WO2023202608A1 - 高抗紫外辐射球孢白僵菌菌株及其定向诱变方法和用途 - Google Patents

高抗紫外辐射球孢白僵菌菌株及其定向诱变方法和用途 Download PDF

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WO2023202608A1
WO2023202608A1 PCT/CN2023/089172 CN2023089172W WO2023202608A1 WO 2023202608 A1 WO2023202608 A1 WO 2023202608A1 CN 2023089172 W CN2023089172 W CN 2023089172W WO 2023202608 A1 WO2023202608 A1 WO 2023202608A1
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beauveria bassiana
uvb
resistant
highly
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杨征宇
王来福
陈培策
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浙江泰达作物科技有限公司
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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    • C12N1/14—Fungi; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
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Definitions

  • the present application relates to the field of biotechnology, specifically to a strain of Beauveria bassiana with high resistance to ultraviolet radiation and its directed mutagenesis method and use.
  • Beauveria bassiana is a broad-spectrum entomopathogenic fungus that mainly reproduces asexually and produces conidia.
  • fungal insecticides formulated from conidia of Beauveria bassiana are used for biological control of pests around the world.
  • my country's use of Beauveria bassiana to control masson pine caterpillars and corn borers is the largest in the world. pest control projects to achieve good continuous pest control results.
  • Beauveria bassiana has 1-2 photolyases.
  • the mechanism of eukaryotic cells to repair DNA damage caused by ultraviolet radiation mainly relies on the photorepair effect of photolyase located in the nucleus.
  • photolyase located in the nucleus.
  • Using this photolyase to quickly repair DNA damage caused by radiation under visible light can make most of the affected cells Rejuvenate damaged cells. Therefore, the expression level of photolyase genes largely determines the ability of fungal cells to photorepair DNA damage.
  • the first is to use exogenous resistance molecular markers to screen for highly expressed target genes.
  • the UV-resistant strains bred by this method inevitably carry exogenous resistance genes and are genetically modified strains, so there are potential ecological safety risks.
  • the second is to use directed mutagenesis and screening without any exogenous resistance molecular markers to select strains with high resistance to UV radiation.
  • the currently selected Beauveria bassiana has limited resistance to UV radiation, making it difficult to better Used for green pest control all day long.
  • this application has bred a strain of Beauveria bassiana that can efficiently express the photolysis gene. This strain has strong anti-ultraviolet radiation effect. Using this The fungal insecticide produced by the strain can be used for green prevention and control of pests around the clock.
  • this application provides a strain of Beauveria bassiana that is highly resistant to ultraviolet radiation, with a deposit number: CGMCC No. 22466, and a deposit date: July 5, 2021.
  • the application's highly UV-resistant Beauveria bassiana strain has conidia that are 53% more resistant to UVB radiation than the starting strain, and the expression level of its key photolyase gene for repairing DNA damage is increased by 98 times.
  • the traits related to biological control potential are completely consistent with the starting strain and do not contain any exogenous resistance molecular markers with potential ecological safety hazards. Therefore, it can be used as a production strain of fungal insecticides with high resistance to ultraviolet radiation and has important application value.
  • the corrected mortality rate of the highly UV-resistant Beauveria bassiana strain is ⁇ 50% on the sixth day after infection through body wall penetration, and the corrected mortality rate on the seventh day after infection through body wall penetration is ⁇ 60%.
  • the insect attachment rate of the highly UV-resistant Beauveria bassiana strain is 92.5-110.3%.
  • the present application provides a directed mutagenesis method for a highly UV-resistant Beauveria bassiana strain.
  • the highly UV-resistant Beauveria bassiana strain is Beauveria bassiana wild strain CGMCC No. 13566.
  • the starting strain was obtained through multiple rounds of repeated stress and directional screening by simulating sunlight's sublethal UVB radiation.
  • the directed mutagenesis method includes the following steps:
  • This application uses the wild strain of Beauveria bassiana as the starting strain and obtains materials from a wide range of sources. It uses multiple rounds of repeated stress and directional screening that simulates sunlight's UVB sub-lethal dose radiation. It is simpler to operate than genetic manipulation or editing methods.
  • the strains screened are molecularly modified strains without any foreign genes and should be regarded as non-GMO strains, and their preparations do not need to pass additional harsh, tedious and expensive environmental safety evaluations.
  • the conidia are dispersed into a spore suspension using sterile water containing 0.01-0.06% Tween-80.
  • Tween-80 is a hydrophilic surfactant, which has a strong effect of destroying cell membranes and causing stress.
  • using a low dose of Tween-80 can increase the conidia membrane.
  • the permeability promotes directional mutagenesis of spores, which can accelerate the breeding efficiency of Beauveria bassiana to a certain extent.
  • the medium in step 2 is Sabouraud's medium.
  • the formula of Sabouraud's medium is mainly yeast extract powder, glucose, peptone and agar, which is a medium commonly used for fungal isolation and culture.
  • the Sabouraud's medium used in this application is sufficient for Beauveria bassiana. It provides basic nutrients for the growth of bacteria. This is the first choice based on the need to screen strains with strong environmental tolerance in this application. It does not require the preparation of additional special culture media and can reduce the cost of strain breeding to a certain extent. At the same time, Effectively screen out mutant strains with excellent performance.
  • the sublethal dose of UVB in step 2 is 0.35-0.40J/cm 2 .
  • the sub-lethal dose can cause the death of about 95% of the conidia, and the few surviving conidia have strong UV radiation resistance, effectively improving the breeding efficiency of Beauveria bassiana.
  • the culture medium after irradiation in step 3 is cultured at a temperature of 22-28°C and a photoperiod of (10-14): (10-14).
  • this application harvests the conidia of the bacterial colonies selected in step 4, soaks the obtained conidia in a trehalose-ethanol aqueous solution, and evenly applies the spore suspension to On the culture plate, irradiate the plate with a sub-lethal dose of UVB in a sunlight simulation radiation box, culture the irradiated surviving spores to grow colonies, select the colonies with strong growth potential and transfer them to the sporulation culture plate, and culture until full production.
  • the conidia obtained were used for UVB resistance determination, and colonies with significantly enhanced UVB resistance based on the best colonies in the previous round were screened out. At the same time, the growth, sporulation and virulence traits were measured for significant changes.
  • Select The strain with ideal UVB resistance in the last round of mutagenesis screening was the highly UV-resistant Beauveria bassiana strain.
