A pesticide for controlling fruit fly based on Beauveria bassiana and its testing method

Abstract

The present invention relates to the field of plant protection technology, and more particularly to a medicament for preventing and treating citrus fruit flies based on Beauveria bassiana and a test method thereof. The medicament is prepared by mixing Beauveria bassiana with an added drug, and the Beauveria bassiana is a Beauveria bassiana Bb‑swu10 strain, and the Beauveria bassiana Bb‑swu10 strain is deposited in the General Microbiological Center of the China Microbiological Culture Collection Committee on May 15, 2024, with a deposit number of CGMCC No.41228, and the added drug is at least one of avermectin and highly effective cypermethrin. A medicament for preventing and treating citrus fruit flies based on Beauveria bassiana provided by the present invention and a test method thereof, which shows a synergistic effect by compounding Beauveria bassiana with avermectin or highly effective cypermethrin, greatly improves the control effect, and can significantly reduce the dosage of chemical pesticides, reduce environmental pollution, and effectively solve the problems that pesticides are prone to resistance and the efficacy is not obvious, and has good application prospects and promotion value.

Description

Technical Field

[0001] The invention relates to the technical field of plant protection, and in particular to a Beauveria bassiana-based agent for preventing and controlling fruit flies and a testing method thereof. Background Art

[0002] Bactrocera dorsalis belongs to the order Diptera and the family Tephritidae. It is an important supplementary quarantine pest in my country. Adult Bactrocera dorsalis mainly lays eggs on fruits and vegetables. The ovipositor pierces the fruit skin, causing mechanical damage, which causes the tissue around the ovipositor to be infected with pathogens, providing conditions for the invasion of other pathogens and causing lesions. The eggs hatch into larvae in the fruit. The newly hatched larvae have the habit of feeding in groups. As the age increases, the larvae's food intake gradually increases, and they feed deep into the flesh, forming tunnels in the flesh tissue, which often causes the fruit to fall or rot and become inedible.

[0003] When the fruit rots, the mature larvae drill out of the fruit and move on the ground by bouncing to find a suitable place to pupate. Some mature larvae that have not left the fruit can also pupate inside the fruit. The larvae hide in the fruit and cause harm, making it difficult for fruit flies to detect them, and then spread over long distances with the damaged fruits.

[0004] At present, the prevention and control of the fruit fly is mainly based on chemical control, combined with the use of sex attractants, fruit bagging and physical and mechanical control. However, the long-term irrational use of chemical pesticides has led to serious pesticide resistance in the wild population of the fruit fly in some areas. The sex attractants currently used mainly have a good effect on male adults; measures such as fruit bagging are not only time-consuming and labor-intensive, but also costly.

[0005] Entomogenous fungi have a wide range of hosts, strong pathogenicity, good control effects, no pollution to the environment, and are harmless to humans, crops, and natural enemies of pests. As a microbial insecticide, it has advantages that other biological agents cannot replace.

[0006] Beauveria bassiana is the most widely used in biological pest control. my country has been using Beauveria bassiana to control agricultural pests such as Ostriniafurnacalis and Leguminivora glycinivorella since the 1950s.

[0007] Deng Chunsheng et al. (1995) reported that the application of Beauveria bassiana emulsifiable concentrate in the field can kill 80% of peanut grubs. Hu Benjin et al. (2013) reported that Beauveria bassiana granules produced by solid fermentation can be used to control corn borers and can show a high control effect. Yang Hongping et al. (1992) reported that the Beauveria bassiana strains with high toxicity to the larvae of the European beetle (Melolontha hippocastani) were isolated and screened from the corpses of the Northeast Black Gill Beetle (Holotrichia dimphoria) and the Dark Gill Beetle (Holotrichia morose). At present, my country has become one of the countries with the largest production and application of Beauveria bassiana.

[0008] To this end, we designed a pesticide for controlling the citrus fruit fly based on Beauveria bassiana and its testing method to provide another technical solution to the above technical problems. Summary of the invention

[0009] Based on this, it is necessary to provide a pesticide for controlling citrus fruit fly based on Beauveria bassiana and a testing method thereof to solve the technical problems raised in the above background technology.

[0010] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0011] A pesticide for controlling citrus fruit fly based on Beauveria bassiana, the pesticide being prepared by mixing Beauveria bassiana and an additive, the Beauveria bassiana being the Beauveria bassiana Bb-swu10 strain, the Beauveria bassiana Bb-swu10 strain being deposited in the General Microbiology Center of the China National Microbiological Culture Collection on May 15, 2024, with a deposit number of CGMCC No. 41228.

[0012] As a preferred embodiment of the Beauveria bassiana-based agent for controlling Bactrocera dorsalis and its testing method provided by the present invention, the added drug is at least one of avermectin and highly effective cypermethrin.

[0013] As a preferred embodiment of the agent for controlling citrus fruit fly based on Beauveria bassiana and its testing method provided by the present invention, the culture of Beauveria bassiana Bb-swu10 is a spore suspension of Beauveria bassiana Bb-swu10, with a concentration of 1×10 5 ~1×10 8 Spores / mL.

