Screening of entomogenous fungi with high pathogenicity to prodenia litura and compound application of entomogenous fungi and tolfenpyrad

Abstract

The invention relates to the field of biological control, and discloses screening of entomogenous fungi with high pathogenicity to prodenia litura and a compound application of the entomogenous fungi and tetraniliprole. The beauveria bassiana strain with high toxicity to the prodenia litura is collected from a tobacco field in polder town in Jianghua County, Yangzhou City, Hunan Province. The invention further provides an efficient, pollution-free and residue-free insecticide combining the tolfenpyrad and the beauveria bassiana, and the insecticide has a synergistic effect. Research on the compounding effect of the tetratolfenpyrad and the beauveria bassiana shows that the insecticide can show a remarkable synergistic effect after being compounded, the prevention and treatment effect is improved, the use dosage of a single chemical pesticide can be obviously reduced, environmental pollution is reduced, and the problems that an existing pesticide is prone to generating resistance, not obvious in pesticide effect and the like are effectively solved.

Description

Technical Field

[0001] The invention relates to the field of plant protection, specifically to the field of biological control, and in particular to the biological control of Spodoptera litura. Background Art

[0002] Since the development of modern agriculture, pesticides have played an extremely important role in agricultural production. However, the correct use of pesticides is also crucial. Improper use of pesticides will cause environmental pollution. Today, the environmental pollution caused by pesticides is becoming increasingly serious. Pesticide pollution not only appears in soil, water and air, but also causes agricultural product pollution, which in turn endangers human health and poses a huge threat to human health (Zhu Cuixia. Pesticide use pollution problems and control measures in crop pest control [J]. Hebei Agricultural Machinery, 2024, (07): 67-69.).

[0003] Agricultural pests pose a huge threat to agriculture and horticulture throughout the world and cause inestimable economic losses. They can directly harm plants by sucking plant sap or other plant tissues, or indirectly harm crops by spreading plant pathogens. Today's pest control relies heavily on the use of chemical pesticides, but this also has a major drawback, such as the development of pesticide resistance and potential toxicity to humans and other non-target organisms. Therefore, there is an urgent need for a new green, healthy and sustainable pest control method. Biological control is one of the promising control methods (Wilberts L, Vuts J, Caulfield JC, Thomas G, Birkett MA, et al. (2022) Impact of endophytic colonization by entomopathogenic fungi on the behavior and lifehistory of the tobacco peach aphid Myzuspersicae var. nicotianae. PLOS ONE 17(9): e0273791.).

[0004] Insect pathogenic fungi can parasitize on the surface and body of insects. There are 100 genera and more than 800 species of insect pathogenic fungi recorded in the world, of which more than 400 species are found in China, including 215 species of insect pathogenic fungi. In recent years, due to the unavoidable harm of chemical control to the environment, people have gradually begun to pay attention to and study biological control. Using insect pathogenic fungi to control pests is one of the important biological control methods (Wang Yudi, Tian Chuanbei. Research progress on the application of insect pathogenic fungi in mite control [J]. Plant Medicine, 2024, 3(01): 8-21.). Insect pathogenic fungi (EPF) are natural enemies that affect insect populations. They have long been considered as biological control agents for many pests and are often regarded as effective biological control agents in integrated pest management (IPM) projects. Pathogenic fungi are commonly used to control pests by flooding or inoculating fungal propagules. Some pathogenic fungi have been commercially used to control plant-sucking pests (Sani I, Jamian S, Saad N, Abdullah S, Mohd HataE, et al. (2023) Inoculation and colonization of the entomopathogenic fungi, Isaria javanica and Purpureocilliumlilacinum, in tomato plants, and their effect on seedling growth, mortality and adult emergence of Bemisia tabaci (Gennadius). PLOS ONE 18(5): e0285666.).

[0005] Spodoptera litura, belongs to the family Noctuidae of the order Lepidoptera. Also known as the tobacco cutworm, it is a species native to Southeast Asia. It spread to India and became an important pest in the Asia-Pacific region. Spodoptera litura is a polyphagous agricultural pest that has a significant economic impact on a variety of crops in countries around the world. The species has more than 120 plant species and has great potential to invade new areas due to its ability to adapt to new climatic and ecological conditions (Tesari, T., Leksono, AS, & Mustafa, I. (2024). Effectiveness of botanical pesticide combined with Beauveria bassiana on mortality, nutritional index and fecundity of Spodoptera litura L. Cogent Food & Agriculture, 10 (1).). The Spodoptera litura is distributed in all regions of my country, north and south, and feeds on 389 host plants including sweet potato, cotton, soybean, tobacco, etc. The larvae have a omnivorous diet and a large appetite, and they have the characteristics of intermittent outbreaks. They are an important pest that causes serious damage to crops and is difficult to control (Zhang Wenmei, Wang Libing, Lu Tian, ​​et al. Study on the control effect of 9 biological pesticides on the Spodoptera litura in tobacco fields [J]. Biological Disaster Science, 2023, 46(04): 438-444.).