  • this application soaks the spores produced corresponding to the strains selected in step 4 with a trehalose-ethanol aqueous solution.
  • the trehalose is used extracellularly, it can not only effectively improve the resistance of the soaked spores.
  • the ultraviolet radiation performance promotes the rapid germination and stable growth of the spores under the sub-lethal dose of UVB, and also promotes the growing strain to have a higher expression level of the key photolyase gene for repairing DNA damage, which may be due to trehalose Attaching to the extracellular side of the strain plays a good protective role, allowing the photolyase gene to be stably expressed in the cell, and the ethanol in it can increase the permeability of the cell wall and cell membrane of the spore to a certain extent, making the nutrients in the culture medium The substance can quickly enter the cell to provide sufficient nutrients for the expression of the photolyase gene.
  • this application provides the application of a strain of Beauveria bassiana with high resistance to ultraviolet radiation in the preparation of fungal insecticides.
  • the fungal insecticides prepared by it overcome the problem of insufficient field stability of existing fungal insecticides. It has a common key technical bottleneck and has good field stability, persistence and pest control effect.
  • Figure 1 shows the colony morphology and microscopic characteristics of the ultraviolet radiation mutagenic strain of the present application.
  • Figure 2 is a comparative diagram of the UVB resistance of wild strains of Beauveria bassiana of the present application and strains resistant to ultraviolet radiation mutagenesis; wherein (A) is the survival trend of conidia as the dose of UVB radiation changes, (B) Calculate the UVB radiation dose LD 50 required for 50% spore inactivation based on the radiation dose-spore survival index fitting curve, (C) is the expression of the photolyase gene phr2 in the mutated strain relative to the wild strain Levels; error bars: standard deviation (SD) of the mean of triplicate experiments.
  • SD standard deviation
  • Figure 3 is a diagram comparing the virulence and sporulation properties of wild strains derived from Beauveria bassiana and strains mutated with resistance to ultraviolet radiation;
  • A is the fifth instar larvae of Galleria mellonella in spore suspension (10 7 spores/ mL) Corrected mortality trend and time LT 50 required for 50% death after 500 spores in body wall penetrating infection (NCI) and single head hemocoel injection infection (CBI).
  • NCI body wall penetrating infection
  • CBI single head hemocoel injection infection
  • C Comparison of the biomass in CDB-BSA culture 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.
  • ECEs extracellular enzymes
  • body wall-degrading enzymes such as Pr1 family proteases required for NCI to successfully penetrate the insect body wall.
  • SDAY Sabouraud's medium
  • D Comparison of the fresh carcasses of Galleria mellonella larvae after death and the growth on the body surface 10 days after death, showing that the growth and sporulation levels of the mutant strain and the wild strain on the carcasses are completely consistent; error bars: average of three repeated experiments Standard deviation (SD), note that there is no significant difference in all tested traits between the mutated strain and the wild strain.
  • SD Standard deviation
  • a directed mutagenesis method for Beauveria bassiana strains with high resistance to ultraviolet radiation including the following steps:
  • the mutagenesis and screening steps of 1, 2 and 3 must be repeated until the UVB resistance of the optimal target colony in the previous round is no longer significantly enhanced in the subsequent round of repeated mutagenesis. Select the final round of mutagenesis screening.
  • the mutant strain that exhibits ideal UVB resistance is the highly UV-resistant Beauveria bassiana strain.
  • the strain submitted for inspection grows quickly on potato glucose culture medium. Under dark conditions at 25°C for 7 days, the diameter of the colony is 30-35cm. The texture is compact, flocculent, white, and slightly raised; the back of the colony is light brown and has no soluble pigment.
  • the conidiophores are not obviously specialized, and the spore-producing cells are flask-shaped, straight or curved, 6.1–35.8 ⁇ 1.5–2.5 ⁇ m, with a slender neck, a zigzag extension at the top, less than 1 ⁇ m in width, solitary or gathered in clusters. ; Conidia are broadly elliptical, nearly spherical, colorless, with smooth walls, 1.5-3.0 ⁇ m, and no sexual sporulation structures are found.
  • the rRNA gene sequence determination results are as follows:
  • the determination method is as follows: apply an equal amount of 60 ⁇ L of conidial suspension (10 7 spores/mL) on a SDAY plate (diameter 9cm), place it on the sample stage of the above-mentioned sunlight ultraviolet radiation simulation box, and perform a gradient dose (0.1 to 0.5 J/cm 2 ) of UVB radiation, the irradiated plate was covered and cultured at 25°C and a photoperiod of 12:12 for 24 hours, and the plate without radiation was used as a control.
  • the determination method is as follows: spread equal amounts of 100 ⁇ L of conidia suspension of the wild strain and the mutated strain on a SDAY plate covered with cellophane, incubate for 3 days at 25°C and a photoperiod of 12:12, and then harvest the culture. After nitrogen grinding, the total RNA of each strain was extracted using the RNAiso Plus Kit (TaKaRa Company, Dalian, China), and then the RNA was reverse transcribed into cDNA using the PrimeScript RT reagent Kit (TaKaRa).
  • the determination method is as follows: using the fifth instar larvae of the model insect Galleria mellonella as test insects, each group of 35 worms is submerged in 40 mL of spore suspension (10 7 /mL) for 10 seconds as a normal body wall inoculation method. Use a microsyringe to inject 5 ⁇ L of spore suspension (10 5 /mL) into each head of the larvae in each group into the hemocoel as a hemocoel injection infection inoculation method. Then, each group of test insects was transferred to a transparent plastic box and placed under the conditions of 25°C and a photoperiod of 12:12. The number of dead and surviving insects was observed and recorded every day until all died.
  • the same amount of 0.02% Tween-80 solution was used as a control, and the corrected mortality rate was calculated daily. Each treatment was repeated three times. The obtained time-mortality curve was analyzed by model fitting, and the time LT 50 value required for 50% of the test insects to die under different inoculation methods of each strain was calculated.
  • the measurement method is as follows: take the hind wings of East Asian migratory locusts (Locusta migratoria manilensis), immerse them in 37% H 2 O 2 aqueous solution for 5 minutes, wash them three times with sterile water, and then stick them on a 0.7% water agar plate. Drop an equal amount of 5 ⁇ L of spore suspension (10 7 /mL) on the center of the hind wing surface, and apply it evenly with a transfer ring. Incubate for 8 hours at 25°C. Immediately remove the hind wings and place them 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 hind wings were then rinsed in sterile water for 30 seconds to remove spores that were not adhered to the hind wing body wall.
