Space mutagenesis beauveria bassiana SCAUHB52 with high pathogenicity to bemisia tabaci and application thereof

Abstract

The invention provides space mutagenesis beauveria bassiana SCAUHB52 with high pathogenicity to bemisia tabaci and application of the space mutagenesis beauveria bassiana SCAUHB52, when UV-B irradiation time reaches 8 h, the colony diameter of the space mutagenesis beauveria bassiana SCAUHB52 is 35.33 + / -0.33 mm, and when UV-B irradiation time reaches 72 h, the germination rate of the space mutagenesis beauveria bassiana SCAUHB52 is 53.33 + / -2.03%; when the UV-B irradiation time reaches 8 hours, the sporulation quantity of the mutagenized SCAUHB52 strain is (10.22 + / -0.13) * 10 < 7 > spores / mL; the strain is used as an active component for preventing and controlling the harm of bemisia tabaci to plants, and has high pathogenicity to bemisia tabaci. The strain has relatively high pathogenicity to bemisia tabaci, has relatively stable characters in various aspects, can be applied to a biological prevention and control technology of bemisia tabaci harms to plants, has very good application prospects and popularization values, and can be subsequently subjected to commercial production.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control of Bemisia tabaci with high pathogenicity to plants. Specifically, it relates to an aerospace-mutagenized Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci and its application. Background Art

[0002] The development of China's pesticide industry started relatively late but has developed rapidly. The output has increased from 330,000 tons in 1983 to over 3.7 million tons in 2014 - 2016, making China the world's largest pesticide producer. According to the statistics of the China Pesticide Industry Association, as of the end of 2022, there were 44,811 pesticide products in a state of valid registration in China, among which 41,935 were pesticides for field use (Yuan Zhili, Ye Wenwu, et al. Research status and development suggestions of green pesticides in China [J]. Science China: Life Sciences, 2023, 53(11): 1643 - 1662.). With the continuous improvement of people's awareness of the safety and health of agricultural products, growing green and high-quality vegetables has become the development direction of the industry, posing more stringent requirements for pesticide management and use. Therefore, it is of great significance to change the traditional method mainly relying on chemical pesticide control, implement the eight-character plant protection principle of "prevention first, comprehensive control", adhere to agricultural control as the main method and biological control as the auxiliary method, and comprehensively manage pests with other control measures, which is of extremely important significance for maintaining sustainable green development (Yang Yunhua, Du Kaishu, Shi Mingwang. Research progress on biological control of entomogenous fungi [J]. Journal of Henan Institute of Science and Technology, 2011, 39(1): 34 - 37).

[0003] As an important group of microorganisms, entomopathogenic fungi are probably the most well-known microorganisms that infect plant pests. It is estimated that they cause the death of about 60% of insects and other arthropods (Bruck D. J. Fungal entomopathogens in the rhizosphere. [J] Biocontrol, 2010, 55: 103–112.). During the infection process, entomopathogenic fungi multiply extensively in the host hemocoel with hyphae, first consuming the nutrients in the host body, reducing the resistance of the host insect, and at the same time producing secondary metabolite toxins, which can inhibit the insect immune system, reduce the activity of detoxifying enzyme systems, disrupt the normal cell morphology and physiological functions of the host, and finally the host dies (Qu S, Wang S. Interaction of entomopathogenic fungi with the host immune system [J]. Developmental & Comparative Immunology, 2018, 83: 96-103.). It is recorded that there are more than 1,000 species in 100 genera of entomogenous fungi known worldwide, and the number of entomogenous fungal species discovered in China has also reached more than 405 species in more than 40 genera. Among them, the main species used as insecticides are Beauveria bassiana, Metarhizium anisopliae, Paecilomyces fumosoroseus, etc. And there are a total of 126 registered and effective biopesticide varieties in China (Liu Chunlai. Application of entomopathogenic fungi in biological control of agricultural and forest pests [J]. Heilongjiang Agricultural Sciences, 2017, (03): 68-73.).