[0006] Beauveria is the first type of insect pathogenic fungus to be discovered and recognized. It is a natural "killer" of insect populations and has been recorded to have more than 700 insect and mite hosts. After the screening and cultivation of a large number of high-quality strains, as well as standardized artificial cultivation and experimental research, the high-efficiency insecticidal performance of Beauveria has been gradually discovered and recognized, and it has begun to be gradually developed into a biological control agent for different pests and mites. Due to the environmental friendliness and human and animal safety characteristics of Beauveria, it is recommended by FAO for promotion and research in pest control. It is the fungal microbial insecticide with the largest number of registrations and the widest control targets today (Nong Xiangqun, Wang Guangjun, Wang Yiyan, et al. Research and development progress of biological pesticide Beauveria insecticide [J]. Journal of Plant Protection, 2024, 51(02): 278-293.). Beauveria bassiana is not only the microbial insecticide with the longest research time and the largest application area in my country, but also an extremely effective biological pesticide for pest control in agriculture and forestry. It mainly controls insects through three invasion pathways: infecting the pest epidermis, digestive tract and respiratory tract (Yang Lei, Wang Xiaofu, Wei Wei, et al. Insect immune response to Beauveria bassiana and its application potential in pest control [J]. Journal of Zhejiang Agricultural Sciences, 2024, 36(04): 825-836.).

[0007] Since Beauveria bassiana has strong specialization and the virulence of different strains varies greatly, when conducting experiments on strain-compound pesticides, different regions and different types of pests need to correspond to different pathogenic fungi and screen for highly toxic strains (Shi Rongrong, Wang Qinglei. Research on the application of Beauveria bassiana to control peanut grubs [J]. Agricultural Science and Technology Communications, 2024, (03): 115-117.).

[0008] Since the use of Beauveria bassiana alone to control pests such as Spodoptera litura often fails to achieve the desired effect, Beauveria bassiana has a slow insecticidal effect, is greatly affected by the environment, and has an unstable effect. The combination of entomogenic fungi and pesticides to control pests has become a hot topic in recent years. Through the combination of fungi and pesticides, the harm of chemical pesticides to the environment and natural enemies can be reduced, the utilization efficiency of biological pesticides can be improved, and suitable chemical agents can be screened for compounding biological control fungi to alleviate the new problem of the rapid increase of resistance to pesticides in Spodoptera litura. Tetrazobium has the characteristics of biological pesticides such as super high efficiency, low toxicity (the preparation is nearly non-toxic), low residue, and pollution-free. The use of Beauveria bassiana and tetrazobium antagonism to control Spodoptera litura can overcome the problem of slow effect of Beauveria bassiana and at the same time delay the effect of pest resistance, thereby achieving a good insecticidal effect. Summary of the invention

[0009] The purpose of the present invention is to develop a biological control of Spodoptera litura in tobacco fields, which is an important demand of agricultural production, and to provide a strain of Beauveria bassiana with high toxicity to Spodoptera litura collected from tobacco fields in Taoxu Town, Jianghua County, Yongzhou City, Hunan Province. At the same time, a highly efficient, pollution-free, and residue-free insecticide of tetrazobacteramide and Beauveria bassiana with synergistic effects is provided. The present invention studies the compounding effect of tetrazobacteramide and Beauveria bassiana and finds that the insecticide can show significant synergistic effects after compounding, improves the control effect, and can significantly reduce the dosage of chemical pesticides, reduce environmental pollution, and effectively solve the problems of existing pesticides being prone to resistance and having unclear efficacy.

[0010] The technical solution of the present invention is as follows:

[0011] The collected, isolated and purified strains were subjected to toxicity determination and analysis to screen out strains with high toxicity to Spodoptera litura. The experiment found that strains of different genera or the same species may have different pathogenicity to Spodoptera litura, and thus have different degrees of pathogenicity. According to the experimental results of the present invention, combined with the comprehensive consideration of the indicators for screening excellent strains, among which pathogenicity is an important indicator for measuring the biological control potential of fungal strains, and the growth rate of colonies or hyphae is another important evaluation indicator for evaluating excellent insect pathogenic fungi, strain GZYKYJH20 was 10 8 The strain GZYKYJH20 had a high pathogenicity to the nymphs of Spodoptera litura at spores / mL. Considering the colony growth rate of the strain, the strain GZYKYJH20 was an excellent strain for causing disease to the nymphs of Spodoptera litura.