  • Three fields of view of the wing surface were again observed under a microscope and the number of remaining spores was counted. Calculate the percentage of the number of spores after rinsing and the number of spores on the forewing surface after rinsing, that is, the attachment rate of conidia on the body wall of the locust's hind wing.
  • the determination method is: inoculate the conidia suspension of each strain into the basic culture medium CDB (3% sucrose, 0.3% NaNO 3 , 0.1% K 2 HPO 4 , 0.05) with 0.3% bovine serum albumin (BSA) as the only nitrogen source. % KCl, 0.05% MgSO 4 and 0.001% FeSO 4 ), the final concentration was 10 4 conidia/mL. After culturing for 3 days with shaking (150r/min) at 25°C, filter and collect the mycelium, dry it at 75°C and measure the biomass; the supernatant of the culture solution is centrifuged at 13,500 ⁇ g for 2 minutes at 4°C, and the supernatant is collected as a crude extract.
  • CDB 3% sucrose, 0.3% NaNO 3 , 0.1% K 2 HPO 4 , 0.05
  • BSA bovine serum albumin
  • proteases used to measure the total enzyme activity of secreted extracellular enzymes (generally known as proteases, chitinases, lipases, etc., abbreviated as ECEs) and Pr1 family proteases.
  • ECEs extracellular enzymes
  • Pr1 protease To measure the total enzyme activity of Pr1 protease, take 100 ⁇ L boiling water bath inactivated (control) or inactivated protein crude extract and 50 ⁇ L reaction substrate with a concentration of 1 mM [succinyl-(alanine)2-proline-phenylalanine-p-nitroanilide] Mix thoroughly with 850 ⁇ L Tris-HCl buffer (15mM, pH 8.5), and let stand at 28°C for 1 hour; add 250 ⁇ L 30% (w/v) acetic acid to terminate the reaction.
  • the reaction system was kept on ice for 15 minutes, centrifuged at 13,000 ⁇ g for 5 minutes at 4°C, and the supernatant was taken to read the absorbance value (OD 410 ) at a wavelength of 410 nm.
  • the enzyme activity unit is defined as the 0.01 increment of the OD 442 or OD 410 reading change during the reaction.
  • the total enzyme activity represents the number of extracellular enzyme activity units per milliliter of culture supernatant (U/mL).
  • the determination method is: refer to the spore number determination method of SB/T 10315-1999.
  • the measurement method is: visual inspection.
  • the mutant strain of Beauveria bassiana obtained by repeated UVB sublethal dose radiation mutagenesis in this application has a 53% increase in conidia resistance to UVB radiation compared with the starting strain (Figure 2B), and it can repair DNA damage.
  • the expression level of the photolyase gene phr2 was significantly increased by 98 times ( Figure 2C). There are no exogenous resistance molecular markers in its cells, so there are no ecological safety risks.
  • the mortality rate of the Beauveria bassiana mutagen strain of this embodiment through body wall penetration infection is higher than that of the wild strain at 3-7 days, in which the corrected death rate on the sixth day after body wall penetration infection is The rate was 52.0% (46.2% for the wild strain), and the corrected mortality rate on the seventh day of infection through body wall penetration was 61.3% (57.0% for the wild strain). It can be seen that the efficiency of infection through body wall penetration of the mutated strain is faster. .
  • the mutant strain has an attachment rate of 101.2% (falling within the range of 94.7-109.8% after adding the standard deviation), while the attachment rate of the wild strain to the insect body is 99.0% (after adding the standard deviation). Falling within the range of 90.3-110.0%), that is, the attachment rate and attachment stability of conidia of the mutated strain to locust wings are higher than those of the wild strain.
  • the Beauveria bassiana mutant strain of this embodiment can be used as a production strain of fungal insecticides resistant to sunlight and ultraviolet radiation, thereby enhancing the resistance of fungal insecticides to ultraviolet radiation and the stability of their control effects on field pests.
  • It also has excellent penetration and infection efficiency through the body wall and attachment rate to the insect body, so it has important application value.
  • the photoperiod of 12:12 is adjusted to 10:14, and the corresponding mutagenized strains are directionally screened.
  • Examples 3-4 are based on the method of Example 1, and the sub-lethal dose is adjusted to 0.38J/cm 2 .
  • Example 3 is specifically 0.3J/cm 2
  • Example 4 is specifically 0.45J/cm 2 .
  • Targeted screening The corresponding mutagen strain was produced.
  • Example 2 According to the performance comparison test steps of the above-mentioned Example 1, the performance of the mutagen strain screened in Example 2-4 and the wild strain were measured. The test results showed that the mutagen strain screened in Example 2-4 was the same as the mutagenic strain screened in Example 1. The colony morphology and microscopic characteristics of the strains were similar. The resistance of their conidia to UVB radiation was increased by 45%, 46% and 49% compared with the starting strain. The expression level of the photolyase gene phr2, which repairs DNA damage, was sequentially increased. 56 times, 67 times and 77 times. The corrected mortality rates on the sixth day of body wall penetrating infection were 50.2%, 50.5% and 51.0% respectively.
  • the corrected mortality rates on the seventh day of body wall penetrating infection were 60.0%. , 60.2% and 60.6%, and the insect attachment rates are 99.1% (all fall within the range of 92.5-110.3% after adding the standard deviation), 100.5% (all fall within the range of 93.7-110.1% after adding the standard deviation) and 100.7% (all fall within the range of 94.1-110.0% after adding standard deviation). It can be seen that the mutagen strain obtained in Example 1 has better anti-ultraviolet radiation effect, infection efficiency through body wall penetration and insect attachment rate. Therefore, this application uses the directed mutagenesis method of Example 1 as a further step. of preference.
  • step 5 which specifically includes the following steps:
  • trehalose-ethanol aqueous solution Harvest the conidia of the colony selected in step 4, and soak the obtained conidia in the trehalose-ethanol aqueous solution for 10-15 minutes.
  • concentration of trehalose is 0.3-0.6g/L, and the volume concentration of ethanol in the ethanol aqueous solution is 5-10%.
  • the spore treatment effect is similar within the above range. If the mutagen strain harvested exceeds the above range, the growth will be poor.
