High UV-resistant Beauveria bassiana strain, its targeted mutagenesis method, and uses

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 biotechnology field, specifically to a highly UV-resistant strain of Beauveria bassiana, a method for targeting its mutagenesis, and its applications. [Background technology]

[0002] Beauveria bassiana is a broad-spectrum entomopathogenic fungus that primarily reproduces asexually, forming conidia. Currently, Beauveria bassiana conidia are prepared as fungal insecticides and used worldwide for biological control of pests. In China in particular, the use of Beauveria bassiana to control Mason pine caterpillars and corn borers has become the world's largest pest control project, demonstrating excellent and sustained pest control effects.

[0003] However, the stability and duration of the pest control effect of formulated Beauveria bassiana are often affected by damage from sunlight ultraviolet radiation during use in fields, which is particularly noticeable in summer when solar radiation is strong and pest outbreaks frequently occur. Therefore, sunlight ultraviolet radiation limits the all-weather use of this fungal insecticide for green pest control. Here, sunlight ultraviolet radiation contains two types of harmful radioactive components: UVB (290-320 nm) and UVA (320-400 nm). Fortunately, the shortest and most harmful UVC rays (<290 nm) are completely filtered and removed by the atmospheric ozone layer before reaching the Earth's surface. Thus, in sunlight, damage to formulated fungal cells by UVB ultraviolet radiation is greatest, while the effects of longer wavelength UVA are very limited.

[0004] Beauveria bassiana possesses 1-2 photolyases. Normally, the mechanism by which eukaryotic cells repair DNA damage caused by ultraviolet radiation relies primarily on the photorepair action of photolyases located in the cell nucleus. These photolyases rapidly repair radiation-induced DNA damage with visible light, thereby restoring activity to a large number of damaged cells. Therefore, the expression level of photolyase genes primarily determines the photorepair capability of fungal cells against DNA damage.

[0005] Currently, there are two methods to improve the expression level of photolyase genes in fungal cells. First, there is a method of selecting highly expressed target genes using foreign resistance molecular markers. However, UV-resistant strains selected in this way are genetically modified strains because they cannot avoid foreign resistance genes, and therefore pose an ecological safety risk. Second, there is a method of mutagenesis and selection that does not use foreign resistance molecular markers, which is a method for selecting highly UV-resistant strains. However, the UV radiation resistance of currently selected Beauveria bassiana is limited, making it difficult to use effectively in all weather conditions for green pest control. [Overview of the project] [Problems that the invention aims to solve]

[0006] To address the problem of the low UV resistance of conventional fungal insecticides, this application allows for the selection of Beauveria bassiana that efficiently expresses photodegradation genes. This strain exhibits strong UV resistance, and the fungal insecticide prepared using this strain can be effectively used in all weather conditions for green pest control. [Means for solving the problem]

[0007] In a first aspect, the present application provides a highly UV-resistant strain of Beauveria bassiana with storage number CGMCC No. 22466 and storage date July 5, 2021.

[0008] The highly UV-resistant Beauveria bassiana strain of this application exhibits 53% greater resistance to UVB radiation in its conidia compared to the starting strain, a 98-fold increase in the expression level of key photolyase genes that repair DNA damage, and completely matches the characteristics of the starting strain in other areas related to biological control potential. Furthermore, it contains no exogenous resistance molecular markers that pose a potential ecological safety risk, making it suitable for use as a production strain for highly UV-resistant fungal insecticides and thus possessing significant application value.

[0009] Preferably, the highly UV-resistant Beauveria bassiana strain has an adjusted mortality rate of ≥50% on day 6 after wall puncture infection and an adjusted mortality rate of ≥60% on day 7 after wall puncture infection.

[0010] Preferably, the insect infestation rate of the highly UV-resistant Beauveria bassiana strain is 92.5 to 110.3%.

[0011] In a second aspect, the present application provides a method for targeted mutagenesis of a highly UV-resistant Beauveria bassiana strain, the said highly UV-resistant Beauveria bassiana strain is obtained by using the wild-type Beauveria bassiana strain CGMCC No. 13566 as the starting strain and undergoing repeated stress by sublethal radiation simulating sunlight UVB and target selection.