[0012] The fungus was isolated from soil collected from tobacco fields in Taoxu Town, Jianghua County, Yongzhou City, Hunan Province. The colony diameter was 63.75 mm and the spore production was 1.237×10 8 spores / ml, and the spore germination rate exceeds 95%. Figure 1 As shown, the colony is velvety in the middle, powdery at the edge, white on the surface, orange-yellow on the back, with a few wrinkles. The spores are oval and nearly spherical. Through molecular biology, it was further verified and identified that it belongs to Beauveria bassiana.

[0013] The present invention is based on the fact that the screened Beauveria bassiana GZYKYJH20 has a certain biological control effect on Spodoptera litura. In order to further seek a better control effect, after a large number of explorations and exploratory experiments, it is found that the combination of tetrazobactam and Beauveria bassiana GZYKYJH20 can produce a significant synergistic effect, has a significant control effect on Spodoptera litura, and finally completes the present invention.

[0014] Therefore, the present invention provides an entomogenous fungus strain with high pathogenicity to Spodoptera litura, which is Beauveria bassiana, and its deposit number is GDMCC NO:65072.

[0015] The present invention further provides application of the strain in biological control of Spodoptera litura.

[0016] The present invention also provides an insecticide containing tetrazobactam and Beauveria bassiana as effective ingredients, wherein the Beauveria bassiana is the strain; the insecticide is used for preventing and controlling Spodoptera litura.

[0017] Specifically, the Beauveria bassiana is in the form of a suspension or spore powder of Beauveria bassiana spores.

[0018] Preferably, the dosage ratio of Beauveria bassiana and tetrazobactam is: the final concentration of Beauveria bassiana is 1×10 4 ~1×10 8 Conidia / mL, preferably 1×10 7 ~1×10 8 conidia / mL, the final concentration of tetrazobactam is 0.01-0.8 mg / L, preferably 0.05-0.5 mg / L.

[0019] More preferably, the ratio of Beauveria bassiana to tetrazobactam is 1×10 4 ~1×10 8 Conidia / mL: 0.05-0.8 mg / L, preferably the ratio of Beauveria bassiana to tetrazobactam is 1×107 ~1.0×10 8 Conidia / mL: 0.2~0.4mg / L.

[0020] More preferably, the ratio of Beauveria bassiana to tetrazobactam is 1×10 8 Conidia / mL: 0.4 mg / L.

[0021] The present invention further provides application of the insecticide in preventing and controlling Spodoptera litura.

[0022] The present invention also provides a method for controlling Spodoptera litura, which comprises applying the strain or the insecticide to crops or environments where Spodoptera litura needs to be controlled, specifically, applying the strain or the insecticide at the early stage of the emergence of Spodoptera litura.

[0023] Biomaterial preservation information: The Beauveria bassiana GZYKYJH20 involved in the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, China) on August 29, 2024, and its preservation number is GDMCC NO: 65072, and its classification name is: Beauveria bassiana. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the colony morphology of GZYKYJH20.

[0025] Figure 2 This is the spore morphology of GZYKYJH20.

[0026] Figure 3 This is the electrophoresis diagram of ITS gene amplification, where M is a marker, the marker of ITS gene is DL2000 marker, and lanes 1-3 are three replicates of Beauveria bassiana strain GZYKYJH20.

[0027] Figure 4 Phylogenetic tree. DETAILED DESCRIPTION

[0028] The following is an introduction through the research and development process and specific implementation methods of the present invention, but does not constitute a limitation of the present invention.

[0029] Example 1: Initial screening of strains

[0030] 1. Test insects

[0031] The eggs of Spodoptera litura were purchased from Keyun Biological Co., Ltd. and were reared with artificial diet in an artificial climate chamber of the Engineering Research Center of Ministry of Education for Biological Control to form a population of Spodoptera litura. The rearing conditions were temperature (27±1)℃, photoperiod L14:D10, and relative humidity (75±5)%.