  • 0.5g/L trehalose-8% ethanol aqueous solution is soaked for 10 minutes to prepare spores. suspension;
  • the mutagen strain with ideal UVB resistance performance in the final round of mutagenesis screening was selected as the highly UV-resistant Beauveria bassiana strain.
  • Example 1 According to the performance comparison test steps of the above-mentioned Example 1, the performance of the mutagen strain corresponding to the screening in Example 5 and the wild strain were measured. The test results showed that the colony morphology and The microscopic characteristics are similar. The resistance of its conidia to UVB radiation is 61% higher than that of the starting strain (53% in Example 1). The expression level of the photolyase gene phr2 that repairs DNA damage is increased by 112 times (implemented).
  • Example 1 is 98 times); the corrected mortality rate on the sixth day of infection through body wall penetration is 54.3% (52.0% in Example 1), and the corrected mortality rate on the seventh day of infection through body wall penetration is 62.5% (implementation Example 1 is 61.3%), and the insect attachment rate is 101.0% (all fall within the interval of 96.0-107.2% after adding the standard deviation) (Example 1 is 101.2% (falling within the interval of 94.7-109.8% after adding the standard deviation) )).
  • the mutagen strains selected by the directed mutagenesis method in Example 1 can obtain anti-ultraviolet radiation effects and penetrate through the body wall. Therefore, the directed mutagenesis method of Example 5 is further preferred in this application.

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Abstract

本申请涉及生物技术领域，具体涉及高抗紫外辐射球孢白僵菌菌株及其定向诱变方法和用途。本申请的高抗紫外辐射球孢白僵菌菌株，保藏编号：CGMCC No.22466，保藏日期：2021年07月05日。该菌株以球孢白僵菌野生株CGMCC No.13566为出发菌株，经模拟阳光UVB亚致死量辐射的多轮反复胁迫和定向筛选而得，具有高抗紫外辐射的性状，采用该菌株制得的真菌杀虫剂能较好地全天候应用于害虫绿色防控。

Description

高抗紫外辐射球孢白僵菌菌株及其定向诱变方法和用途 技术领域
本申请涉及生物技术领域，具体涉及高抗紫外辐射球孢白僵菌菌株及其定向诱变方法和用途。
背景技术
球孢白僵菌(Beauveria bassiana)是广谱性昆虫病原真菌，主要进行无性繁殖，产生分生孢子。目前，将球孢白僵菌分生孢子制成剂型化的真菌杀虫剂，在世界范围被用于害虫生物防治，其中我国使用球孢白僵菌防治马尾松毛虫和玉米螟是国际上最大的害虫防治项目，取得良好的害虫持续控制效果。
但是，剂型化的球孢白僵菌在田间应用中常遭受阳光紫外辐射的损伤而影响害虫防治效果的稳定性和持效性，突出表现在害虫种群经常暴发和阳光辐射尤其强烈的夏季，故阳光紫外辐射限制了真菌杀虫剂对害虫绿色防控的全天候应用。其中，阳光紫外辐射包含UVB(290-320nm)和UVA(320-400nm)两种有害射线成分，所幸最短最有害的UVC射线(<290nm)在阳光抵达地球表面之前被大气臭氧层全部过滤去除。因此，太阳光中以UVB紫外辐射对剂型化真菌细胞的损伤最大，而较大波长UVA的影响则相当有限。
球孢白僵菌有1-2个光裂合酶。通常真核细胞修复紫外辐射所致DNA损伤的机制主要靠定位于细胞核的光裂合酶的光修复作用，利用该光裂合酶在可见光下快速修复辐射所致DNA损伤，可使大部分受损细胞恢复活力。因此，光裂合酶基因的表达水平在很大程度上决定了真菌细胞对DNA损伤的光修复能力。
目前，提高光裂合酶基因在真菌细胞中表达水平的途径有两条。一是利用外源抗性分子标记筛选高表达目标基因的途径，由此选育的抗紫外菌株不可避免地携带外源抗性基因，属于转基因菌株，因而存在生态安全性隐患。二是不用任何外源抗性分子标记的定向诱变和筛选途径，选育高抗紫外辐射菌株的途径，但目前选育的球孢白僵菌对紫外辐射的抗性有限，难以较好地全天候应用于害虫绿色防控。