[0004] Beauveria bassiana is an important entomopathogenic fungus belonging to the Deuteromycetes class. It causes Beauveria disease in insects such as whiteflies, aphids, thrips, grasshoppers, and beetles (Sinha, K.K., Choudhary, A.K., Kumari, P. Ecofriendly Pest Management for Food Security Entomopathogenic Fungi. Academic Press, 2016.). Multiple reports have shown that Beauveria bassiana has no particular host preference and can be used as a broad-spectrum fungicide (Uma Devi, K., Padmavathi, J., Uma Maheswara Rao, C., et al. A study of host specificity in the entomopathogenic fungus Beauveria bassiana (Hypocreales, Clavicipitaceae). Biocontrol Science and Technology, 2008. 18(10), 975-989.). It has been successfully used in crops such as tomatoes, potatoes, cabbages, beans, coffee, and corn. The fungal spores are sprayed on the affected crops in the form of wettable powders or emulsifiable suspensions, paralyzing the pests (Sandhu, S.S., Sharma, A.K., Beniwal, V., et al. Myco-biocontrol of insect pests: factors involved, mechanism, and regulation. Journal of Pathogens, 2012, 1-10.). In recent years, more and more studies have been conducted on the secondary metabolites of Beauveria bassiana. Among them, beauvercin and beauverolides are the main components of the active secondary metabolites of Beauveria bassiana and can be used for antibacterial, insecticidal, and tumor cell inhibition. It has been used in the control of agricultural pests and diseases such as insects and can also be used as raw materials for medical drugs and industrial production, showing great potential in future production and applications.

[0005] Space breeding is a breeding method that uses recoverable satellites or spacecraft to carry biological materials (plants, seeds, strains, etc.) into space, mutagenize the biological materials using the special environment in space, and then, after returning to the ground, select new materials and new varieties. It is also called space mutation breeding or aerospace mutation breeding, and is also known as space breeding (Ma Cheng, Ma Weichao, An Jianping, etc. Application and research progress of aerospace mutation breeding of microorganisms in China [J]. Hunan Agricultural Sciences, 2012(19): 5-8.). Special space flight conditions include long-term exposure to microgravity, radiation, and high vacuum environments, which enable microorganisms to adapt to the extreme space environment through their own phenotypic and genetic changes. Under long-term adaptation, favorable traits of strains can be improved, favorable variations such as creating new varieties can occur, and quantitative changes related to yield can also be induced, such as enhanced stress resistance and pathogenicity, and accelerated growth rate, thus obtaining excellent stress-resistant strains that cannot be produced on the ground. In addition, the characteristics of aerospace mutagenesis are that the variations are diverse, the mutation frequency is high, and the breeding period is shortened. The genetic resources created are superior to those of conventional breeding and can be developed into microbial products (Su, X.Y., Schmitz, G., Zhang, M.L., et al. Heterologous gene expression in filamentous fungi. Adv. Appl. Microbiol. 2012, 81, 1–61.; Liu Luxiang. Innovation of aerospace engineering breeding technology and current situation of industrial development [J]. Vegetables, 2012(09): 1-4.). It is reported that after mutagenesis treatment of Beauveria bassiana carried by the Shenzhou VIII spacecraft, 2 highly pathogenic strains with significantly higher mortality rates of the 4th instar larvae of Apriona germari than the original strain were screened out (Wang Xizhuo, Wang Laifa, Ma Jianwei, etc. Screening of highly virulent aerospace mutagenesis strains of Beauveria bassiana against Monochamus alternatus [J]. Acta Entomologica Sinica, 2014, 57(11): 1299-1305.).