[0014] As a preferred embodiment of the agent for controlling Bactrocera dorsalis based on Beauveria bassiana and its testing method provided by the present invention, the mixed solution of the Beauveria bassiana spore suspension and avermectin is obtained according to the following ratio:

[0015] 5mL concentration is 2.5×10 5 spores / mL of Beauveria bassiana spore suspension + 1mL of avermectin with a concentration of 9.664 mg / L.

[0016] As a preferred embodiment of the agent for controlling Bactrocera dorsalis based on Beauveria bassiana and its testing method provided by the present invention, a mixed solution of the Beauveria bassiana spore suspension and high-efficiency cypermethrin is obtained according to the following ratio:

[0017] 5mL concentration is 2.5×10 5 spores / mL of Beauveria bassiana spore suspension + 1mL of 150mg / L high-efficiency cypermethrin.

[0018] A testing method for a pesticide for controlling Bactrocera dorsalis based on Beauveria bassiana, used for any of the above items, comprising the following steps:

[0019] Spray the spore suspension of Beauveria bassiana Bb-swu10 onto the adult bodies of Bactrocera dorsalis;

[0020] The larvae of Bactrocera dorsalis were impregnated with a spore suspension of Beauveria bassiana Bb-swu10;

[0021] The spore suspension of Beauveria bassiana Bb-swu10 was impregnated into the pupae of Bactrocera dorsalis.

[0022] As a preferred embodiment of the testing method of the agent for controlling Bactrocera dorsalis based on Beauveria bassiana provided by the present invention, a spore suspension of Beauveria bassiana Bb-swu10 is sprayed on adult insects of Bactrocera dorsalis, and the steps are as follows:

[0023] Pick out 3-day-old adults of Bactrocera dorsalis, freeze them in a -20℃ refrigerator for 1.5 min, and transfer them to ice after they become unconscious, so that they remain in a continuous coma state.

[0024] Arrange the adults of Bactrocera dorsalis neatly in a culture dish, place the culture dish on the stage of the spray tower, take 1 mL of different concentrations of pesticides for spraying, the spraying time is 1 min, and 30 heads are treated each time;

[0025] Place the infected test insects in a 10cm×10cm×10cm cubic mesh cage and leave them at room temperature for 30 minutes to wake up;

[0026] After the fruit fly wakes up, place the cube mesh cage in a constant temperature incubator for observation;

[0027] The survival rate of the citrus fruit fly was counted every 24 hours, and the dead insects were removed in time, and the statistics were continued for 8 days.

[0028] As a preferred embodiment of the testing method of the Bactrocera dorsalis agent based on Beauveria bassiana provided by the present invention, the larvae of Bactrocera dorsalis are impregnated with a spore suspension of Beauveria bassiana Bb-swu10, and the steps are as follows:

[0029] Pick out the third-instar larvae of Bactrocera dorsalis that have just jumped out of the glass culture dish and are about to pupate;

[0030] The larvae were placed in a sterile petri dish containing 3 mL of spore suspension and immersed for 1 min, with 30 larvae treated each time;

[0031] Take out the immersed larvae and dry them, then put them into a new sterile culture dish, and finally put the culture dish into the incubator for observation.

[0032] Larval mortality was counted every 24 hours, and the dead insects were removed promptly. The mortality was continued until the larvae pupated and emerged.

[0033] As a preferred embodiment of the testing method of the agent for controlling Bactrocera dorsalis based on Beauveria bassiana provided by the present invention, the spore suspension of Beauveria bassiana Bb-swu10 is impregnated with Bactrocera dorsalis pupae, and the steps are as follows:

[0034] Pupae were picked out 24h, 48h, 96h, and 144h after pupation, washed with sterile water, dried for 30min, and placed in a 1.5mL centrifuge tube containing 1mL of spore suspension for 1min. 30 pupae were treated each time.

[0035] Take out the immersed pupae and dry them, then put them into a new sterile culture dish, and finally put the culture dish into the incubator for observation.

[0036] The mortality rate of pupae was counted every 24 h and continued until 12 days after pupae emerged.

[0037] It can be seen without a doubt that the above-mentioned technical solution of the present application can definitely solve the technical problem to be solved by the present application.

[0038] At the same time, through the above technical solutions, the present invention has at least the following beneficial effects:

[0039] The invention provides a pesticide for controlling citrus fruit fly based on Beauveria bassiana and a testing method thereof. The pesticide is compounded with avermectin or high-efficiency cypermethrin to show a synergistic effect, which greatly improves the control effect, can significantly reduce the dosage of chemical pesticides, reduce environmental pollution, and effectively solve the problems of easy resistance to pesticides and unclear efficacy. The pesticide has good application prospects and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0041] Figure 1 This is a colony morphology diagram of Bb-swu10 of the present invention;

[0042] Figure 2 This is a morphological diagram of conidia and hyphae of Bb-swu10 of the present invention under an optical microscope;

[0043] Figure 3 This is a morphological diagram of Bb-swu10 of the present invention infecting Bactrocera dorsalis;

[0044] Figure 4 It is a schematic diagram of molecular identification of the present invention;

[0045] Figure 5 This is a schematic diagram of the phylogenetic tree of Bb-swu10 based on rDNA-ITS sequence of the present invention. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0047] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings.