[0032] 2. Experimental methods

[0033] 1) Collection of soil samples and isolation and purification of fungi

[0034] Soil samples were collected. When sampling, about 100g of soil was taken from 10 to 15cm below the surface soil, which was sealed in a plastic bag and taken back to the laboratory for processing. The soil samples were sieved to remove stones and debris, and then 10g of clean soil was suspended in 90mL of 0.1% Tween 80 solution, shaken, and allowed to stand for 15min. 2mL of the supernatant was diluted in 8mL of 0.05% Tween 80 to prepare a soil suspension. 0.1mL of the soil suspension was inoculated on a Bengal red agar medium plate, and the suspension was evenly pushed under the surface of the plate with a triangular glass scraper. It was cultured at 25℃ for 3 to 7d, and then a single colony was cut with an inoculation loop and inoculated on a PDA plate for further culture to obtain the Beauveria bassiana strain GZYKYJH20. The mycelium block was cut and transplanted to the PDA slope, and cultured continuously and stored in a refrigerator at 4℃.

[0035] 2) Observation of biological characteristics of strains

[0036] Preparation of potato glucose medium: 200g potato, 20g glucose, 20g agar, 1000ml water, inoculate the purified strain on PDA medium and culture for 7-10 days, observe the color, texture, size, height, surface decoration, edge, exudate, etc. of the front and back of the colony and take pictures. Determination of colony growth rate: Inoculate the purified white bassiana strain on the PDA plate, place it in a 26℃ incubator and culture it for 7-10 days for use, use a 5mm puncher to punch holes in the cultured medium, then inoculate it into a new PDA plate, seal it with a sealing film, and culture it in a 26℃ constant temperature incubator, repeat 5 times. Use the cross method to measure the colony diameter and record it, and calculate the average daily growth.

[0037] Determination of spore production: Use a pipette to transfer 500 μL of a 1×10 4 The spore suspension of spores / mL was inoculated on a 90mm culture dish. The culture dish was rotated at a constant speed to make the liquid evenly distributed on the surface of the culture medium, and then sealed with a sealing film and placed in a 26°C incubator for constant temperature culture. Repeat 5 times. After 15 days of culture, 5 colonies with uniform growth in the culture medium were taken with a 10mm puncher, added to 20mL of 0.5‰ Tween-80 solution, and stirred at medium speed for 20 minutes using a constant temperature magnetic stirrer to prepare a spore suspension. The content was determined using a hemocytometer.

[0038] Determination of spore germination rate: Take 1 mL of spores with a concentration of 1×10 5 Spore suspensions with a concentration of spores / mL were added to 10 mL of SDY medium and cultured at 26°C and 220 r / min for 24 h, repeated 5 times. An appropriate amount of bacterial liquid was dropped on a glass slide, placed under a microscope, and the germination of spores was observed and recorded. Spores with a germ tube length greater than or equal to the spore diameter were considered to have germinated. No less than 50 spores were examined under the microscope for each treatment, and the examination was repeated 5 times.

[0039] The calculation formula is: Spore germination percentage (%) = (number of germinated spores in the field of view / total number of spores in the field of view) × 100

[0040] 3) Molecular biological identification

[0041] The purified strains were identified by molecular biology.

[0042] DNA was extracted using the CTAB method, which included the following steps:

[0043] (a) After Beauveria bassiana strain GZYKYJH20 was cultured on a PDA plate for one week, mycelium was carefully scraped into a mortar and quickly and thoroughly ground by adding liquid nitrogen;

[0044] (b) Take an appropriate amount of ground mycelium and quickly transfer it to a centrifuge tube, add 300 μL of DNA extraction solution preheated at 65°C, mix thoroughly, add an equal volume of chloroform / isoamyl alcohol (24 / 1) mixture, mix well, incubate in a water bath at 65°C for 1 h, shake gently once every 10 min, and centrifuge at 12,000 rpm for 5 min at room temperature;

[0045] (c) Slowly aspirate the supernatant into another new centrifuge tube, slowly add an equal volume of chloroform / isoamyl alcohol (24 / 1) mixture for re-extraction, and centrifuge at 12000 rpm for 5 min at room temperature;

[0046] (d) Slowly pipette the supernatant into another new centrifuge tube, add 1 / 10 volume of sodium acetate and an equal volume of pre-cooled isopropanol, and let stand at room temperature for 15 min;

[0047] (e) adding 70% ethanol to wash the precipitate twice;

[0048] (f) Slowly suck out the ethanol with a pipette close to the tube wall and place it in a clean bench to dry;

[0049] (g) The obtained precipitate was dissolved in 50 μL of TE;

[0050] (h) The extract was detected by 1% agarose gel electrophoresis and the remaining extract was stored at -20°C.

[0051] The primers used in the PCR of fungal DNA were universal primers, and the amplification conditions were as follows. The PCR products were subjected to 1% agarose gel electrophoresis (containing EB), the buffer was 1×TAE, the voltage was 150V, the current was 220mA, and Marker DL2000 was used as a molecular weight standard reference. After the electrophoresis, the gel was detected on a gel imager and photographed and recorded.