[0006] The whitefly, Bemisia tabaci (Gennadius), belongs to the genus Bemisia in the family Aleyrodidae of the order Hemiptera. It is recognized by the Food and Agriculture Organization of the United Nations (FAO) as the second most harmful pest in the world and is also the only agricultural pest ever to be labeled a "super pest". It is listed as one of the 100 most dangerous invasive species (Wang Zehua, Shi Baocai, Wei Shujun, et al. Identification and control of Bemisia tabaci [J]. China Vegetables, 2012, 000(015): 27 - 28.). In the mid - 1990s, the large - scale invasion and spread of the "B - biotype" whitefly gradually developed into a major pest on crops such as vegetables, flowers, and tobacco in China and has drawn attention as a major pest (Luo Chen, Yao Yuan, Wang Rongjiang, et al. Identification of the biotypes of Bemisia tabaci in China using mtDNA COⅠ gene sequences [J]. Acta Entomologica Sinica, 2002, 45(006): 759 - 763.). Bemisia tabaci causes direct damage mainly by feeding on the leaves of host plants. Both nymphs and adults of Bemisia tabaci can pierce and suck the phloem of plants to extract sap, which can lead to leaf curling and chlorosis, greatly affecting the growth and development of plants and causing poor development of host plants. At the same time, it indirectly harms by secreting honeydew, which can cause sooty mold on host plants, affect photosynthesis, reduce fruit quality, and decrease crop yields (Oliveira M, Recursos E, Biotecnologia G, et al. Current status and collaborative research projects for Bemisia tabaci [J]. Crop Protection, 2001, 20(9): 709 - 723.). In addition, Bemisia tabaci is a vector for many plant viruses. It has the strongest ability to transmit geminiviruses on cucurbitaceae, leguminosae, euphorbiaceae, malvaceae, and solanaceae. Diseased plants show symptoms such as yellowing, dwarfing, and leaf curling (Hunter W B, Polston J E. Development of a continuous whitefly cell line [Homoptera: Aleryrodidae: Bemisia tabaci (Gennadius)] for the study of begomovirus [J]. Journal of Invertebrate Pathology, 2001, 77(1): 33 - 36.). Currently, the control of Bemisia tabaci mainly relies on chemical control techniques. However, due to abuse, improper use, and the increase in application doses, the problem of drug resistance is becoming increasingly serious (Lu Dingyihui, Liu Yong, Ge Daqing, et al. Damage characteristics of Bemisia tabaci and its integrated control techniques [J]. Yangtze River Vegetables, 2020(18): 69 - 71.).

[0007] On the other hand, due to the long-term use of chemical agents, a series of environmental health and food safety problems have emerged. In particular, the long-term application of a single drug can cause pests to develop drug resistance. So far, Bemisia tabaci has developed resistance to 68 active substances (https: / / www.pesticideresistance.org). Therefore, it is particularly important to use new, green and highly efficient biological control methods. Summary of the Invention

[0008] In view of this, on the one hand, the present invention provides an aerospace-mutagenized Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci. The preservation number is GDMCC No: 65962, the taxonomic name is Beauveria bassiana, the preservation address is the Guangdong Provincial Microbial Culture Collection Center, and the preservation time is February 28, 2025.

[0009] Furthermore, for the aerospace-mutagenized Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci, when the UV-B irradiation time reaches 8 h, the colony diameter is 35.33 ± 0.33 mm; when the UV-B irradiation time reaches 72 h, the germination rate is 53.33 ± 2.03%; when the UV-B irradiation time reaches 8 h, the sporulation amount of the SCAUHB52 strain is (10.22 ± 0.13) × 10 7 spores / mL.

[0010] Specifically, compared with the original strain SCAUSB53, the aerospace-mutagenized Beauveria bassiana SCAUHB52 has significant changes in colony diameter, spore germination rate and sporulation amount growth. The sporulation amount, colony diameter and spore germination rate of the mutagenized strain are significantly higher than those of the original strain. On the 15th day of growth, there are significant differences in the colony diameters of the original strain SCAUSB53 and the aerospace-mutagenized strain SCAUHB52 under different UV-B irradiation time treatments. When the UV-B irradiation time reaches 8 h, the original strain is more sensitive to UV-B, and the colony diameter grows to 22 mm, while the colony diameter of the mutagenized strain SCAUHB52 is 35.33 mm.

[0011] When the UV-B irradiation time reaches 8 h, the differences in spore germination rates between the original strain and the aerospace-mutagenized strain are also relatively obvious at 24 h, 48 h and 72 h. The spore germination rates of the aerospace-mutagenized strain are higher than those of the original strain at 24 h, 48 h and 72 h. The germination rate of the aerospace-mutagenized strain SCAUHB52 reaches 53.33% at 72 h, which is significantly higher than the spore germination rate of the original strain.

[0012] There are also significant differences in the sporulation amounts between the original strain and the space-mutagenized strain after UV-B irradiation treatment. When the UV-B irradiation time reaches 8 h, the sporulation amount of the original strain on the 15th day is 2.52×10 7 spores / mL, while the sporulation amount of strain SCAUHB52 reaches 10.22×10 7 spores / mL. The results of the biological characteristics of the strains show that the space-mutagenized strain is significantly higher than the original strain SCAUSB53 in terms of sporulation amount, colony diameter, and spore germination rate, indicating that the space-mutagenized strain has strong spore viability, belongs to excellent biocontrol bacteria, and has application potential.