发明内容
为了解决现有真菌杀虫剂抗紫外辐射性能差的问题，本申请选育出一株能高效表达光裂合基因的球孢白僵菌，该菌株具有较强的抗紫外辐射效果，采用该菌株制得的真菌杀虫剂能较好地全天候应用于害虫绿色防控。
第一方面，本申请提供一种高抗紫外辐射球孢白僵菌菌株，保藏编号：CGMCC No.22466，保藏日期：2021年07月05日。
本申请的高抗紫外辐射球孢白僵菌菌株，其分生孢子对UVB辐射的抗性较出发菌株提高53％，其修复DNA损伤的关键光裂合酶基因的表达水平上调98倍，其余生物防治潜能相关性状与出发菌株完全一致，不含任何具潜在生态安全隐患的外源抗性分子标记，因而可作为高抗紫外辐射真菌杀虫剂的生产菌株，具有重要的应用价值。
优选的，所述高抗紫外辐射球孢白僵菌菌株经体壁穿透感染第六天的校正死亡率≥50％，经体壁穿透感染第七天的校正死亡率≥60％。
优选的，所述高抗紫外辐射球孢白僵菌菌株的虫体附着率为92.5-110.3％。
第二方面，本申请提供一种高抗紫外辐射球孢白僵菌菌株的定向诱变方法，所述高抗紫外辐射球孢白僵菌菌株以球孢白僵菌野生株CGMCC No.13566为出发菌株，经模拟阳光UVB亚致死量辐射的多轮反复胁迫和定向筛选而得。
优选的，所述定向诱变方法包括以下步骤：
①以球孢白僵菌野生株CGMCC No.13566为出发菌株，配制其分生孢子的孢子悬液；
②将孢子悬液均匀涂布于培养基平板上，在阳光模拟辐射箱中以UVB的亚致死剂量对平板进行辐射；
③培养经辐射的存活孢子至长出菌落，挑选生长势旺盛的菌落转移至产孢培养平板上，培养直至充分产孢，所获分生孢子用于UVB抗性测定，筛选出在前一轮最佳菌落基础上UVB抗性进一步显著增强的菌落，并同时测定生长产孢和毒力性状有无显著变化；
④反复重复①、②和③的诱变与筛选步骤，直到前一轮最优目标菌落的UVB抗性在后一轮重复诱变中不再显著增强为止，选择最后一轮诱变筛选中UVB抗性表现理想的菌株即为高抗紫外辐射球孢白僵菌菌株。
本申请以球孢白僵菌野生株为出发菌株，材料获取来源广泛，其采用模拟阳光UVB亚致死剂量辐射的多轮反复胁迫和定向筛选，相对于基因操作或编辑手段操作更为简便，由此筛选的菌株是无任何外源基因的分子定向改良菌株，应视为非转基因菌株，其制剂产品无需通过额外苛刻繁琐且昂贵的环境安全性评价。
优选的，步骤①中分生孢子使用含0.01-0.06％吐温-80的无菌水分散为孢子悬液。
通过采用上述技术方案，吐温-80为亲水性表面活性剂，具有较强的破坏细胞膜的作用而引起应激性，在本申请中使用低剂量的吐温-80能增加分生孢子膜的通透性，促使孢子发生定向诱变，在一定程度上可加快球孢白僵菌的选育效率。
优选的，步骤②中培养基为萨氏培养基。
通过采用上述技术方案，萨氏培养基的配方主要为酵母浸出粉、葡糖糖、蛋白胨和琼脂，是常规用于真菌分离培养的培养基，本申请使用萨氏培养基足以为球孢白僵菌的生长提供基础的营养成分，这是基于本申请需要筛选环境耐受性强的菌株而做出的优选，其无需额外配制专属培养基，能在一定程度上降低菌株的选育成本，同时有效筛选出性能优异的突变株。
优选的，步骤②中UVB的亚致死剂量为0.35-0.40J/cm2。
通过采用上述技术方案，上述亚致死剂量能导致95％左右的分生孢子死亡，少数存活下来的分生孢子则具有较强的抗紫外辐射性能，有效提高球孢白僵菌选育效率。
优选的，步骤③中经辐射后的培养基置于温度为22-28℃、光周期为(10-14):(10-14)的条件下进行培养。
辐射温度和光周期是影响菌株变异的关键因素，采用上述温度和光周期培养的诱变菌株有助于筛选出本申请所述的高抗紫外辐射球孢白僵菌菌株适应田间应用环境。
优选的，本申请在上述定向诱变方法的基础上，收获步骤④中挑选的菌落的分生孢子，将所获分生孢子浸泡于海藻糖-乙醇水溶液中，将孢子悬液均匀涂布于培养基平板上，在阳光模拟辐射箱中以UVB的亚致死剂量对平板进行辐射，培养经辐射的存活孢子长出菌落，挑选生长势旺盛的菌落转移至产孢培养平板上，培养直至充分产孢，所获分生孢子用于UVB抗性测定，筛选出在前一轮最佳菌落基础上UVB抗性进一步显著增强的菌落，并同时测定生长产孢和毒力性状有无显著变化，选择最后一轮诱变筛选中UVB抗性表现理想的菌株即为高抗紫外辐射球孢白僵菌菌株。
通过采用上述技术方案，本申请将步骤④挑选的菌株对应产出的孢子用海藻糖-乙醇水溶液浸泡，其中的海藻糖虽然用于胞外，但其不但能有效提高经浸泡后的孢子的抗紫外辐射性能，促使该孢子在UVB的亚致死量下快速萌发并稳定生长，还能促使生长的菌株具有更高的修复DNA损伤的关键光裂合酶基因的表达水平，这可能是由于海藻糖附着在菌株胞外起到良好的防护作用，使光裂合酶基因在胞内稳定表达，而其中的乙醇能在一定程度上增加孢子的细胞壁和细胞膜的通透性，使培养基中的营养物质能快速进入胞内为光裂合酶基因表达提供充足的养分。
第三方面，本申请提供一种高抗紫外辐射球孢白僵菌菌株在制备真菌杀虫剂中的应用，其制得的真菌杀虫剂克服了现有真菌杀虫剂田间稳定性不足这一共性关键技术瓶颈，具有良好的田间稳定性、持效性和害虫防治效果。
附图说明
图1是本申请抗紫外辐射诱变菌株的菌落形态及显微特征。
图2是本申请球孢白僵菌出发野生菌株与抗紫外辐射诱变菌株的UVB抗性比较示图；其中，(A)为分生孢子随UVB辐射剂量变化的存活趋势， (B)为基于辐射剂量-孢子存活指数拟合曲线计算孢子失活50％所需的UVB辐射剂量LD50，(C)为光裂合酶基因phr2在诱变株中相对于野生株的表达水平；误差线：三次重复实验平均值的标准差(SD)。
图3是球孢白僵出发野生菌株与抗紫外辐射诱变菌株的毒力与产孢性状比较示图；其中，(A)为大蜡螟五龄幼虫在孢子悬液(107个孢子/mL)经体壁穿透侵染(NCI)和单头血腔注射侵染(CBI)500个孢子之后的校正死亡率趋势及死亡50％所需时间LT50。(B)为启动NCI所需分生孢子在蝗虫后翅体壁上的附着率比较。(C)为CDB-BSA培养物中生物量与NCI成功穿透昆虫体壁所需的胞外酶(ECEs)和Pr1家族蛋白酶等体壁降解酶在培养物上清液中的总酶活比较。(D)在萨氏培养基(SDAY)平板上用孢子悬液涂板接种后正常培养期间的分生孢子产量比较。(D)为大蜡螟幼虫病死新鲜虫尸与死后10天体表长出物比较，显示诱变株与野生株在虫尸上的生长产孢水平完全一致；误差线：三次重复实验平均值的标准差(SD)，注意诱变株与野生株的所有考查性状均无显著差异。