[0052] The specific PCR products were sequenced by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. Sequence alignment and phylogenetic tree construction: The sequencing results were searched for related sequences with high homology using BLAST, and the Cluster W in MEGA5.1 software was used for sequence alignment and multiple sequence alignment. A Neighbour-joining (NJ) phylogenetic tree was constructed in the software.

[0053] 4) Toxicity determination and analysis on second-instar larvae of Spodoptera litura

[0054] a. Preparation of Beauveria bassiana spore suspension

[0055] Take Beauveria bassiana that has been cultured for about 10-14 days and add distilled water containing 0.05% Tween-80 in a clean bench to scrape off the colonies, wash out the spores in the culture dish, transfer to a sterilized triangular flask, and stir at medium speed for 20 minutes using a constant temperature magnetic stirrer to evenly disperse it to prepare a mother solution. Dilute the bacterial solution 100 times according to the 10-fold dilution method, count the number of conidia with a hemocytometer under a microscope, and repeat 5 plates. Then dilute the conidia concentration to 1×10 8 Spores / mL for later use.

[0056] b. Toxicity test of Beauveria bassiana against Spodoptera litura

[0057] The feed was fed at a concentration of 1×10 8 The larvae were soaked in a spore suspension of spores / mL for about 30 seconds, and then fed with it. Sterilized 0.05% Tween-80 water was used as a control, 30 larvae were treated in each treatment, and each treatment was repeated 3 times. After the inoculation was completed, all culture dishes (with a diameter of 12 cm) were ventilated with insect-proof nets after opening holes and placed in a biochemical incubator for breeding. Regular observations and records were made every day for 5 consecutive days, and the dead insects were removed in time. The dead insects were placed in a moisturizing culture at 26°C and confirmed to be effectively lethal based on the characteristics of the growths on the body surface of the 2nd instar larvae of the dead insects. The infected dead insects were dead insects.

[0058] c. Perform concentration gradient analysis on highly virulent strains

[0059] The spore powder of the strain with better virulence effect was taken and prepared into a concentration of 1×10 8The spore suspension was diluted in a certain gradient, and the final dilution concentration was 1×10 8 spores / mL, 1×10 7 spores / mL, 1×10 6 spores / mL, 1×10 5 spores / mL, 1×10 4 The mortality rate of the second-instar larvae of Spodoptera litura at different concentrations of Beauveria bassiana strain GZYKYJH20 was determined by feeding method.

[0060] Experimental Results

[0061] 1. Isolation of eight strains of Beauveria bassiana

[0062] The soil sample was sieved to remove stones and debris, and then 10g of clean soil was suspended in 90mL of 0.1% Tween 80 solution, shaken, and allowed to stand for 15 minutes. 2mL of the supernatant was diluted in 8mL of 0.1% Tween 80 to prepare a soil suspension. The soil suspension was then diluted 10 times. 0.5mL of the soil suspension and the dilution were inoculated on a Bengal red agar medium plate, repeated 4 times, and the suspension was evenly spread under the surface of the plate with a triangular glass scraper. The suspension was cultured at 25℃ for 3-7 days, and then a single colony was cut with an inoculation loop and inoculated on a PDA plate for further culture. If there was contamination, the inoculation was continued until a single colony grew in the culture medium.

[0063] 2. Biological characteristics of GZYKYJH20 strain

[0064] On the PDA plate, at 26°C, the colony diameter was 63.75 mm and the spore production was 1.237×10 8 spores / ml, and the spore germination rate exceeds 95%. Figure 1 As shown, the colony is velvety in the middle, powdery at the edge, white on the surface, orange-yellow on the back, with a few wrinkles. The spores are oval and nearly spherical.

[0065] 3. Molecular Biology Identification

[0066] (1) Figure 3 As shown, the electrophoresis bands in each lane are relatively clear, and the gene fragment of the strain can be accurately judged to be about 600 bp based on the migration distance of the PCR product. Lanes 1-3 are three repeats of the test strain GZYKYJH20.

[0067] (2) The obtained ITS sequence of the strain genotype was combined with the sequences of four Beauveria bassiana strains downloaded from GenBank to construct a molecular phylogenetic tree. The results are as follows: Figure 4As shown, the strain GZYKYJH20 belongs to Beauveria bassiana, which is a Beauveria bassiana. The comprehensive identification results of morphological characteristics and physiological and biochemical characteristics showed that the strain GZYKYJH20 belongs to the genus Beauveria bassiana.