[0013] The second aspect of the present invention also discloses an application of the space-mutagenized Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci, which is applied to the prevention and control of the harm of Bemisia tabaci to plants.

[0014] Furthermore, the application concentration of the space-mutagenized Beauveria bassiana SCAUHB52 is 1×10 4 ~1×10 8 conidia / mL.

[0015] Furthermore, the application concentration of the space-mutagenized Beauveria bassiana SCAUHB52 is 1×10 6 ~1×10 8 conidia / mL.

[0016] Furthermore, the application concentration of the space-mutagenized Beauveria bassiana SCAUHB52 is 1×10 8 conidia / mL.

[0017] Specifically, there are significant differences in the lethality rates of the conidia suspensions of different strains to Bemisia tabaci, and the pathogenicity of strain SCAUHB52 is significantly higher than that of other strains. Each treatment also shows significant differences in terms of time, and the mortality rates after three days of treatment with each strain are obvious. Generally speaking, at the same concentration of different strains, there are also differences in the mortality rates of Bemisia tabaci, and it is found that they will increase with the increase of the treatment time. When the concentration is 1×10 8 spores / mL, the mortality rate of strain SCAUHB52 on the 7th day reaches 100%, which is significantly higher than that of other mutagenized strains and the original strain, and it belongs to excellent biocontrol bacteria. Among them, from the 1st day to the 7th day of treatment, the lethality rate of the strain is higher than that of other strains.

[0018] To further evaluate the dose effect of strain SCAUHB52, a concentration gradient of 1×10 4 ~1×10 8 spores / mL was also set to verify the dose effect of strain SCAUHB52. When its spore concentration ≥ 1×10 6When the concentration was [X] spores / mL, the cumulative mortality rate of Bemisia tabaci exceeded 60% within 7 days, and showed a significant concentration-dependence (p < 0.05). At the concentration of 1×10 8 spores / mL, the lethality rate of strain SCAUHB52 was 100%, which was significantly higher than that of other concentrations (31.67% - 80.33%), indicating that the highly virulent strain SCAUHB52 had a dose-response advantage.

[0019] The third aspect of the present invention also discloses a microbial agent of the space-mutated Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci, which contains the space-mutated Beauveria bassiana SCAUHB52 strain with high pathogenicity to Bemisia tabaci.

[0020] Furthermore, the microbial agent of the space-mutated Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci is repeatedly applied every 5 - 7 days.

[0021] Furthermore, the application of the microbial agent of the space-mutated Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci. When the microbial agent is applied during the early nymph stage of Bemisia tabaci, the lethality rate to Bemisia tabaci can be increased.

[0022] Furthermore, the microbial agent of the space-mutated Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci contains a spore suspension of the space-mutated Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci.

[0023] Furthermore, the microbial agent of the space-mutated Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci contains spore powder of the space-mutated Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci.

[0024] The present invention also provides a method for obtaining the space-mutated Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci, including:

[0025] Inoculating the original strain, inoculating the required test strain in a cryotube, verifying the safety and reliability of the container and its contents during flight, and loading the test sample into the cabin;

[0026] Conducting in-orbit tests on the test strain. After the spaceship passes the tests, it is launched. The test strain undergoes in-orbit tests, the retrieved samples of the test strain carried in space are inoculated and sealed in PE tubes, and then placed in a test chamber and loaded into the cabin;

[0027] Space mutation. At a preset altitude in space for a preset time, the test strain is irradiated by the high-energy particle radiation belt to generate mutations, and the SCAUHB52 strain is obtained.