[0048] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features and technical solutions in the embodiments may be combined with each other.

[0049] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0050] Reference Figure 1-Figure 5 , a pesticide for controlling the citrus fruit fly based on Beauveria bassiana and a testing method thereof.

[0051] Example 1: Screening of highly toxic strains

[0052] 1. Preparation of spore suspension

[0053] The strain was inoculated on a potato dextrose agar (PDA) culture dish for activation, sealed with a sealing film, and placed in an inverted culture in a constant temperature incubator at a temperature of 26±1°C, a photoperiod of L:D=0h:24h, and a relative humidity of 75±5%. Take the entomogenous fungus that has been cultured for about 10 to 14 days, add distilled water containing 0.05% Tween-80 in the clean bench to scrape off the colonies, wash out the spores in the dish, transfer to a 15mL sterile centrifuge tube, and shake on a vortex shaker for 10 minutes until the conidia are completely shaken off the hyphae and dispersed. Filter the spore suspension after the shaking with gauze to remove the culture medium fragments and hyphae in the suspension and prepare it into 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 3 plates. Then the conidia concentration is configured to 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 The concentration of spores / mL was set aside.

[0054] LT of 13 strains against Bactrocera dorsalis 50 Determination

[0055] Pick 3-5 day old adults of Bactrocera dorsalis, freeze them in -20℃ refrigerator for 1.5min, transfer them to ice after they become unconscious, and keep them in a continuous coma state; arrange adults of Bactrocera dorsalis neatly in a culture dish, place the culture dish on the stage of the spray tower, take 1mL (1×10 8 spores / mL) spore suspension was sprayed for 1 minute, and 30 insects were treated each time; the infected test insects were placed in a 10cm×10cm×10cm cube mesh cage and left at room temperature for 30 minutes to wake up; after the orange fruit fly woke up, the cube mesh cage was placed in a constant temperature incubator (temperature 27±1℃, photoperiod L:D=14h:10h, relative humidity 75±5%) for cultivation and observation. The survival rate of the orange fruit fly was counted every 24 hours, and the insect corpses were removed in time, and the statistics were continued for 8 days.

[0056] Calculate the number of 13 strains in 1×10 8 LT of 3-5-day-old adults of Bactrocera dorsalis at spore / mL concentration 50 The results showed that Bb-swu03, Bb-swu04, Bb-swu08, Bb-swu10, and Bb-swu18 had a good lethal rate against 3- to 5-day-old adults of the citrus fruit fly, and their infectivity was significantly better than other strains, making them potential excellent biocontrol bacteria.

[0057] Table 113 strains against 3-5 day old adults of Bactrocera dorsalis LT 50

[0058]

[0059] LC of 3 and 5 strains against Bactrocera dorsalis 50 Determination

[0060] Pick 3-5 day old adults of Bactrocera dorsalis, freeze them in -20℃ refrigerator for 1.5min, transfer them to ice after they become unconscious, and keep them in a continuous coma state; arrange adults of Bactrocera dorsalis neatly in a culture dish, place the culture dish on the stage of the spray tower, and take 1mL of different concentrations (1×10 5 ~1×10 9 spores / mL) spore suspension was sprayed for 1 minute, and 30 insects were treated each time; the infected test insects were placed in a 10cm×10cm×10cm cube mesh cage and left at room temperature for 30 minutes to wake up; after the orange fruit fly woke up, the cube mesh cage was placed in a constant temperature incubator (temperature 27±1℃, photoperiod L:D=14h:10h, relative humidity 75±5%) for cultivation and observation. The survival rate of the orange fruit fly was counted every 24 hours, and the insect corpses were removed in time, and the statistics were continued for 8 days.

[0061] Calculate the expression of 5 strains at different concentrations (1×10 5 ~1×10 9 LC of 5.34 spores / mL for 3- to 5-day-old adults of Bactrocera dorsalis 50 The results showed that the lethal concentration of the strains to Bactrocera dorsalis gradually decreased with the increase of time. Among them, Bb-swu10 had the strongest toxicity, with a lethal concentration of only 3.73×10 5 spores / mL, significantly better than other strains.

[0062] Table 2 LC of 5 strains against 3-5 day old adults of Bactrocera dorsalis 50 (Spores / mL)

[0063]

[0064] In the present invention, the Beauveria bassiana Bb-swu10 strain was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on May 15, 2024, with a deposit number of CGMCC No.41228.

[0065] Example 2: Identification of strain Bb-swu10

[0066] The strain is an entomogenous fungus isolated from peach aphid (number Bb-swu10). The strain was identified by morphological and molecular identification under laboratory conditions. Figure 4Figure 1 shows the sequence diagram of PCR amplification in molecular identification (Figure A) and the electrophoresis diagram of PCR amplification products (Figure B).