[0068] 4. Screening and toxicity analysis of highly toxic strains of Spodoptera litura

[0069] The virulence of the eight strains was tested by dipping the larvae into a concentration of 1×10 8 The larvae were immersed in a spore suspension of spores / mL for about 5 seconds, and then moved into a culture dish containing feed for observation. Sterile 0.05% Tween-80 water was used as a control, 30 larvae were treated in each treatment, and each treatment was repeated 3 times. After the inoculation was completed, all culture dishes (12 cm in diameter) were sealed with plastic wrap and punctured for ventilation, and then placed in a biochemical incubator for breeding. Regular observations and records were made every day for 5 consecutive days, and the insect corpses were removed in time. The insects were placed in a moisturizing culture at 26°C and confirmed to be effectively killed based on the characteristics of the growths on the body surface of the second-instar larvae of the Spodoptera litura. The infected insect corpses were dead insects. The experimental results showed that at the same concentration, the pathogenicity of the eight entomogenous fungi varied. When the spore suspension concentration was 1×10 8 spores / ml, after 5 days of treatment, the corrected mortality rates of the eight strains against the second-instar larvae of Spodoptera litura were between 17.31% and 57.84%, and the strain GZYKYJH20 had the highest corrected mortality rate of 57.84%, which was significantly higher than that of the other strains. This also showed that the strain GZYKYJH20 had a stronger pathogenicity than the other strains.

[0070] The results showed that the strain GZYKYJH20 had a good lethal effect on the second-instar larvae of Spodoptera litura, and the pathogenic effect of GZYKYJH20 was significantly better than other strains, indicating that it is a potential excellent biocontrol bacterium.

[0071] Example 2: Compatibility study of highly toxic strains and tetrazobactam and screening of combined doses

[0072] 1. Effect of tetrazobactam on spore germination rate of strain GZYKYJH20

[0073] During the test, the strain to be tested was first transferred to a PDA plate and cultured at 26±1°C. After conidia were produced, the spores were washed with 0.05% Tween-80 sterile water, stirred with a magnetic stirrer, filtered, and counted by hemocytometer. The spores were then prepared into 1×10 6 spores / mL spore suspension for later use. Dissolve tetrazolyl thiocarb in acetone to prepare a 10 mg / L agent for later use. Add tetrazolyl thiocarb to 50 mL SDA liquid culture medium to prepare SDA liquid culture medium with tetrazolyl thiocarb concentrations of 0.05, 0.25, 0.5, 1.0 and 2.0 mg / L.6 Spores / mL spore suspension was added to the culture medium with different concentrations of tetrazolyl amide, and placed in a shaker at 180rpm and 25℃ for shaking culture. Samples were taken with a pipette at 24h, 48h and 72h, and the spore germination of each treatment was examined under a microscope on a blood cell counting plate, and the spore germination rate was calculated. Each treatment was repeated 3 times, and the liquid culture medium with sterile water was used as a blank control.

[0074] 2. Effect of tetrazobactam on the mycelial growth of strain GZYKYJH20

[0075] During the test, tetrazobium amide corresponding to the concentration of 2.5.1 was added to each PDA culture dish (7.5 cm in diameter), and the drug solution was evenly applied to the surface of the culture medium with a high-temperature sterilized triangular glass rod to prepare drug-containing PDA culture medium plates of different concentrations. Take a Beauveria bassiana culture dish with uniform mycelium distribution, punch the bacterial cake with a 5 mm diameter puncher on the clean bench, place the bacterial cake in the center of the drug-containing PDA plate for inoculation, and culture it in a light incubator at 26±2℃. Use the cross method to measure the colony diameter on the 3rd, 5th and 7th days, respectively, in mm. Each treatment was repeated 5 times, and the PDA culture medium with sterile water was used as a control.

[0076] 3. Toxicity test of strain GZYKYJH20 and tetrazobactam combination against Spodoptera litura

[0077] a. Toxicity test of tetrazobactam to the second-instar larvae of Spodoptera litura

[0078] Use sterile water to prepare tetrazobactam agents with concentrations of 0.05, 0.1, 0.2, 0.4, and 0.8 mg / mL, and the remaining steps are the same as above.

[0079] b. Toxicity test of the combination of strain GZYKYJH20 and tetrazobactam against the second-instar larvae of Spodoptera litura

[0080] According to the combination in Table 1, indoor toxicity test was carried out, and the experimental method and steps were the same as above.

