia; (2) A step of uniformly applying the spore suspension onto a culture medium plate and irradiating the plate with a sub - lethal dose of UVB in a solar radiation simulation box; (3) A step of culturing the irradiated individual surviving spores to grow colonies, selecting the actively growing colonies and transferring them to a spore - forming culture plate, culturing until sufficient spore formation, using the obtained conidia for UVB resistance measurement, selecting colonies with a further significantly increased UVB resistance on top of the previous highest colonies, and measuring whether there are significant changes in spore formation and pathogenicity characteristics; (4) A step of repeating the mutagenesis and selection steps of (1), (2) and (3) until the UVB resistance of the previous optimal target colonies no longer significantly increases in the next repeated mutagenesis, and selecting a strain showing ideal UVB resistance in the last mutagenesis selection as the Beauveria bassiana strain with high UV resistance. The method for targeted mutagenesis according to claim 4, characterized by the above.

6. In step (1), the conidia are dispersed as a spore suspension using sterile water containing 0.01 - 0.06% polysorbate 80 The method for targeted mutagenesis according to claim 4, characterized by the above.

7. In the project (2), the sub-lethal dose of UVB is 0.35 to 0.40 J / cm ２ is The method for targeted mutagenesis according to claim 4, characterized in that...

8. Culturing the medium irradiated in step (3) under the conditions of a temperature of 22 to 28 °C and a photoperiod of (10 to 14):(10 to 14). The method for targeted mutagenesis according to claim 4, characterized in that...

9. Obtaining conidia of the colonies selected in step (4), immersing the obtained conidia in an aqueous solution of trehalose and ethanol, uniformly applying the spore suspension to a culture medium plate, irradiating the plate with a sublethal dose of UVB in a solar simulation radiation box, culturing the surviving spores irradiated, growing colonies, selecting the colonies that grow actively and transferring them to a spore formation culture plate, culturing until sufficient spore formation, using the obtained conidia for UVB resistance measurement, screening for colonies with a further significantly increased UVB resistance on top of the previous highest colonies, measuring whether there are significant changes in spore formation and pathogenicity characteristics, and selecting a strain showing ideal UVB resistance in the last mutagenesis screening as a highly UV-resistant Beauveria bassiana strain. The method for targeted mutagenesis according to claim 4, characterized in that...

10. Use of the highly UV-resistant Beauveria bassiana strain according to any one of claims 1 to 3 or the strain obtained by the method for targeted mutagenesis according to any one of claims 4 to 9 in the preparation of a fungal insecticide.

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