具体实施方式
以下结合附图、实施例和对比例对本申请作进一步详细说明。
原料来源
本申请的原料均购自市售产品，具体参见下表一。
表一 本申请的原料来源

实施例1
一种高抗紫外辐射球孢白僵菌菌株的定向诱变方法，包括以下步骤：
①以球孢白僵菌野生菌株CGMCC No.13566(简称野生菌株)为出发菌株，将其在萨氏培养基(SDAY)平板上正常培养所产的分生孢子悬浮于含0.02％吐温-80(允许在0.01-0.06％的范围内波动)的无菌水中，配制成107个孢子/mL的悬液；
②在无菌条件下，用上述孢子悬液60μL等量均匀涂布SDAY平板。空气干燥数分钟后(使涂布的孢子悬液干燥)，将平板置于阳光紫外辐射模拟箱Bio-Sun++UV Chamber(Vilber Lourmat,Marne-la-Vallée,France)的样品台(12cm×16cm)上，按亚致死剂量0.38J/cm2(允许在0.35-0.4J/cm2范围内波动)进行UVB辐射，导致95％左右的孢子死亡；
③涂有孢子的平板经辐射后即刻加盖，在温度为25℃、光周期为12:12(允许在22-28℃、(10-14):(10-14)的范围内波动)的条件下培养，直到少数存活孢子长出菌落，挑选生长势旺盛的菌落转移至新的SDAY平板培养直到充分产孢，所获孢子用于UVB抗性测定，筛选出在上一轮最佳菌落基础上UVB抗性进一步显著增强的菌落，并同时测定生长产孢和毒力性状有无显著变化；
④反得重复①、②和③的诱变与筛选步骤，直到前一轮最优目标菌落的UVB抗性在后一轮重复诱变中不再显著增强为止，选择最后一轮诱变筛选中UVB抗性表现理想的诱变株即为高抗紫外辐射球孢白僵菌菌株。
诱变株的生物学鉴定(菌落形态及显微特征、基因序列测定)
本申请将上述诱变筛选的高抗紫外辐射球孢白僵菌菌株送入中国科学院微生物研究所进行生物鉴定及保藏，送检时间为2021年7月5日，菌株编号为TICZJU618(中国微生物菌种保藏中心保藏号CGMCC No.22466)，鉴定结果为球孢白僵菌(Beauveria bassiana)。
菌落形态及显微特征(参见图1)如下：
送检菌种在马铃薯葡糖糖培养基上生长较快，25℃黑暗条件下7天，菌落直径30-35cm，质地紧密，絮状，白色，略隆起；菌落背面浅褐色，无可溶性色素。
分生孢子梗特化不明显，产孢细胞烧瓶状，直或弯曲，6.1–35.8×1.5–2.5μm，颈部细长，顶端“之”字形延伸，宽度不足1μm，单生或聚集成簇；分生孢子宽椭圆形、近球形，无色，壁光滑，1.5-3.0μm，未见有性态产孢结构。
rRNA基因序列测定结果如下：
包括18SrRNA片段，ITS1、5.8SrRNA、ITS2的全序列及28S区序列片段，序列如SEQ ID NO.1所示。
定向诱变方法的重复再现性
选择三个实验员按实施例1的定向诱变方法分别进行试验，分别进行上述生物学鉴定，可以得到三个试验员所获诱变株的发菌落形态、显微特征和保守基因序列均相同，可见本申请的定向诱变方法具有重复再现性，能够稳定诱导选育出本申请的诱变株。
诱变株与野生株的性能对比试验
选择在最后一轮诱变筛选中UVB抗性表现理想的诱变株与野生株进行如下比对实验。
1、UVB抗性测定
测定方法为：将分生孢子悬液(107个孢子/mL)60μL等量涂布于SDAY平板(直径9cm)上，置于上述阳光紫外辐射模拟箱样品台上进行梯度剂量(0.1～0.5J/cm2)的UVB辐射，经辐射的平板加盖后置于25℃和光周期12:12的条件下培养24小时，以不辐射的平板作为对照。培养期间，从第4小时开始每隔2小时显微镜检3个视野的总孢子数和萌发孢子数，计算孢子存活指数(百分率除以100)，然后对梯度剂量下的存活指数进行模型拟合分析，获得UVB的致死中剂量LD50。各剂量实验设三个独立重复。
检测结果参见图2A和图2B。
2、关键光裂合酶基因的表达水平测定
测定方法为：将野生株与诱变株的分生孢子悬液100μL等量涂布于贴有玻璃纸的SDAY平板上，在25℃和光周期12:12的条件下培养3天后收获培养物，液氮研磨后用RNAiso Plus Kit试剂盒(中国大连TaKaRa公司)提取各菌株的总RNA，再用PrimeScript RT reagent Kit试剂盒(TaKaRa)将RNA反转录为cDNA。以所获cDNA为模板，在SYBR Premix Ex Taq酶(TaKaRa)作用下进行实时定量PCR分析，测定光裂合酶基因phr2在各菌株cDNA中的转录水平，以β-actin基因作为内参。目标基因在诱变株中相对于野生株中的表达水平采用2-△△CT方法进行试算。各菌株重复测定3个cDNA独立样本。
检测结果参见图2C。
3、经体壁和血腔注射侵染的毒力测定
测定方法为：以模式昆虫大蜡螟五龄幼虫作为试虫，每35头一组没入40mL孢子悬液(107个/mL)中浸渍10秒钟，作为正常体壁侵染接种方式。对各组幼虫的每一头用微注射器注入5μL孢子悬液(105个/mL)至血腔中，作为血腔注射侵染接种方式。然后，将每组试虫转入透明塑料盒中，置于25℃和光周期12:12的条件下，每天观察记录死亡和存活虫数，直到全部死亡为止。以0.02％吐温-80液的等量浸渍或注射处理作为对照，逐日计算校正死亡率。各处理重复3次。所获时间-死亡率曲线经模型拟合分析，计算各菌株不同接种方式下试虫死亡50％所需的时间LT50值。
检测结果参见图3A。
4、决定正常体壁侵染成功率的分生孢子附着昆虫体表能力的测定
测定方法为：取东亚飞蝗(Locusta migratoria manilensis)后翅，在37％H2O2水溶液中浸渍消毒5分钟，无菌水清洗三次后贴在0.7％水琼脂平板上。孢子悬液(107个/mL)5μL等量滴在后翅表面中央，用移菌环涂抹均匀后。在25℃下培养8小时，即刻取下后翅置于载玻片上，在显微镜下观察3个视野，计数各视野中的分生孢子数。观察的后翅随后在无菌水中漂洗30秒钟去除未粘附在后翅体壁上的孢子，再次在显微镜下观察翅面的3个视野并计数存留的孢子数。计算漂洗后孢子数与漂洗前翅面上的孢子数百分比，即得分生孢子在蝗虫后翅体壁上的附着率。