[0081] Table 1. Combination of strain GZYKYJH20 and tetrazolylpyrim

[0082]

[0083]

[0084] c.Data statistics and analysis

[0085] SPSS 19.0 software was used to process and analyze the experimental data. One-way ANOVA was used to analyze the results of the mortality rate of the second-instar larvae of Spodoptera litura, and Tukey's test was used to test the significance of the difference. The data calculation formula was based on Huang et al. (2013), and the specific formula is as follows:

[0086] Me=Ma+Mb×(1-Ma)

[0087]

[0088] In the formula, the meanings of each item are as follows:

[0089] Ma: actual corrected mortality of tetrazobactam alone against the second-instar larvae of Spodoptera litura;

[0090] Mb: actual corrected mortality of the highly virulent strain against the second-instar larvae of Spodoptera litura alone;

[0091] Mab: Actual corrected mortality of fungicide combination against second instar larvae of Spodoptera litura.

[0092] Experimental results:

[0093] 1. Virulence analysis of GZYKYJH20

[0094] The mortality of the second-instar larvae of Spodoptera litura infected with different concentrations of Beauveria bassiana spore suspensions after 5 days is shown in Table 2. The results showed that the spore concentration was lower than 1×10 6 spores / ml, the lethality of Beauveria bassiana to the second-instar larvae of Spodoptera litura was less than 5%, and the spore concentration was 1×10 7 The lethality of spores / ml is 20%, and the spore concentration is 1×10 8 The lethality rate of spores / ml was 53.33%. As the spore concentration increased, the lethality rate to Spodoptera litura also increased.

[0095] Table 2 Corrected mortality of spore suspensions of different concentrations against second-instar larvae of Spodoptera litura (%)

[0096] Treatment (spore concentration) / ml Corrected mortality rate (%) <![CDATA[1×10 8 ]]> 54.46±1.67 <![CDATA[1×10 7 ]]> 43.67±5.0 <![CDATA[1×10 6 ]]> 21.33±1.67 <![CDATA[1×10 5 ]]> 12.67±1.67 <![CDATA[1×10 4 ]]> 5.91±1.67 TW-80 Sterile Water 0.00

[0097] 2. Effects of tetrazobactam on spore germination rate and mycelial growth of strain GZYKYJH20

[0098] The test results in Table 3 show that when treated for 24 hours, the spore germination rates of strain GZYKYJH20 were 96.32%, 95.98%, 95.64%, 94.21%, and 90.72% respectively at 0.05, 0.10, 0.20, 0.4, and 0.8 mg / L concentrations, which were significantly different from the blank control (98.82%). When treated for 48 hours, the spore germination rates of strain GZYKYJH20 were 98.86%, 98.46%, 98.08%, 97.83%, and 97.66% respectively at 0.05, 0.10, 0.20, 0.4, and 0.8 mg / L concentrations of tetrazolyl thiamethoxam, which were not significantly different from the sterile water blank control (98.87%). When treated for 72 hours, the effect of different concentrations of tetrazolyl thiocarb on the spore germination rate of strain GZYKYJH20 showed no significant difference compared with the sterile water blank control. In the treatments of 0.05, 0.10, 0.20, 0.4, and 0.8 mg / L tetrazolyl thiocarb, the spore germination rates of strain GZYKYJH20 were 98.84%, 98.70%, 98.11%, 98.01%, and 97.83%, respectively, which were not significantly different from the blank control (98.98%).

[0099] The test results in Table 3 show that when the concentrations of tetrazolyl thiocarbamide were 0.05 and 0.1 mg / L, the colony diameters of strain GZYKYJH20 on the 7th day were 43.95 mm and 43.85 mm, which were not significantly different from the blank control (43.60 mm). The colony diameters of strain GZYKYJH20 treated with tetrazolyl thiocarbamide at concentrations of 0.2, 0.4 and 0.8 mg / L were 41.04 mm, 39.98 mm and 36.54 mm on the 7th day, which were significantly different from the blank control (44.46 mm).

[0100] The results showed that low and medium concentrations of tetrazobactam had good compatibility with Beauveria bassiana GZYKYJH20 and could be compounded in subsequent tests and practical applications.

[0101] Table 3 Effects of tetrazobactam on spore germination rate and mycelial growth of strain GZYKYJH20

[0102]

[0103] Note: According to Tukey test, different lowercase letters in the same column indicate significant differences in pathogenicity between different strains (P < 0.05)

[0104] 3. Toxicity test of strain GZYKYJH20 and tetrazobactam combination against Spodoptera litura

[0105] 1) Results of the toxicity test of tetrazobactam against the second-instar larvae of Spodoptera litura

[0106] The corrected mortality rates of the second-instar larvae of Spodoptera litura treated with different concentrations of tetrazobactam were different and the differences were significant. With the increase of tetrazobactam concentration, the corrected mortality rate of the second-instar larvae of Spodoptera litura also increased. When the tetrazobactam concentration was 0.05-0.8 mg / L, the corrected mortality rate of the second-instar larvae of Spodoptera litura after treatment was 15.00%-98.82%.