[0067] 1. Observation of biological characteristics of strain Bb-swu10

[0068] The strain Bb-swu10 was inoculated on PDA medium and cultured for 10 days. The colony color and matrix color, colony morphology, size, etc. were observed and photographed.

[0069] Determination of colony growth rate: 1 μL of a concentration of 1×10 6 A spore suspension of spores / mL was dropped in the center of the culture dish, sealed with a sealing film, and placed in an incubator (temperature 26±1°C, photoperiod L:D=0h:24h, relative humidity 75±5%) for 5 times. The diameter was measured by the cross method at 5d and 10d.

[0070] Determination of spore germination rate: Add spore suspension into SDY medium and dilute to 1×10 6 The spores were placed in a 1.5 mL centrifuge tube at a concentration of 100 μL / mL and placed in a constant temperature shaker at 26°C and 200 r / min for 24 h, repeated 5 times. 10 μL of bacterial solution 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.

[0071] Determination of spore production: 1 μL spore concentration is 1×10 6 Spore suspension of spores / mL was dropped in the center of the culture dish, sealed with a sealing film and placed in an incubator (temperature 26±1°C, photoperiod L:D=0h:24h, relative humidity 75±5%) for cultivation, and repeated 5 times. After 10 days, a 10mm punch was used to take a uniformly growing colony in the culture medium, add 4mL of 0.05% Tween-80 solution, and oscillate on a vortex oscillator for 10 minutes until the conidia were completely shaken off the hyphae and dispersed. The spore suspension after the shaking was filtered with gauze to remove the culture medium fragments and hyphae in the suspension and prepare a mother solution. The content was determined using a hemocytometer.

[0072] The experimental results showed that on the PDA plate, at 26°C, after 10 days of culture, the colony diameter was 3.57 mm, the spore germination rate exceeded 75%, and the spore production was 5.075×10 8 Spores / mL. The growth rate of colonies or mycelium and the spore production of strains are important evaluation indicators for evaluating excellent insect pathogenic fungi. Good growth rate and spore production characteristics are conducive to large-scale industrial fermentation production in the later stage.

[0073] like Figure 1As shown, the colonies are powdery, the colonies are light yellow, and the matrix is ​​yellow. The spores are oval and nearly spherical (such as Figure 2 (shown)

[0074] Table 3 Colony diameter, spore germination rate and spore production of strain Bb-swu10

[0075]

[0076] 2. Molecular Biology Identification

[0077] Molecular biological identification was performed on the strain Bb-swu10. DNA was extracted using the CTAB method, including the following steps:

[0078] (a) After Beauveria bassiana Bb-swu10 was cultured on a PDA plate for one week, the mycelium was carefully scraped into a 1.5 mL centrifuge tube and quickly and thoroughly ground by adding liquid nitrogen;

[0079] (b) Add 200 μL of CTAB extract (with reducing agent added) preheated to 65°C into the centrifuge tube.

[0080] (c) Place the centrifuge tube in a 65°C water bath for 30 to 60 minutes, gently shaking the tube every 10 minutes.

[0081] (d) After the water bath is completed, cool for 2 minutes, add 150 μL of chloroform-isoamyl alcohol mixture, shake vigorously to mix, and centrifuge at 10000-12000 rpm for 5-10 minutes.

[0082] (e) Transfer the supernatant from the upper centrifuge tube to a new centrifuge tube.

[0083] (f) Add an equal volume of isopropanol to the supernatant, mix thoroughly by inversion, place at -20°C for precipitation for more than 30 min, centrifuge at 12,000 rpm for 10 min, and discard the supernatant.

[0084] (g) The precipitate was washed once with 75% ethanol, centrifuged at 12000 rpm for 3-5 min, the supernatant was discarded, the precipitate was naturally dried and dissolved in 50 μL of deionized water or TE buffer, and incubated at 37°C for 30 min.

[0085] (h) Detection was performed by electrophoresis using 1% agarose gel, and the remaining extracts were stored at -20°C

[0086] The primers for PCR amplification of fungal DNA were universal primers: ITS1: 5'-CCGTAGGTGAACCTGCGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3'. Amplification conditions: 94℃ pre-denaturation for 5min; 95℃ 20s, 60℃ 30s, 72℃ 30s, 35 cycles; 72℃ 5min.

[0087] The target fragment was amplified by PCR and sent to Qingke Biotechnology for sequencing; the fungal ITS sequence was compared with known sequences in the NCBI GenBank database using BLAST to identify the strain, and the phylogenetic tree was constructed using the maximum likelihood method. Figure 5 .