[0012] Preferably, the targeted mutagenesis method is (1) Using wild strain Beauveria bassiana CGMCC No. 13566 as the starting strain, the process involves preparing a spore suspension of its conidia, (2) A step of uniformly spreading the spore suspension onto a culture plate and irradiating the plate with a sublethal dose of UVB in a sunlight-simulating radiation box, (3) The process involves culturing the released surviving spores to grow colonies, selecting actively growing colonies and transferring them to a spore-forming culture plate, culturing them until sufficient spore formation occurs, using the obtained conidia for UVB resistance measurement, selecting colonies that show a further significant increase in UVB resistance above the previous best colony, and measuring whether there are any significant changes in spore formation and pathogenicity characteristics. (4) The process includes repeating the mutagenesis and selection steps of (1), (2), and (3) until the UVB resistance of the previously optimal target colony no longer significantly increases in the next mutagenesis, and selecting a strain that exhibits ideal UVB resistance in the final mutagenesis and selection as a highly UV-resistant Beauveria bassiana strain.

[0013] This application uses wild-type Beauveria bassiana as the starting strain, has a wide range of material sources, employs multiple repeated stresses and target selection using sublethal doses of radiation simulating sunlight UVB, is more convenient than genetic manipulation or editing techniques, and the strains selected by this method are molecularly targeted strains that do not contain foreign genes and should be considered as non-genetically modified strains, and the resulting formulations do not require additional rigorous, complicated, and expensive 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 employing the above technical solution, polysorbate 80, being a hydrophilic surfactant, has a strong effect of disrupting cell membranes and causing irritation. In this application, using a low dose of polysorbate 80 increases the permeability of the meristem membrane, promoting targeted mutagenesis in spores and, to some extent, improving the selection efficiency of Beauveria bassiana.

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

[0017] By adopting the above technical solution, the formulation of the Sabouraud medium mainly consists of 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 nutritional components necessary for the growth of Beauveria bassiana can be sufficiently provided, which is the optimal choice based on the purpose of this application to select strains with strong environmental tolerance. There is no need to additionally prepare a dedicated medium, which can reduce the strain selection cost to a certain extent and enable the 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 is.

[0019] By adopting the above technical solution, about 95% of the conidia die due to the above sublethal dose, and the surviving minority 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 mutant 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, using the above-described targeted mutagenesis method, conidia are obtained from the colonies selected in step (4), the obtained conidia are immersed in a trehalose-ethanol aqueous solution, the spore suspension is uniformly spread onto a culture plate, the plate is irradiated with a sublethal dose of UVB in a sunlight-simulating radiation box, the irradiated surviving spores are cultured to grow colonies, actively growing colonies are selected and transferred to a spore-forming culture plate, and cultured until sufficient spore formation occurs. The obtained conidia are used for UVB resistance measurement, colonies showing a further significant increase in UVB resistance above the previous best colony are selected, and whether there are significant changes in spore formation and pathogenicity characteristics is measured. In the final mutagenesis selection, a strain showing ideal UVB resistance is selected as a highly UV-resistant Beauveria bassiana strain.

[0023] By adopting the above technical solution, in this application, the spores produced by the selected strain in step (4) are immersed in a trehalose-ethanol aqueous solution. Herein, the trehalose is used extraspores, but it effectively improves the ultraviolet resistance of the spores after immersion, promoting rapid germination and stable growth of the spores at sublethal doses of UVB. Furthermore, it can also promote the grown strain to have high expression levels of the major photolyase genes that repair DNA damage. This is because the trehalose adheres to the outside of the strain and exerts a good protective effect, which may lead to the stable expression of photolyase genes within the cells. In addition, the ethanol improves the permeability of the spore cell wall and cell membrane to some extent, allowing nutrients in the culture medium to rapidly enter the cells and supply the nutrients necessary for the expression of photolyase genes.