[0028] The present invention provides a space-mutated Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci, its application and microbial agent. The strain has high pathogenicity to Bemisia tabaci, and the traits of the strain are relatively stable in all aspects. The microbial agent containing the strain can be applied to the biological control technology of the damage of Bemisia tabaci to plants, and has good application prospects, promotion value, and can be commercially produced subsequently. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] By reading the following detailed description of the preferred embodiments, various other advantages and benefits will become clear to those of ordinary skill in the art. The drawings are only for the purpose of showing the preferred embodiments, and are not considered to be a limitation of the present invention. Moreover, throughout the drawings, the same reference numerals are used to represent the same components. In the drawings:

[0030] Figure 1 It is a comparison chart of the colony diameters of the original strain SCAUSB53 and the space-mutated SCAUHB52 strain provided by the embodiment of the present invention under different UV-B irradiation times; in the figure, (A) and (B) respectively represent the front view and the back view of the colony growth of the strain after being irradiated with UV-B for different times;

[0031] Figure 2 It is a spore colony diameter chart of the original strain SCAUSB53 and the space-mutated SCAUHB52 strain provided by the embodiment of the present invention;

[0032] Figure 3 It is a sporulation amount chart of the original strain SCAUSB53 and the space-mutated SCAUHB52 under different UV-B irradiation times provided by the embodiment of the present invention;

[0033] Figure 4 It is a curve chart showing the influence of different concentrations of spore suspension (SCAUHB52) on the cumulative mortality of the second-instar nymphs of Bemisia tabaci provided by the embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] The present invention will be further described below in conjunction with the drawings of the specification and specific embodiments. These embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out according to the conventional conditions in the art or according to the conditions recommended by the manufacturer. Unless otherwise defined, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art.

[0035] The sources of some materials involved in this application are as follows:

[0036] Original strain Beauveria bassiana SCAUSB53: It is an entomogenous fungus sub-cultured and preserved by the Engineering Research Center of Biocontrol, Ministry of Education, South China Agricultural University. It was collected under a tree in Dinghushan in September 2009.

[0037] Mutagenized strains, numbered SCAUSB17, SCAUSB19, SCAUSB26, SCAUSB33, SCAUSB44, SCAUSB48, SCAUHB52, SCAUHB60, SCAUHB65, SCAUHB90: All are derived from space mutagenesis treatment.

[0038] Example 1: Space mutagenesis breeding

[0039] Test strains

[0040] The test strain Beauveria bassiana (B. bassiana) SCAUSB53 of the present invention is an entomogenous fungus sub-cultured and preserved by the Engineering Research Center of Biocontrol, Ministry of Education, South China Agricultural University. It was collected under a tree in Dinghushan in September 2009. Since it was found in previous experiments that this strain has a fast growth rate, is easy to culture, and has a certain control effect on Bemisia tabaci, this strain was selected for space mutagenesis treatment.

[0041] Experimental method

[0042] Inoculate the required test strains in a freezing tube containing PDA. After the successful microgravity ground simulation test, according to the characteristics of the payload, screen and finalize the payload resources of the primary container and the secondary container on the spacecraft. It is also necessary to verify the safety and reliability of the container and its contents during flight, so as to conduct tests on indicators such as vibration and noise. Then, put the sample container into the test chamber and load it into the cabin; after the spaceship passes the test, it is launched. The test strains are subjected to on-orbit tests. The return capsule of the spaceship is recovered, and the sample of the test strains carried is retrieved. Inoculate the original strain SCAUSB53 into 5 PE tubes, seal them with sealing film, and put them into the test chamber and load them into the cabin. Conduct on-orbit tests in space through the spacecraft carried by the Long March 5B carrier rocket. The on-orbit altitude is about 300 - 8000 kilometers, the space flight is about 67 hours, and it passes through the high-energy particle radiation belt many times. Finally, return to the ground for sample collection.

[0043] Experimental results

[0044] A total of 5 mutagenized PE tubes of Beauveria bassiana were obtained from this space mutation. The 5 tube samples were placed in the Engineering Research Center of Ministry of Education for Biological Control, South China Agricultural University, stored in a refrigerator at 4°C, and used for subsequent experiments. One of the mutagenized strains, the space-mutagenized Beauveria bassiana SCAUHB52, was deposited, with the deposit number GDMCC65962, the deposit address being the Guangdong Provincial Culture Collection of Microorganisms, and the deposit date being March 7, 2025.

[0045] Example 2: Bioassay of the mutagenized strain SCAUHB52

[0046] Test insects

[0047] Test insects: Bemisia tabaci biotype B, reared in the Engineering Research Center of Ministry of Education for Biological Control, South China Agricultural University, and multi-generationally propagated using the host plant cotton. The temperature conditions for laboratory rearing were set as: temperature (27±2)°C, photoperiod L:D = 14:10, and relative humidity (45±10)%.