[0088] Experimental Example 3: Further analysis of the virulence of strain Bb-swu10

[0089] 1. Toxicity test of Beauveria bassiana strain Bb-swu10 against the third instar larvae of Bactrocera dorsalis

[0090] Pick out the third-instar larvae of Bactrocera dorsalis that have just jumped out of the glass culture dish and are about to pupate, and place the larvae in 3 mL (1×10 8 The larvae were immersed in a sterile culture dish (9 cm × 1.5 cm) containing spore suspension (100 spores / mL) for 1 min, and 30 larvae were treated each time; the immersed larvae were taken out and the excess water was sucked, and then placed in a new sterile culture dish, and finally the culture dish was placed in an incubator (temperature 27 ± 1 ° C, photoperiod L: D = 14h: 10h, relative humidity 75 ± 5%) for cultivation and observation. The larval mortality rate was counted every 24 hours, and the dead insects were removed in time, and the statistics were continued until the larvae pupated and emerged.

[0091] The infection of Bb-swu10 on the third instar larvae of Bactrocera dorsalis is shown in Table 4. As can be seen from Table 4, the strain has little effect on the third instar larvae of Bactrocera dorsalis, but after pupation, it will significantly affect its emergence rate. The morphological picture of Bb-swu10 infected with Bactrocera dorsalis is shown in Figure 3 ; Figure A is a morphological picture of Bb-swu10 infecting citrus fruit fly larvae for 5 days; Figure B is a morphological picture of Bb-swu10 infecting citrus fruit fly pupae for 5 days; Figure C is a morphological picture of Bb-swu10 infecting citrus fruit fly adults for 1 day; Figure D is a morphological picture of Bb-swu10 infecting citrus fruit fly adults for 5 days.

[0092] Table 4 Results of infection of the third instar larvae of Bactrocera dorsalis by strain Bb-swu10

[0093]

[0094] 2. Determination of the toxicity of strain Bb-swu10 to pupae of different ages of Bactrocera dorsalis

[0095] Pupae were selected after 24h, 48h, 96h, and 144h of pupation, washed with sterile water, and dried for 30min. 8 The pupae were immersed in a 1.5 mL centrifuge tube containing spore suspension (spores / mL) for 1 min, and 30 pupae were treated each time; the pupae were taken out after immersion and the excess water was sucked off, and then placed in a new sterile culture dish, and finally the culture dish was placed in an incubator (temperature 27±1℃, photoperiod L:D=14h:10h, relative humidity 75±5%) for cultivation and observation. The mortality rate of pupae was counted every 24 hours, and the statistics were continuously counted until 12 days after the pupae emerged.

[0096] The infection of Bb-swu10 on pupae of different ages of Bactrocera dorsalis is shown in Table 5. As can be seen from Table 5, the mortality rate of the pupae is six-day-old pupae> one-day-old pupae> two-day-old and four-day-old pupae, that is, the infection of Bb-swu10 has a greater impact on the late-stage pupae of Bactrocera dorsalis; as for the mortality rate of Bactrocera dorsalis adults after emergence, the mortality rate increases with the increase of pupal age, that is, six-day-old pupae> four-day-old pupae> two-day-old pupae> one-day-old pupae.

[0097] Table 5 Results of strain Bb-swu10 infecting pupae of different ages of Bactrocera dorsalis

[0098]

[0099] Experimental Example 4: Toxicity determination of two pesticides against 3-day-old adults of Bactrocera dorsalis

[0100] The spray method was used to prepare different concentration gradients of pesticides; 3-day-old adults of Bactrocera dorsalis were selected and frozen in a -20℃ refrigerator for 1.5 minutes. After the test insects were unconscious, they were transferred to ice to keep them in a continuous coma state; the adults of Bactrocera dorsalis were neatly arranged in a culture dish, the culture dish was placed on the stage of the spray tower, 1mL of different concentrations of pesticides were sprayed, the spray time was 1 minute, and 30 insects were treated each time; the infected test insects were placed in a 10cm×10cm×10cm cube gauze cage and left at room temperature for 30 minutes to wake up; after the Bactrocera dorsalis woke up, the cube gauze cage was placed in a constant temperature incubator (temperature 27±1℃, light cycle L:D=14h:10h, relative humidity 75±5%) for cultivation and observation. The survival rate of Bactrocera dorsalis was counted every 24 hours, and the insect corpses were removed in time, and the statistics were continuously counted for 48 hours.

[0101] SPSS19.0 software was used to process and analyze the experimental data.

[0102] The results showed that after the two pesticides with different concentrations were used to treat the 3-day-old adults of Bactrocera dorsalis, the mortality rate increased with the increase of concentration; among them, avermectin had the strongest toxicity to the 3-day-old adults of Bactrocera dorsalis, and LC 50 Only 9.664mg / L. The LC of avermectin will be used in the future.50 and LC of cypermethrin 30 Mixed with Beauveria bassiana.