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

[0025] [Figure 1] Figure 1 shows the colony morphology and microscopic characteristics of the UV-resistant mutagenic strain of the present application. [Figure 2] Figure 2 is a comparison of UVB resistance between the wild-type and UV-resistant mutagenesis strains of Beauveria bassiana according to this application, where (A) is the conidial survival trend with respect to changes in UVB radiation dose, (B) is the UVB radiation dose LD50 required for 50% spore inactivation calculated from the radiation dose-spore survival index fitting curve, and (C) is the expression level of the photolyase gene phr2 in the mutagenesis strain compared to the wild-type strain. Error bars represent the standard deviation (SD) of the mean of three replicate experiments. [Figure 3] Figure 3 shows a comparison of the toxicity and spore formation characteristics of wild-type and UV-resistant mutagenic strains of Beauveria bassiana, where (A) is the adjusted mortality trend and time LT50 (50% mortality) required for 50% mortality in Galleria melonella 5th instar larvae to enter the body wall via nonconductive inoculation (NCI) and single-headed Hemocoel injection (CBI). (B) is a comparison of the attachment rate of conidia to the hindwing body wall of grasshoppers, which is necessary for the initiation of NCI. (C) is a comparison of biomass in CDB-BSA cultures and the total enzyme activity in the culture supernatant of extracellular enzymes (ECEs) and body wall-degrading enzymes such as Pr1 family proteases required for NCI to successfully penetrate the insect body wall. (D) is a comparison of conidial production during the normal culture period after inoculation using spore suspension on Sabolow agar (SDAY) plates. (D) shows that the growth levels on the body surface of fresh dead Galleria melonella larvae and those grown 10 days after death were identical between the mutagenic strain and the wild-type strain. The error bars represent the standard deviation (SD) of the mean of three replicated experiments, and it should be noted that there were no significant differences in any of the observed characteristics between the mutagenic strain and the wild-type strain. [Modes for carrying out the invention]

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

[0027] Source of raw materials The raw materials used in this application are all commercially available products; please refer to Table 1 below for specific details.

[0028] Table 1. Sources of raw materials for this application [Table 1]

[0029] Example 1 A method for targeted mutagenesis of highly UV-resistant Beauveria bassiana strains, (1) Beauveria bassiana wild strain CGMCC No. 13566 (abbreviated as wild strain) is the starting strain. The conidia produced by normal culture on Sabolow agar (SDAY) plates are suspended in sterile water containing 0.02% polysorbate 80 (to which variation in the range of 0.01-0.06% is acceptable) and 10 7 The process involves preparing a suspension of individual spores / mL, (2) The process includes the step of uniformly spreading 60 μL of the above spore suspension onto an SDAY plate under sterile conditions. After the air has dried for several minutes (to dry the spread spore suspension), the plate is placed in a Bio-Sun UV radiation simulation box. ++ The sample was placed on a 12cm x 16cm sample stage in a UV Chamber (Vilber Lourmat, Marne-la-Vallee, France) and measured a sublethal dose of 0.38 J / cm³. 2 (0.35~0.4J / cm 2 The process involves emitting UVB radiation (which is allowed to vary within a certain range), thereby killing about 95% of the spores. (3) Immediately after irradiating the plate coated with spores, the plate is covered and cultured under conditions of a temperature of 25°C and a photoperiod of 12:12 (allowed to fluctuate within the range of 22-28°C, (10-14):(10-14)). Until a small number of surviving spores form colonies, actively growing colonies are selected and transferred to a new SDAY plate, where they are cultured until sufficient spore formation occurs. The obtained spores are used for UVB resistance measurement, and colonies showing a further significant increase in UVB resistance above the previous best colony are selected. The process also involves measuring whether there are significant changes in spore formation and pathogenicity characteristics. (4) The process includes repeating the mutagenesis and selection steps of (1), (2), and (3) until the UVB resistance of the previously optimal target colony no longer significantly increases in the next mutagenesis, and selecting a mutagenesis strain that exhibits ideal UVB resistance in the final mutagenesis and selection as a highly UV-resistant Beauveria bassiana strain.