检测结果参见图3B。
5、主要昆虫体壁降解酶的总酶活测定
测定方法为：将各菌株分生孢子悬液接种于以0.3％牛血清蛋白(BSA)作为唯一氮源的基础培养液CDB(3％蔗糖、0.3％NaNO3、0.1％K2HPO4、0.05％KCl、0.05％MgSO4及0.001％FeSO4)中，终浓度为104个分生孢子/mL。在25℃下振荡(150r/min)培养3天后过滤收集菌丝，75℃烘干后测定生物量；培养液的上清在4℃下13,500×g离心2min，收集上清液作为粗提物，用于测定分泌的胞外酶(蛋白酶、几丁质酶、脂酶等总称，缩写为ECEs)和Pr1家族蛋白酶的总酶活。胞外酶总酶活的测定，取100μL浓度为5mg/mL的偶氮蛋白(溶于50mM Tris-HCl,pH 8.0)溶液，与100μL经沸水浴15min变性的蛋白粗提液(对照组)或不变性的蛋白粗提液(实验组)充分混匀，37℃下避光孵育1小时后，加入400μL 10％(w/v)三氯乙酸终止反应。12,000×g离心5min后，吸取上清液转移至新离心管中，与700μL 525mM NaOH充分混匀，在442nm波长下读取吸光值(OD442)。Pr1蛋白酶总酶活的测定，取100μL沸水浴灭活(对照)或不灭活蛋白粗提液与50μL浓度为1mM的反应底物[succinyl-(alanine)2-proline-phenylalanine-p-nitroanilide]和850μL Tris-HCl缓冲液(15mM，pH 8.5)充分混匀，28℃下静置1小时；加入250μL 30％(w/v)乙酸终止反应。将反应体系冰浴15分钟，4℃下13,000×g离心5分钟，取上清液在410nm波长下读取吸光值(OD410)。酶活单位定义为反应期间OD442或OD410读数变化0.01的增量，总酶活表示每毫升培养液上清所含的胞外酶活单位数(U/mL)。
检测结果参见图3C。
6、正常生长产孢水平的测定
测定方法为：参照SB/T 10315-1999的孢子数测定法进行测定。
检测结果参见图3D。
7、虫尸体表生长产孢水平的观察
测定方法为：目测。
检测结果参见图3E。
综上，本申请经反复UVB亚致死剂量辐射诱变获得的球孢白僵菌诱变株，其分生孢子对UVB辐射的抗性较出发菌株提高53％(图2B)，其修复DNA损伤的光裂合酶基因phr2的表达水平大幅上调98倍(图2C)，其细胞中无任何源外抗性分子标记，因而无生态安全隐患。
由于野外杀菌对虫体一一进行血腔注射耗时耗力，因此通常采用体壁穿透感染方式进行，况且野外环境具有不可控性，因此球孢白僵菌的体壁穿透感染效率越快、对虫体的附着率越高，则能使球孢白僵菌更好地发挥杀虫效果。参见图3A和3B，本实施例的球孢白僵菌诱变株通过体壁穿透感染的死亡率在3-7天高于野生株，其中经体壁穿透感染第六天的校正死亡率为52.0％(野生株为46.2％)，经体壁穿透感染第七天的校正死亡率为61.3％(野生株为57.0％)，可见诱变株经体壁穿透感染的效率更快。对虫体附着率方面，诱变株对虫体附着率为101.2％(加标准差后落在94.7-109.8％区间内)，而野生株对虫体的附着率为99.0％(加标准差后落在90.3-110.0％区间内)，即诱变株的分生孢子在蝗翅上的附着率以及附着稳定性高于野生株。
除此之外，本实施例收获的球孢白僵菌诱变株的生长、产孢和毒力性状与出野生株一致(图3C-E)。
因此，本实施例的球孢白僵菌诱变株可作为抗阳光紫外辐射真菌杀虫剂的生产菌株，增强真菌杀虫剂对紫外辐射的抵抗力和对田间害虫防治效果的稳定性，此外还具有优异的经体壁穿透感染效率以及对虫体附着率，因而具有重要的应用价值。
实施例2
本实施例在实施例1的方法基础上，将光周期12:12调整为10:14，定向筛选出对应的诱变株。
实施例3-4
实施例3-4在实施例1的方法基础上，将亚致死剂量0.38J/cm2进行调整，实施例3具体为0.3J/cm2，实施例4具体为0.45J/cm2，定向筛选出对应的诱变株。
按照上述实施例1的性能对比试验步骤，测定实施例2-4对应筛选的诱变株与野生株的性能，检测结果显示实施例2-4筛选的诱变株与实施例1筛选的诱变株菌落形态及显微特征相近，其分生孢子对UVB辐射的抗性较出发菌株依次提高为45％、46％和49％，其修复DNA损伤的光裂合酶基因phr2的表达水平依次上调56倍、67倍和77倍，经体壁穿透感染第六天的校正死亡率依次为50.2％、50.5％和51.0％，经体壁穿透感染第七天的校正死亡率依次为60.0％、60.2％和60.6％，对虫体附着率依次为99.1％(加标准差后均落在92.5-110.3％区间内)、100.5％(加标准差后均落在93.7-110.1％区间内)和100.7％(加标准差后均落在94.1-110.0％区间内)。由此可见，实施例1获得的诱变株具有更为优异的抗紫外辐射效果、经体壁穿透感染效率以及对虫体附着率，因此本申请将实施例1的定向诱变方法作为进一步的优选。
实施例5
本实施例在实施例1的定向诱变方法的基础上，还设有步骤⑤，具体包括如下步骤：
收获步骤④中挑选的菌落的分生孢子，将所获分生孢子浸泡于海藻糖-乙醇水溶液中10-15min，海藻糖的浓度为0.3-0.6g/L，乙醇水溶液中乙醇的体积浓度为5-10％，上述范围内对孢子处理效果相近，超过上述范围其收获的诱变株长势欠佳，本实施例中具体采用0.5g/L海藻糖-8％乙醇水溶液浸泡10min，制得孢子悬液；
用上述孢子悬液60μL等量均匀涂布SDAY平板，空气干燥数分钟后(使涂布的孢子悬液干燥)，将平板置于阳光紫外辐射模拟箱Bio-Sun++UV Chamber(Vilber Lourmat,Marne-la-Vallée,France)的样品台(12cm×16cm)上，按亚致死剂量0.38J/cm2(允许在0.35-0.4J/cm2范围内波动)进行UVB辐射；
将涂有孢子的平板经辐射后即刻加盖，在温度为25℃、光周期为12:12(允许在22-28℃、(10-14):(10-14)的范围内波动)的条件下培养，直到存活孢子长出菌落，挑选生长势旺盛的菌落转移至新的SDAY平板培养直到充分产孢，所获孢子用于UVB抗性测定，筛选出在上一轮最佳菌落基础上UVB抗性进一步显著增强的菌落，并同时测定生长产孢和毒力性状有无显著变化；