[0103] Table 6 The median lethal concentration (LC) of avermectin and cypermethrin against 3-day-old adults of Bactrocera dorsalis 50 )

[0104]

[0105] Experimental Example 5: Compatibility study of strain Bb-swu10 with two drugs

[0106] 1. Effects of the two agents on the spore germination rate of strain Bb-swu10

[0107] The spore suspension was added to SDY medium and diluted to 2×10 6 Conidia / mL concentration, the prepared spore suspension was mixed with avermectin at a ratio of 1:1 to prepare 1x, 0.5x, and 0.2x LC 50 Concentration: 1:1 with highly effective cypermethrin to make 1x, 0.5x, 0.2x LC 30 Concentration; the mixed solution of each concentration was placed in a 1.5mL centrifuge tube and cultured in a constant temperature shaker at 26℃ and 200r / min for 24h, repeated 5 times. 10μL of bacterial solution 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 in a single microscopic examination for each treatment, and the microscopic examination was repeated 5 times.

[0108] The experimental results in Table 7 show that avermectin has an inhibitory effect on the germination of Bb-swu10 spores, and the inhibitory effect increases with the increase of concentration; highly effective cypermethrin has a certain inhibitory effect on the germination of Bb-swu10 spores, but at 0.2 times LC 30 There was basically no inhibitory effect at this concentration.

[0109] 2. Effects of the two agents on the mycelial growth of strain Bb-swu10

[0110] The spore suspension was diluted to 1 × 10 6 Concentration of spores / mL; dilute the agent with 0.05% Tween-80 solution, and dilute avermectin to 1 times, 0.5 times, and 0.2 times LC 50 Concentration: highly effective cypermethrin diluted to 1 times, 0.5 times, 0.2 times LC 30 Concentration: 100 μL of diluted solution was evenly spread on the surface of PDA culture medium plate, and 1 μL of 1×10 6The spore suspension with a concentration of spores / mL was placed in the center of the infected plate; the control group was smeared with 0.05% Tween-80; it was sealed with a sealing film and placed in an incubator for dark culture, repeated 5 times; the diameter was measured by the cross method at 5d and 10d, and the mycelial growth inhibition rate was calculated.

[0111] The experimental results in Table 7 show that on the 5th day, avermectin was 1 and 0.5 times LC 50 The concentration of 0.2 times LC 50 concentration; on the 10th day, avermectin promoted the growth of Bb-swu10 hyphae, and the promotion effect showed a trend of first increasing and then weakening with the increase of concentration; on the 5th day, highly effective cypermethrin had a certain inhibitory effect on the growth of Bb-swu10 hyphae; on the 10th day, highly effective cypermethrin also had an inhibitory effect on the growth of Bb-swu10 hyphae, and at 0.5 times LC 30 The inhibitory effect was strongest at this concentration.

[0112] 3. Effects of the two agents on the spore production of strain Bb-swu10

[0113] The spore suspension was diluted to 1 × 10 6 Concentration of spores / mL; dilute the agent with 0.05% Tween-80 solution, and dilute avermectin to 1 times, 0.5 times, and 0.2 times LC 50 Concentration: highly effective cypermethrin diluted to 1 times, 0.5 times, 0.2 times LC 30 Concentration: 100 μL of diluted solution was evenly spread on the surface of PDA culture medium plate, and 1 μL of 1×10 6 Spore suspension at a concentration of spores / mL was placed in the center of the infected plate; the control group was smeared with 0.05% Tween-80; it was sealed with a sealing film and placed in an incubator for dark culture, repeated 5 times; at 10 days, a 10mm borer was used to take a uniformly growing colony in the culture medium, and 4mL of 0.05% Tween-80 solution was added, and it was shaken on a vortex shaker for 10 minutes until the conidia were completely shaken off the hyphae and dispersed, and the spore suspension after the shaking was filtered with gauze to remove the culture medium fragments and hyphae in the suspension and prepare a mother solution. The blood cell counting plate was used to determine its

[0114] content.

[0115] The experimental results in Table 7 show that avermectin has a certain inhibitory effect on the spore production of Bb-swu10, and the inhibitory effect increases with the increase of concentration; highly effective chlorpyrifos has a significant promoting effect on the spore production of Bb-swu10, and the promoting effect is strongest at 75 mg / L.

[0116] The results showed that lower concentrations of avermectin and highly effective cypermethrin were compatible with strain Bb-swu10 and could be mixed in subsequent experiments and actual use.

[0117] Table 7 Compatibility of strain Bb-swu10 and two drugs

[0118]

[0119] Example 6. Ratio of Beauveria bassiana spore suspension to avermectin and high-efficiency cypermethrin

[0120] The mixed solution of Beauveria bassiana spore suspension and avermectin is obtained according to the following ratio:

[0121] 5mL concentration is 2.5×10 5 spores / mL of Beauveria bassiana spore suspension + 1mL of avermectin with a concentration of 9.664 mg / L.

[0122] 2mL concentration is 2.5×10 5 spores / mL of Beauveria bassiana spore suspension + 1mL of avermectin with a concentration of 9.664 mg / L.

[0123] 1mL concentration is 2.5×10 5 spores / mL of Beauveria bassiana spore suspension + 1mL of avermectin with a concentration of 9.664 mg / L.

[0124] 1mL concentration is 2.5×10 5 spores / mL of Beauveria bassiana spore suspension + 2mL of avermectin with a concentration of 9.664 mg / L.