[0107] Table 4 The results of toxicity test of different concentrations of tetrazobactam to the second instar larvae of Pseudomonas littoralis

[0108]

[0109]

[0110] Note: According to Tukey's test, different lowercase letters in the same column indicate significant differences in pathogenicity between different strains (P < 0.05). 2) Results of toxicity test of strain GZYKYJH20 and tetrazopyram against the second-instar larvae of Spodoptera litura

[0111] The toxicity of strain GZYKYJH20 and tetrazolyl thiocarb to the 2nd instar larvae of Spodoptera litura was as follows: when different concentrations of tetrazolyl thiocarb (0.2 and 0.4 mg / L) were used alone to treat the 2nd instar larvae of Spodoptera litura, the corrected mortality rates after the 5th day were 39.67% and 53.50%, respectively; when different concentrations of strain GZYKYJH20 (1×10 7 , 1×10 8 spores / mL) were used to treat the 2nd instar larvae of Spodoptera litura, the corrected mortality rates on the 3rd day were 23.91% and 28.33%, and on the 5th day were 43.67% and 54.46%, respectively.

[0112] The mortality rate of the combination of different concentrations of tetrazobacteramide and different concentrations of strain GZYKYJH20 on the second instar larvae of Spodoptera litura on the 5th day was significantly higher than that of each single agent. In the toxicity test of the combination of tetrazobacteramide and strain GZYKYJH20 on the second instar larvae of Spodoptera litura, the combination T6 (1×10 8 +0.2), T7(1×10 8 +0.4), T8(1×10 7 +0.2), T9(1×10 7 +0.4) showed synergistic effects 1 to 5 days after treatment, with the highest corrected mortality rates of 93.26%, 100%, 88.76%, 95.04%, and 100%, respectively, and the differences were not significant. These four combinations all had good synergistic effects. Combined with the dosage and actual lethal effect, 1×10 8The best combination was the spores / ml Beauveria bassiana GZYKYJH20 and 0.4mg / L tetrazobactam.

[0113] Table 5 Corrected mortality of the second instar larvae of Spodoptera litura by synergistic effect of strain GZYKYJH20 and tetrazobactam

[0114]

[0115] Note: The data in the table are mean ± standard error. After Tukey test, different lowercase letters indicate significant differences between different combination concentrations at the same time (P < 0.05). The values ​​in brackets represent the expected mortality rate and the calculated chi-square value χ 2 , * indicates a combination with synergistic effect.

[0116] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. An entomogenous fungus strain with high pathogenicity to Spodoptera litura, characterized in that: It is Beauveria bassiana ( Beauveria bassiana ), the deposit number is GDMCC NO: 65072.

2. Use of the strain as claimed in claim 1 in biological control of Spodoptera litura.

3. An insecticide containing tetrazobactam and Beauveria bassiana as active ingredients, wherein the Beauveria bassiana is the strain as claimed in claim 1; the insecticide is used for controlling Spodoptera litura.

4. The insecticide according to claim 3, characterized in that The Beauveria bassiana is in the form of a suspension or spore powder of Beauveria bassiana spores.

5. The insecticide according to claim 4, characterized in that The dosage ratio of Beauveria bassiana and tetrazobactam is as follows: the final concentration of Beauveria bassiana is 1×10 4 ~1×10 8 Conidia / mL, preferably 1×10 7 ~1×10 8 conidia / mL, the final concentration of tetrazobactam is 0.01~0.8 mg / L, preferably 0.05~0.5 mg / L.

6. The insecticide according to claim 3, characterized in that The ratio of Beauveria bassiana to tetrazobactam is 1×10 4 ~1×10 8 Conidia / mL: 0.05~0.8mg / L, preferably the ratio of Beauveria bassiana to tetrazobactam is 1×10 7 ~1.0×10 8 Conidia / mL: 0.2~0.4mg / L.

7. The insecticide according to claim 3, characterized in that The ratio of Beauveria bassiana to tetrazobactam is 1×10 8 Conidia / mL: 0.4 mg / L.

8. Use of the insecticide according to any one of claims 3 to 7 in controlling Spodoptera litura.

9. A method for controlling Spodoptera litura, characterized in that: Apply the strain according to claim 1 or the insecticide according to any one of claims 3 to 7 to crops or environments where Spodoptera litura needs to be controlled.

10. The method according to claim 9, characterized in that Apply at the early stage of Spodoptera litura emergence.

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