[0048] Test strains

[0049] From the space-mutagenized strains obtained in Example 1, the SCAUHB52 strain was selected. At the same time, multiple other mutagenized strains and the original strain SCAUSB53 were selected as control strains. 10 single-spore conidia were picked from each strain for a high-virulence experiment on Bemisia tabaci.

[0050] Experimental method

[0051] (1) Preparation of spore suspension

[0052] Each strain was inoculated onto PDA medium and cultured in an incubator at 26±1°C, L:D = 16:8 h for 10 d. After sufficient sporulation upon maturity, the mycelium was scraped into a 0.05% Tween-80 solution with a small spatula and stirred thoroughly with a magnetic stirrer to evenly disperse the conidia, which was then filtered through lens paper for oil immersion lenses. Counting was performed using a hemocytometer. The filtrate was dropped onto the counting area of the hemocytometer, covered with a cover slip, and observed under an inverted microscope. Five middle grids were selected using the five-point sampling method.

[0053] Calculation formula for spore suspension concentration: Concentration = 5 × number of middle grids × 5 × 10 4 (spores / mL). Finally, a suspension with a concentration of 1.0×10 8 spores / mL was prepared.

[0054] (2) Virulence determination of entomogenous fungi against Bemisia tabaci

[0055] Select cotton plants with basically the same growth vigor, many healthy young leaves. Select 3 to 5 true leaves from each cotton plant and connect the insect-receiving box. Introduce 100 pairs of Bemisia tabaci adults into each insect-receiving box, place them under room temperature conditions, and suck out the adults and put them back into the insect-rearing cage after 24 h of oviposition. When the eggs on the leaves hatch and develop to the second instar, pick off the leaves and immerse the leaves in the prepared spore suspension of 1.0×10 8 spores / mL for 10 s, and let them air-dry naturally at room temperature. Lay the treated leaves flat on a petri dish with 2% water agar for moisturizing. Mark 100 insects under a stereomicroscope, then seal the petri dish with plastic wrap and punch holes in the plastic wrap to keep it ventilated. Place the petri dish in a climate chamber at a temperature of 26±1°C, a relative humidity of 75 - 80%, and a photoperiod of L:D = 14:10 h. Observe and record the number of dead Bemisia tabaci under a stereomicroscope every day for 7 days. Treat 100 insects per strain, set 3 biological replicates, and soak them in 0.05% Tween-80 solution as a control.

[0056] (3) Data processing

[0057] Use SPSS 25.0 software to process and analyze the data, perform one-way ANOVA on each result, and use Tukey test to test for significant differences.

[0058] Experimental results

[0059] The experimental results show that there are significant differences in the lethal rates of conidial suspensions of different strains against Bemisia tabaci. The pathogenicity of the mutagenized strain SCAUHB52 is significantly higher than that of other strains, as shown in Table 1.

[0060] Table 1 Mortality rates (%) of tested entomogenous fungal strains (1×10 8 spores / mL) against Bemisia tabaci

[0061]

[0062] Each treatment also showed significant differences in terms of time, and the mortality rates after three days of treatment with each strain were obvious. Generally speaking, at the same concentration of different strains, there were also differences in the mortality rates of Bemisia tabaci, and it was found that they increased with the increase of treatment time. And when the mutagenized strain SCAUHB52 was at the same spore concentration, its mortality rate against Bemisia tabaci was higher than the lethal rates of other strains against Bemisia tabaci. Refer to the data in Table 1. When the concentration was 1×10 8 spores / mL, the mortality rate of strain SCAUHB52 reached 100% on the 7th day, which was significantly higher than that of other mutagenized strains, and it belonged to an excellent biocontrol bacterium. Among them, from the first day to the seventh day of treatment, the lethal rate of the strain was higher than that of other strains. To further evaluate the dose effect of strain SCAUHB52, this study set up 1×10 4 ~1×108 Concentration gradient of spores / mL.