[0125] 1mL concentration is 2.5×10 5 spores / mL of Beauveria bassiana spore suspension + 5mL of avermectin with a concentration of 9.664 mg / L.

[0126] Preferably, the most preferred ratio of Beauveria bassiana Bb-swu10 and avermectin is 5 mL with a concentration of 2.5×10 5 spores / mL of Beauveria bassiana spore suspension + 1mL of avermectin with a concentration of 9.664 mg / L.

[0127] The mixed solution of Beauveria bassiana spore suspension and highly effective cypermethrin is obtained according to the following ratio:

[0128] 5mL concentration is 2.5×10 5 spores / mL of Beauveria bassiana spore suspension + 1mL of 150mg / L high-efficiency cypermethrin.

[0129] 2mL concentration is 2.5×10 5 spores / mL of Beauveria bassiana spore suspension + 1mL of 150mg / L high-efficiency cypermethrin.

[0130] 1mL concentration is 2.5×10 5 spores / mL of Beauveria bassiana spore suspension + 1mL of 150mg / L high-efficiency cypermethrin.

[0131] 1mL concentration is 2.5×10 5 spores / mL of Beauveria bassiana spore suspension + 2mL of 150mg / L of highly effective cypermethrin.

[0132] 1mL concentration is 2.5×10 5 spores / mL of Beauveria bassiana spore suspension + 5mL of 150mg / L of highly effective cypermethrin.

[0133] Preferably, the most preferred ratio of Beauveria bassiana Bb-swu10 and highly effective cypermethrin is 5 mL with a concentration of 2.5×10 5 spores / mL of Beauveria bassiana spore suspension + 1mL of 150mg / L high-efficiency cypermethrin.

[0134] Experimental Example 7: Toxicity determination of the mixture of strain Bb-swu10 and two pesticides against 3-day-old adults of Bactrocera dorsalis

[0135] The strain Bb-swu10 was used to isolate the LC of 3-day-old adults of Bactrocera dorsalis on the 12th day. 50 The LC of 3-day-old adults of Bactrocera dorsalis and avermectin on the second day 50 , LC of cypermethrin against 3-day-old adults of Bactrocera dorsalis on the second day 30 Prepare mixed solutions; mix them at volume ratios of 5:1, 2:1, 1:1, 1:2, and 1:5, and determine the toxicity of each ratio to 3-day-old adults of Bactrocera dorsalis; calculate the co-toxicity factor based on the expected mortality rate and the actual mortality rate. A co-toxicity factor above 20 indicates a synergistic effect, below -20 indicates an antagonistic effect, and between -20 and 20 indicates an additive effect.

[0136] Co-toxicity factor = [measured mortality of the mixture (%) - expected mortality of the mixture (%)] / expected mortality of the mixture (%) / expected mortality of the mixture (%).

[0137] 1. Toxicity test of Bb-swu10 and avermectin mixture against 3-day-old adults of Bactrocera dorsalis

[0138] Bb-swu10 was diluted with 0.05% Tween-80 to obtain the LC against 3-day-old adults of Bactrocera dorsalis. 50 Concentration (2.5×10 5spores / mL); Avermectin was diluted with 0.05% Tween-80 to LC against 3-day-old adults of Bactrocera dorsalis. 50 concentration (9.664 mg / L); then mixed according to the volume ratio of 5:1, 2:1, 1:1, 1:2, 1:5, and measured the mortality rate. The co-toxicity factor was calculated based on the expected mortality rate and the actual mortality rate.

[0139] The results in Table 8 show that Bb-swu10 and avermectin have a synergistic effect at a volume ratio of 5:1; and only have an additive effect at a volume ratio of 2:1, 1:1, 1:2, and 1:5.

[0140] Table 8 Toxicity of different ratios of Bb-swu10 and avermectin mixtures to 3-day-old adults of Bactrocera dorsalis

[0141]

[0142] 2. Toxicity determination of the mixture of Bb-swu10 and highly effective cypermethrin to 3-day-old adults of Bactrocera dorsalis

[0143] Bb-swu10 was diluted with 0.05% Tween-80 to obtain the LC against 3-day-old adults of Bactrocera dorsalis. 50 Concentration (2.5×10 5 spores / mL); highly effective cypermethrin was diluted with 0.05% Tween-80 to LC against 3-day-old adults of Bactrocera dorsalis. 30 concentration (150mg / L); then mixed according to the volume ratio of 5:1, 2:1, 1:1, 1:2, 1:5, and measured the mortality rate. The co-toxicity factor was calculated based on the expected mortality rate and the actual mortality rate.

[0144] The experimental results in Table 9 show that Bb-swu10 and high-efficiency cypermethrin have synergistic effects at volume ratios of 5:1, 1:2, and 1:5, and the synergistic effect is strongest at 5:1; at volume ratios of 2:1 and 1:1, they only have additive effects;

[0145] Table 9 Toxicity of different ratios of Bb-swu10 and highly effective cypermethrin mixture to 3-day-old adults of Bactrocera dorsalis

[0146]

[0147] Based on the dosage and experimental data, it is recommended to use a mixed solution of Bb-swu10 and avermectin, or Bb-swu10 and highly effective chlorpyrifos, in a volume ratio of 5:1.