[0030] Biological identification of mutagenic strains (measurement of colony morphology and microscopic characteristics, and gene sequence).

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

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

[0033] The test strain grew rapidly on potato glucose medium, reaching a colony diameter of 30-35 cm in 7 days under dark conditions at 25°C. The colonies were dense, fibrous, white, slightly raised, with a light brown underside and no soluble pigments observed.

[0034] The specialization of the conidiophores is not clear. The spore-forming cells are beaker-shaped, linear or curved, measuring 6.1–35.8 × 1.5–2.5 μm, with an elongated neck and a curved tip, less than 1 μm wide, existing individually or in clusters. The conidia are broadly elliptical to nearly spherical, colorless, with smooth walls, measuring 1.5–3.0 μm, and no sexual spore-forming structures have been observed.

[0035] The results of the rRNA gene sequence measurement are as follows:

[0036] The complete sequences of 18S rRNA, ITS1, 5.8S rRNA, and ITS2, as well as a 28S region sequence fragment, are shown in Sequence ID No. 1.

[0037] Reproducibility of targeted mutagenesis

[0038] Following the targeted mutagenesis method of Example 1, three experimenters were selected and tested, and the above-mentioned biological analysis was performed. The bacterial morphology, microscopic characteristics, and conserved gene sequences of the mutagenesis strains obtained by the three experimenters were all identical. As can be seen from this, the targeted mutagenesis method of this application is reproducible and can be used to stably select the mutagenesis strains of this application.

[0039] Performance comparison test between mutagenic strain and wild-type strain

[0040] In the final mutagenesis selection, we will select a mutagenesis strain that exhibits ideal UVB resistance and conduct the following comparative experiment with the wild-type strain.

[0041] 1. UVB resistance measurement Measurement method: 60 μL of conidia suspension (10 7 The same amount of spores (0.1~0.5 J / cm²) was spread onto an SDAY plate (9 cm in diameter), and the sample stage of the solar ultraviolet radiation simulation box was placed to measure the gradient dose (0.1~0.5 J / cm²). 2Perform UVB irradiation of ), 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 50 of UVB. Each dose experiment is repeated independently three 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 using the -△△CT method with respect to the expression level in the wild strain in the mutagenized strain. For each strain, measure three independent cDNA samples repeatedly.

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

[0045] 3. Measurement of pathogenicity by injection infection of the body wall and hemolymph 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 Immerse in (10 spores / mL) for 10 seconds. As a blood-injection inoculation method, microinject 5 μL of spore suspension (10 5 Inject (100 pylori / mL) into Hemocoel. Then, transfer each group of test insects into a transparent plastic box and, under conditions of 25°C and a photoperiod of 12:12, observe and record the number of dead and living insects daily, stopping until all insects have died. Calculate the corrected mortality rate for each day using equal volume immersion or injection treatment with 0.02% polysorbate 80 as a control. Each treatment is repeated three times. The obtained time-mortality curves are model-fitted and analyzed to determine the time (LT) required for 50% mortality of test insects for each strain by different inoculation methods. 50 Calculate the value.

[0046] See Figure 3A for the detection results.

[0047] 4. Measuring the ability of conidia to adhere to the insect body surface to determine the success rate of infection of normal body walls. Measurement method: Remove the hindwings of the migratory locust (Locusta migratoria manilensis), sterilize by immersion in a 37% H2O2 aqueous solution for 5 minutes, wash three times with sterile water, and then mount on a 0.7% agar plate. 5 μL of spore suspension (10 7 Apply an equal amount of (1 spores / mL) to the center of the surface of the hindwing and evenly coat it with the inoculation loop. Incubate at 25°C for 8 hours, then immediately remove the hindwing and place it on a glass slide. Observe three fields of view under a microscope and count the number of conidia in each field of view. The observed hindwing is then washed with sterile water for 30 seconds to remove spores attached to the hindwing body wall, and again observe three fields of view of the wing surface under a microscope and count the number of remaining spores. Calculating the percentage of spores attached to the hindwing body wall of the grasshopper from the number of spores on the wing surface before washing gives the percentage of conidia attached to the grasshopper hindwing body wall.