[0063] As Figure 4 shown, when the spore concentration ≥ 1×10 6 spores / mL, the cumulative mortality rate of Bemisia tabaci within 7 days exceeded 60% and showed a significant concentration dependence (p < 0.05). At the concentration of 1×10 8 spores / mL, the lethality rate of strain SCAUHB52 was significantly higher than that of other concentrations. When the concentration was 1×10 8 spores / mL, the lethality rate of Bemisia tabaci was 100%, and the lethality rates of other concentrations were 31.67% - 80.33%, indicating that its high pathogenicity characteristics had a dose-response advantage.

[0064] Note: Attached Figure 4 in, the data were the cumulative mortality rates (%) of Bemisia tabaci at different time periods under each concentration treatment, that is, concentrations of 1×10 4 -1×10 8 spores / mL and the control. The error bars represent the standard error.

[0065] Note: For the mortality rates of Bemisia tabaci by each tested strain, the mortality rates of Bemisia tabaci at 2, 3, 5, and 7 days were selected. The data in the table were the mean ± standard error. After Tukey test, different lowercase letters indicated that there were significant differences in the mortality rates of Bemisia tabaci by each tested strain at the same concentration (p < 0.05).

[0066] Example 3: Biological characteristics of the strain before and after mutagenesis in the UV-B ultraviolet environment

[0067] Tested strains

[0068] Original strain SCAUSB53, space mutagenized strain SCAUHB52.

[0069] Experimental method

[0070] (1) Effect of UV-B on the germination rate of mutagenized strains

[0071] The highly virulent mutagenized strain was studied for its resistance to UV-B. The concentration of the highly virulent strain was prepared as 1×10 8 spores / mL. A 35W UV-B lamp was placed 35 cm above the liquid. After the bacterial liquid was irradiated for different ultraviolet times (0 h, 1 h, 2 h, 4 h, and 8 h), the sample was shaken at regular intervals. 1 mL of the bacterial liquid irradiated with ultraviolet for different times was added to 18 mL of Sabouraud medium and cultured in an incubator at 130 rpm and 26 °C. After culturing for 24 h, 48 h, and 72 h respectively, the spore germination rate of the strain was calculated by microscopic examination using a hemocytometer. There were 3 replicates for one treatment, and the irradiation with 0 h UV-B was used as the test control.

[0072] (2) Test the effect of UV-B irradiation on the colony growth of the strain

[0073] Using a pipette gun in a laminar flow hood, take 5 μL of the bacterial liquid treated with UV-B for different times and drop it in the center of a PDA plate, then seal it with plastic film. Set the temperature condition in the incubator to 25 ± 2 °C and continuously culture for 15 d. Measure and record the colony diameter by the cross method. Set 3 replicates for each treatment and use the irradiation of 0 h UV-B as the control.

[0074] (3) Test the effect of UV-B on the sporulation amount of the strain

[0075] After culturing the above-mentioned strain colonies for 15 d, scrape the mycelium into a conical flask containing 0.05% Tween-80, shake well to make a conidial suspension, and calculate the spore yield by taking five-point samples with a hemocytometer. Set 3 replicates for each treatment and use the irradiation of 0 h UV-B as the control. After treating the highly virulent strain after space mutation with UV-B according to the above test method, screen out the mutant strains with both highly virulent characteristics and high UV resistance based on the growth indexes of the strains for the next experimental study.

[0076] Test results

[0077] The test results show that the space-mutated strain SCAUHB52 compared with the original strain shows in the growth test indexes of colony diameter, spore germination rate and sporulation amount: the sporulation amount, colony diameter and spore germination rate of the space-mutated strain are significantly higher than those of the original strain. On the 15th day of growth, there are significant differences in the colony diameters of the original strain SCAUSB53 and the space-mutated strain SCAUHB52 under different UV-B irradiation times, as Figure 1 shown, when the UV-B irradiation time reaches 8 h, the original strain is more sensitive to UV-B, and the colony growth is 22 mm, while the colony diameter of the mutant strain SCAUHB52 is 35.33 mm.

[0078] When the UV-B irradiation time reaches 8 h, the differences in the spore germination rates of the original strain and the space-mutated strain are also obvious at 24 h, 48 h and 72 h. As shown in Table 2, the spore germination rates of the space-mutated strain at 24 h, 48 h and 72 h are all higher than those of the original strain. The germination rate of the space-mutated strain SCAUHB52 reaches 53.33% at 72 h, which is significantly higher than the spore germination rate of the original strain.