[0148] The results showed that the Beauveria bassiana Bb-swu10 strain has great application potential in the prevention and control of the citrus fruit fly. It can kill the adult citrus fruit fly at a lower concentration and has a killing effect on different insect stages of the citrus fruit fly. When the Beauveria bassiana Bb-swu10 strain is mixed with avermectin and highly effective chlorpyrifos in a certain proportion, the control effect is far better than using a single agent, and the mixed application significantly reduces the amount of chemical pesticides used, which is beneficial to environmental safety and helps to delay or even overcome the development of drug resistance.

[0149] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the present invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A pesticide for controlling Bactrocera dorsalis based on Beauveria bassiana, characterized in that: The medicament is prepared by mixing Beauveria bassiana and an additive, wherein the Beauveria bassiana is the Beauveria bassiana Bb-swu10 strain, and the Beauveria bassiana Bb-swu10 strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on May 15, 2024, with a deposit number of CGMCC No.41228; The added drug is at least one of avermectin and high-efficiency cypermethrin; The culture of Beauveria bassiana Bb-swu10 is a spore suspension of Beauveria bassiana Bb-swu10, with a concentration of 1×10 5 Spores / mL ~ 1×10 8 Spores / mL; The mixed solution of the Beauveria bassiana spore suspension and avermectin is obtained according to the following ratio: 5 mL concentration is 2.5 × 10 5 spores / mL of Beauveria bassiana spore suspension + 1 mL of avermectin with a concentration of 9.664 mg / L; The mixed solution of the Beauveria bassiana spore suspension and high-efficiency cypermethrin is obtained according to the following ratio: 5 mL concentration is 2.5 × 10 5 spores / mL of Beauveria bassiana spore suspension + 1 mL of 150 mg / L of highly effective cypermethrin.

2. A testing method for a pesticide for controlling the fruit fly based on Beauveria bassiana, used for the pesticide for controlling the fruit fly based on Beauveria bassiana according to claim 1, characterized in that: Here are the steps: Spray the spore suspension of Beauveria bassiana Bb-swu10 onto the adult bodies of Bactrocera dorsalis; The larvae of Bactrocera dorsalis were impregnated with a spore suspension of Beauveria bassiana Bb-swu10; The spore suspension of Beauveria bassiana Bb-swu10 was impregnated into the pupae of Bactrocera dorsalis.

3. The method for testing a Bactrocera dorsalis-control agent based on Beauveria bassiana according to claim 2, characterized in that: Spray the spore suspension of Beauveria bassiana Bb-swu10 on the adult insects of Bactrocera dorsalis in the following steps: Pick out 3-day-old adults of Bactrocera dorsalis, freeze them in a -20℃ refrigerator for 1.5 min, and transfer them to ice after they become unconscious, so that they remain in a continuous coma state. Arrange the adults of Bactrocera dorsalis neatly in a petri dish, place the petri dish on the stage of the spray tower, take 1 mL of different concentrations of pesticides for spraying, the spraying time is 1 min, and 30 heads are treated each time; Place the infected test insects in a 10 cm × 10 cm × 10 cm cubic mesh cage at room temperature for 30 min to allow them to wake up; After the fruit fly wakes up, place the cube mesh cage in a constant temperature incubator for observation; The survival rate of Bactrocera dorsalis was counted every 24 hours, and the dead insects were removed promptly for 8 consecutive days.

4. The method for testing a Bactrocera dorsalis-control agent based on Beauveria bassiana according to claim 2, characterized in that: The spore suspension of Beauveria bassiana Bb-swu10 is impregnated with Bactrocera dorsalis larvae in the following steps: Pick out the third-instar larvae of Bactrocera dorsalis that have just jumped out of the glass culture dish and are about to pupate; The larvae were placed in a sterile petri dish containing 3 mL of spore suspension and immersed for 1 min, with 30 larvae treated each time; Take out the immersed larvae and dry them, then put them into a new sterile culture dish, and finally put the culture dish into the incubator for observation. Larval mortality was counted every 24 h, and the dead insects were removed promptly. The mortality was continued until the larvae pupated and emerged.

5. The method for testing a Bactrocera dorsalis-control agent based on Beauveria bassiana according to claim 2, characterized in that: The spore suspension of Beauveria bassiana Bb-swu10 is impregnated with Bactrocera dorsalis pupae in the following steps: Pupae were selected after 24 h, 48 h, 96 h, and 144 h of pupation, washed with sterile water, and dried for 30 min. Pupae were placed in a 1.5 mL centrifuge tube containing 1 mL of spore suspension and immersed for 1 min. Thirty pupae were treated each time. Take out the impregnated pupae and dry them, then put them into a new sterile culture dish, and finally put the culture dish into the incubator for observation; The mortality rate of pupae was counted every 24 h until 12 days after pupae emerged.

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