[0048] See Figure 3B for the detection results.

[0049] 5. Measurement of total enzyme activity of body wall-degrading enzymes in major insects. Measurement method: Conidial suspensions of each bacterial strain were 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 measured at 10 4 The concentration is 1 conidia / mL. After shaking at 25°C (150 r / min) and culturing for 3 days, the mycelium is collected by filtration, dried at 75°C, and the biomass is measured. The supernatant of the culture medium is centrifuged at 13,500 × g for 2 minutes at 4°C, and the supernatant is collected as a crude extract. This is used to measure the total enzyme activity of secreted extracellular enzymes (a general term for proteases, chitinases, lipases, etc., abbreviated as ECE) and Pr1 family proteases. For the measurement of total extracellular enzyme activity, 100 μL of a 5 mg / mL azoprotein solution (dissolved in 50 mM Tris-HCl, pH 8.0) was taken and thoroughly mixed with 100 μL of either a protein extract denatured by a boiling water bath for 15 minutes (control group) or an undenatured crude protein extract (experimental group). After incubation at 37°C in the dark for 1 hour, the reaction was stopped by adding 400 μL of 10% (w / v) trichloroacetic acid. After centrifugation at 12,000 × g for 5 minutes, the supernatant was aspirated and transferred to a new centrifuge tube. This was thoroughly mixed with 700 μL of 525 mM NaOH, and the absorbance value (OD) was measured at a wavelength of 442 nm. 442 ) is read. For the measurement of total Pr1 protease enzyme activity, take 100 μL of boiling water bath inactivated (control group) or uninactivated crude protein extract, mix thoroughly and homogeneously with 50 μL of 1 mM reaction substrate (succinyl-(alanine)2-proline-phenylalanine-p-nitroanilide) and 850 μL of Tris-HCl buffer (15 mM, pH 8.5), stand at 28°C for 1 hour, add 250 μL of 30% (w / v) acetic acid to stop the reaction. Ice bath the reaction system for 15 minutes, centrifuge at 13,000 × g for 5 minutes at 4°C, take the supernatant and measure the absorbance value (OD) at a wavelength of 410 nm. 410 ) is read. As for enzyme activity units, the reaction period is OD 442 or OD 410The reading is defined as an increment of 0.01, and the total enzyme activity represents the number of extracellular enzyme activity units contained in 1 ml of culture supernatant (U / mL).

[0050] See Figure 3C for the detection results.

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

[0052] See Figure 3D for the detection results.

[0053] 7. Observation of spore production levels on the insect body surface. Measurement method: Visual measurement.

[0054] See Figure 3E for the detection results.

[0055] In summary, this application describes how mutagenic strains of Beauveria bassiana, obtained by repeated sublethal UVB radiation, exhibit a 53% improvement in conidial resistance to UVB radiation compared to the starting strain (Figure 2B), a significant 98-fold increase in the expression level of the photolyase gene phr2 (Figure 2C), and the complete absence of exogenous resistance molecules within their cells, thus posing no ecological safety risk.

[0056] In outdoor sterilization, injecting insects one by one into Hemocoel is time-consuming and labor-intensive, so it is usually carried out using a body wall puncture infection method. Furthermore, since the outdoor environment is uncontrollable, the faster the efficiency of body wall puncture infection with Beauveria bassiana, the higher the insect attachment rate and the better the insecticidal effect on Beauveria bassiana. Referring to Figures 3A and 3B, the Beauveria bassiana mutagenesis strain in this example has a higher mortality rate from body wall puncture infection at 3-7 days compared to the wild type, with an adjusted mortality rate of 52.0% on day 6 after body wall puncture infection (compared to 46.2% for the wild type) and an adjusted mortality rate of 61.3% on day 7 after body wall puncture infection (compared to 57.0% for the wild type). As can be seen from this, the efficiency of body wall puncture infection in the mutagenesis strain is faster. In terms of insect attachment rate, the mutagenetic strain had an attachment rate of 101.2% (with the standard deviation added, it fell within the range of 94.7-109.8%), while the wild-type strain had an attachment rate of 99.0% (with the standard deviation added, it fell within the range of 90.3-110.0%). In other words, the conidia of the mutagenetic strain had a higher attachment rate and attachment stability on grasshopper wings than the wild-type strain.