[0079] Table 2 Spore germination rates (%) of the strain under different UV-B irradiation treatment times

[0080]

[0081] There are also significant differences in the spore production of the original strain and the space-mutagenized strain under UV-B irradiation treatment. When the UV-B irradiation time reaches 8 h, the spore production of the original strain on the 15th day is 2.52×10 7 spores / mL, while the spore production of the SCAUHB52 strain reaches 10.22×10 7 spores / mL. The results of the biological characteristics of the strains show that the space-mutagenized strain is significantly higher than the original strain SCAUSB53 in terms of spore production, colony diameter, and spore germination rate, indicating that the space-mutagenized strain has strong spore viability, belongs to an excellent biocontrol bacterium, and has application potential.

[0082] In summary, the space-mutagenized Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci, its application, and the microbial agent disclosed in this embodiment have high pathogenicity to Bemisia tabaci. The various traits of this strain are relatively stable and can be applied to the biological control technology of the damage of Bemisia tabaci to plants, having good application prospects, promotion value, and can be commercially produced subsequently.

[0083] It should be noted that in the description of the present invention, the terms indicating directions or positional relationships such as "up", "down", "left", "right", "inside", "outside", etc. are based on the directions or positional relationships shown in the drawings. This is only for convenience of description and does not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention.

[0084] In addition, it should also be noted that in the description of the present invention, unless otherwise clearly specified and limited, the terms "installation", "connection", and "connection" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0085] Obviously, those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. An aerospace-mutagenized Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci, characterized in that, The preservation number is GDMCC65962.

2. The Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci obtained by space mutation as claimed in claim 1, characterized in that, When the UV-B irradiation time reaches 8 h, the colony diameter is 35.33 ± 0.33 mm. When the UV-B irradiation time reaches 72 h, the germination rate is 53.33 ± 2.03%. When the UV-B irradiation time reaches 8 h, the spore production of the mutagenized SCAUHB52 strain is (10.22 ± 0.13) × 10 7 spores / mL.

3. Use of the spaceflight mutagenesis Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci as described in claim 1 or 2, characterized in that, It is applied to the prevention and control of the harm of Bemisia tabaci to plants.

4. Use of the spaceflight-induced Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci as described in claim 3, characterized in that, The application concentration of the space-mutated Beauveria bassiana SCAUHB52 is 1×10 4 ~1×10 8 conidia / mL; preferably, 1×10 6 ~1×10 8 conidia / mL.

5. Use of the spaceflight mutagenesis Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci as described in claim 4, characterized in that, The application concentration of the space mutation Beauveria bassiana SCAUHB52 is 1×10 8 conidia / mL.

6. The microbial agent of the space-mutated Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci according to claim 1 or 2, characterized in that, It contains the spaceflight-induced Beauveria bassiana strain SCAUHB52 with high pathogenicity to Bemisia tabaci.

7. The microbial agent of Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci obtained by space mutation as claimed in claim 6, wherein It contains a spore solution or spore powder of the spaceflight-induced Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci.

8. Use of the microbial agent of Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci obtained by space mutation as claimed in claim 6, characterized in that, The said microbial agent is repeatedly applied every 5 - 7 days.

9. Use of the microbial agent of the space mutagenesis Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci as claimed in claim 6, characterized in that, The said microbial agent is applied during the early nymph stage of Bemisia tabaci.

10. The method for obtaining the space mutagenized Beauveria bassiana SCAUHB52 with high pathogenicity to Bemisia tabaci according to claim 1 or 2, characterized in that, It includes: Inoculating the original strain, inoculating the required test strain in a cryotube, verifying the safety and reliability of the container and its contents during flight, and loading the test sample into the cabin; Conducting on-orbit tests on the test strain, launching after the spacecraft passes the test, conducting on-orbit tests on the test strain, retrieving the samples of the test strain carried in space, inoculating the test strain into a sealed PE tube, and placing it in a test chamber and loading it into the cabin; Spaceflight mutagenesis, at a preset altitude in space and for a preset time of navigation, the test strain is irradiated by the high-energy particle radiation belt to produce mutagenesis, and the SCAUHB52 strain is obtained.

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