选择最后一轮诱变筛选中UVB抗性表现理想的诱变株即为高抗紫外辐射球孢白僵菌菌株。
按照上述实施例1的性能对比试验步骤，测定实施例5对应筛选的诱变株与野生株的性能，检测结果显示实施例5筛选的诱变株与实施例1筛选的诱变株菌落形态及显微特征相近，其分生孢子对UVB辐射的抗性较出发菌株提高为61％(实施例1为53％)，其修复DNA损伤的光裂合酶基因phr2的表达水平上调112倍(实施例1为98倍)；经体壁穿透感染第六天的校正死亡率为54.3％(实施例1为52.0％)，经体壁穿透感染第七天的校正死亡率为62.5％(实施例1为61.3％)，对虫体附着率为101.0％(加标准差后均落在96.0-107.2％区间内)(实施例1为101.2％(加标准差后落在94.7-109.8％区间内))。
由此可见，经实施例1的定向诱变方法筛选出的诱变株，再用海藻糖-乙醇水溶液浸泡处理后再培养产出的孢子，能够获得抗紫外辐射效果、经体壁穿透感染效率以及对虫体附着率更为优异的诱变株，因此本申请将实施例5的定向诱变方法作为进一步的优选。
本具体实施例仅仅是对本申请的解释，其并不是对本申请的限制，本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改，但只要在本申请的权利要求范围内都受到专利法的保护。

Claims (10)

  1. 一种高抗紫外辐射球孢白僵菌菌株，其特征在于，保藏编号：CGMCC No.22466，保藏日期：2021年07月05日。
  2. 根据权利要求1所述的高抗紫外辐射球孢白僵菌菌株，其特征在于，所述高抗紫外辐射球孢白僵菌菌株经体壁穿透感染第六天的校正死亡率≥50％，经体壁穿透感染第七天的校正死亡率≥60％。
  3. 根据权利要求1所述的高抗紫外辐射球孢白僵菌菌株，其特征在于，所述高抗紫外辐射球孢白僵菌菌株的虫体附着率为92.5-110.3％。
  4. 权利要求1所述的高抗紫外辐射球孢白僵菌菌株的定向诱变方法，其特征在于，所述高抗紫外辐射球孢白僵菌菌株以球孢白僵菌野生株CGMCC No.13566为出发菌株，经模拟阳光UVB亚致死量辐射的多轮反复胁迫和定向筛选而得。
  5. 根据权利要求4所述的定向诱变方法，其特征在于，包括以下步骤：
    ①以球孢白僵菌野生株CGMCC No.13566为出发菌株，配制其分生孢子的孢子悬液；
    ②将孢子悬液均匀涂布于培养基平板上，在阳光模拟辐射箱中以UVB的亚致死剂量对平板进行辐射；
    ③培养经辐射的个别存活孢子长出菌落，挑选生长势旺盛的菌落转移至产孢培养平板上，培养直至充分产孢，所获分生孢子用于UVB抗性测定，筛选出在前一轮最佳菌落基础上UVB抗性进一步显著增强的菌落，并同时测定生长产孢和毒力性状有无显著变化；
    ④反复重复①、②和③的诱变与筛选步骤，直到前一轮最优目标菌落的UVB抗性在后一轮重复诱变中不再显著增强为止，选择最后一轮诱变筛选中UVB抗性表现理想的菌株即为高抗紫外辐射球孢白僵菌菌株。
  6. 根据权利要求4所述的定向诱变方法，其特征在于：步骤①中分生孢子使用含0.01-0.06％吐温-80的无菌水分散为孢子悬液。
  7. 根据权利要求4所述的定向诱变方法，其特征在于：步骤②中UVB的亚致死剂量为0.35-0.40J/cm2。
  8. 根据权利要求4所述的定向诱变方法，其特征在于：步骤③中经辐射后的培养基置于温度为22-28℃、光周期为(10-14):(10-14)的条件下进行培养。
  9. 根据权利要求4所述的定向诱变方法，其特征在于：收获步骤④中挑选的菌落的分生孢子，将所获分生孢子浸泡于海藻糖-乙醇水溶液中，将孢子悬液均匀涂布于培养基平板上，在阳光模拟辐射箱中以UVB的亚致死剂量对平板进行辐射，培养经辐射的存活孢子长出菌落，挑选生长势旺盛的菌落转移至产孢培养平板上，培养直至充分产孢，所获分生孢子用于UVB抗性测定，筛选出在前一轮最佳菌落基础上UVB抗性进一步显著增强的菌落，并同时测定生长产孢和毒力性状有无显著变化，选择最后一轮诱变筛选中UVB抗性表现理想的菌株即为高抗紫外辐射球孢白僵菌菌株。
  10. 权利要求1-3任一项所述的高抗紫外辐射球孢白僵菌菌株或权利要求4-9任一项所述的定向诱变方法制得的菌株在制备真菌杀虫剂中的应用。
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YANFANG SHANG; ZHIBING DUAN; WEI HUANG; QIANG GAO; CHENGSHU WANG;: "Improving UV resistance and virulence ofby genetic engineering with an exogenous tyrosinase gene", JOURNAL OF INVERTEBRATE PATHOLOGY, SAN DIEGO, CA, US, vol. 109, no. 1, 7 October 2011 (2011-10-07), US , pages 105 - 109, XP028355516, ISSN: 0022-2011, DOI: 10.1016/j.jip.2011.10.004 *
金丹等 (JIN, DAN ET AL.): "噻唑合成酶BbThI在球孢白僵菌紫外辐射损伤耐受中的作用和机制 (Non-official translation: Function and Mechanism of BbThI in UV Radiation Damage Tolerance in Beauveria Bassiana)", 重庆微生物学会第九届会员代表大会暨学术年会论文摘要集 (NON-OFFICIAL TRANSLATION: ABSTRACTS OF THE NINTH CONGRESS AND ANNUAL MEETING OF THE CHONGQING SOCIETY FOR MICROBIOLOGY), 18 December 2009 (2009-12-18) *

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