[0057] In addition, the growth, spore formation, and pathogenicity characteristics of the Beauveria bassiana mutagenesis strains obtained in this embodiment are consistent with those of the wild-type strain (Figure 3C-E).

[0058] Therefore, the mutagenetic strain of Beauveria bassiana in this embodiment has significant application value as a production strain of UV-resistant fungal insecticide, as it can enhance the resistance of fungal insecticides to UV radiation and the stability of their field pest control effect, and also possesses excellent body wall puncture infection efficiency and insect attachment rate.

[0059] Example 2 This embodiment is based on the method of Example 1, but adjusts the photoperiod from 12:12 to 10:14 and selects the corresponding mutagenic strain as the target.

[0060] Examples 3-4 Examples 3 and 4 were based on Example 1, with a sublethal dose of 0.38 J / cm³. 2 Adjust the value to specifically 0.3 J / cm² in Example 3. 2In Example 4, the specific value is 0.45 J / cm². 2 Therefore, the corresponding mutagenic strains are targeted and selected.

[0061] Following the performance comparison test procedure of Example 1 described above, the performance of the mutagenic strains and wild-type strains selected in Examples 2-4 was measured. As shown in the detection results, the colony morphology and microscopic characteristics of the mutagenic strains selected in Examples 2-4 and the mutagenic strain selected in Example 1 were similar. Their conidia's resistance to UVB radiation improved by 45%, 46%, and 49% respectively compared to the starting strain, and the expression level of the photolyase gene phr2, which repairs DNA damage, improved by 56, 67, and 77 times, respectively. The corrected mortality rates on day 6 after wall puncture infection were 50.2%, 50.5%, and 51.0%, respectively, and the corrected mortality rates on day 7 after wall puncture infection were 60.0%, 60.2%, and 60.6%, respectively. The parasite attachment rates were 99.1% (all within the range of 92.5-110.3% when standard deviation is added), 100.5% (all within the range of 93.7-110.1% when standard deviation is added), and 100.7% (all within the range of 94.1-110.0% when standard deviation is added). As can be seen from this, the mutagenic strain obtained in Example 1 has superior UV resistance, wall puncture infection efficiency, and parasite attachment rate, therefore, in this application, the targeted mutagenic method of Example 1 is preferred.

[0062] Example 5 This embodiment is based on the targeted mutagenesis method of Example 1, with step (5) further defined, and specifically, In step (4), conidia are harvested from the selected colonies, and the obtained conidia are immersed in a trehalose-ethanol aqueous solution for 10-15 minutes, with a trehalose concentration of 0.3-0.6 g / L and a volume concentration of ethanol in the ethanol aqueous solution of 5-10%. Within this range, the effect of treating the spores is close, and if it exceeds this range, the growth of the harvested mutagenic strain is poor. In this example, specifically, a 0.5 g / L trehalose-8% ethanol aqueous solution is used to prepare a spore suspension by immersing the conidia for 10 minutes. Apply 60 μL of the above spore suspension uniformly to the SDAY plate, allow the air to dry for several minutes (to dry the applied spore suspension), and then place the plate in the Bio-Sun solar ultraviolet radiation simulation box. ++ The sample was placed on a 12cm x 16cm sample stage in a UV Chamber (Vilber Lourmat, Marne-la-Vallee, France) and measured a sublethal dose of 0.38 J / cm³. 2 (0.35~0.4J / cm 2 A process of emitting UVB radiation (to be allowed to vary within the range), Immediately after irradiating the plate coated with spores, the plate is covered and cultured under conditions of 25°C and a photoperiod of 12:12 (allowed to fluctuate within the range of 22-28°C, (10-14):(10-14)). Until the surviving spores form colonies, actively growing colonies are selected and transferred to a new SDAY plate, where they are cultured until sufficient spore formation occurs. The obtained spores are then used for UVB resistance measurement, and colonies showing a further significant increase in UVB resistance above the previous best colony are selected. The process also involves measuring whether there are significant changes in spore formation and pathogenicity characteristics. The process includes selecting a mutagenesis strain that exhibits ideal UVB resistance in the final mutagenesis selection as a highly UV-resistant Beauveria bassiana strain.

[0063] Following the performance comparison test procedure of Example 1 described above, the performance of the mutagenic strains and wild-type strains selected in Example 5 was measured. As shown in the detection results, the colony morphology and microscopic characteristics of the mutagenic strains selected in Example 5 and Example 1 were similar, and the conidial resistance to UVB radiation was 61% higher than that of the starting strain (53% higher in Example 1), as was the expression level of the photolyase gene phr2, which repairs DNA damage. The improvement was 112 times (98 times in Example 1), the adjusted mortality rate on day 6 after wall puncture infection was 54.3% (52.0% in Example 1), the adjusted mortality rate on day 7 after wall puncture infection was 62.5% (61.3% in Example 1), and the parasite attachment rate was 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, by further immersing the mutagenic strains selected by the targeted mutagenic method of Example 1 in a trehalose-ethanol aqueous solution and culturing the resulting spores, it is possible to obtain mutagenic strains with superior UV resistance, body wall puncture infection efficiency, and insect attachment rate. Therefore, this application further favors the targeted mutagenic method of Example 5.

[0065] This specific embodiment is merely a description of the present application and does not limit it. Those skilled in the art may, after reading this specification, make amendments to this embodiment as necessary, without making any creative contribution, but all such amendments will be protected under patent law as long as they are within the scope of the claims of this application.

Claims

1. This is a highly UV-resistant strain of Beauveria bassiana. The storage number is CGMCC No. 22466, and the storage date is July 5, 2021. A highly UV-resistant strain of Beauveria bassiana characterized by these features.

2. The aforementioned highly UV-resistant Beauveria bassiana strain exhibits an adjusted mortality rate of ≥50% on day 6 after wall puncture infection and an adjusted mortality rate of ≥60% on day 7 after wall puncture infection. The highly UV-resistant Beauveria bassiana strain according to feature 1.

3. The insect infestation rate of the aforementioned highly UV-resistant Beauveria bassiana strain is 92.5-110.3%. The highly UV-resistant Beauveria bassiana strain according to feature 1.

4. A method for targeting mutagenesis of a highly UV-resistant Beauveria bassiana strain according to claim 1, (1) Using wild strain Beauveria bassiana CGMCC No. 13566 as the starting strain, the conidia produced by normal culture on a Saboro medium (SDAY) plate are suspended in sterile water containing 0.02% polysorbate 80 to prepare a spore suspension, (2) Under sterile conditions, 60 μL of the above spore suspension is uniformly spread onto an SDAY plate in the same amount, dried, and then subjected to a sublethal dose of 0.38 J / cm² in a sunlight-simulating radiation box. ２ The process involves emitting UVB radiation, (3) Immediately after irradiating the plate coated with spores, the lid is attached and the cells are cultured at a temperature of 25°C and a photoperiod of 12:12 to grow colonies. Actively growing colonies are selected and transferred to a spore-forming culture plate, where they are cultured until sufficient spores are formed. The obtained conidia are used for UVB resistance measurement, and colonies with even more significant increases in UVB resistance are selected above the previous best colony. The process also involves measuring whether there are any significant changes in spore formation and pathogenicity characteristics. A targeted mutagenesis method characterized by comprising: (4) repeating the mutagenesis and selection steps (1), (2), and (3) until the UVB resistance of the previously optimal target colony no longer significantly increases in the next repeated mutagenesis, and selecting a mutagenesis strain that exhibits ideal UVB resistance in the final mutagenesis selection as a highly UV-resistant Beauveria bassiana strain.

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

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