A high pathogenicity entomopathogenic composition of beauveria bassiana against bactrocera dorsalis
The combination of Beauveria bassiana 20251932 and 2008658 as an insecticide has solved the problems of chemical resistance and environmental risks in the control of oriental fruit fly, achieving efficient, safe and low-cost control, which is in line with the concept of green plant protection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- QINGDAO AGRI UNIV
- Filing Date
- 2025-10-13
- Publication Date
- 2026-07-03
AI Technical Summary
Existing chemical agents have resistance problems in controlling the oriental fruit fly and pose potential risks to the environment and non-target organisms, making it difficult to meet the concept of green plant protection. The control effect of using Beauveria bassiana alone is limited.
A compound insecticidal composition of Beauveria bassiana 20251932 and Beauveria bassiana 2008658 was used to form a highly pathogenic insecticidal composition by optimizing the spore ratio. This composition was applied by spraying to control the citrus fruit fly.
It significantly improves pathogenicity against the oriental fruit fly, reduces the amount used, lowers costs, enhances the stability of the control effect, and has no chemical residues, making it highly safe and compliant with organic agriculture requirements.
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Figure CN121312642B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to an insecticide with high pathogenicity against the oriental fruit fly, specifically to an insecticidal composition of Beauveria bassiana with high pathogenicity against the oriental fruit fly, its preparation method and application, belonging to the field of pest control technology. Background Technology
[0002] Oriental fruit fly ( Bactrocera dorsalis Hendel (Fruit Fly) belongs to the genus Hendel in the family Tetranychidae of the order Diptera. The larvae feed inside the fruit, often causing the fruit to turn yellow and fall off before ripening. After emerging from the fruit, the larvae pupate in the soil. It is a Class II quarantine pest in my country. This insect can damage more than 250 species of fruits (mango, guava, custard apple, apple, star fruit, loquat, etc.) and vegetables (eggplant, cucumber, tomato, etc.), causing a significant impact on the yield and quality of fruit and vegetable products. In severely affected areas, yields can be reduced by more than 80%, or even result in total crop failure.
[0003] Currently, the main pesticides used for the control of oriental fruit flies are pyrethroids, neonicotinoids, and organophosphates. However, oriental fruit fly populations in many areas have developed resistance to these commonly used pesticides, leading to reduced efficacy. Furthermore, these pesticides have high residue levels and are highly toxic, posing potential risks to beneficial organisms such as bees and aquatic life, as well as the ecological environment. Long-term use of these pesticides may result in pesticide residues, environmental pollution, and food safety issues.
[0004] Beauveria bassiana is an entomopathogenic fungus belonging to the ascomycetes. The main species include Beauveria bassiana and Beauveria bryonis. It usually reproduces asexually by producing conidia. The hyphae have septa and segments. Beauveria bassiana can invade the bodies of more than 200 kinds of insects and mites in 6 orders and 15 families and multiply in large quantities. At the same time, it produces beauveria bassiana (a non-ribosomal polypeptide toxin), oosporin (a benzoquinone toxin), and calcium oxalate crystals. These substances can cause insect poisoning, disrupt metabolism, and lead to death.
[0005] Over the years, Beauveria bassiana has been successfully used in China to control nearly 40 kinds of agricultural and forestry pests. Currently, the main pests used in production include pine caterpillars, corn borers, white grubs, locusts, potato beetles, pine sawyer beetles, termites, tea green leafhoppers, and orchard fruit moths. Although some studies have attempted to use Beauveria bassiana to control the oriental fruit fly, most focus on strategies involving the mixing of fungi with chemical agents, using Beauveria bassiana as a synergistic component of chemical agents to slightly reduce pesticide dosage. This approach still struggles to avoid the potential stress of chemical pesticides on field ecosystems and non-target organisms, and falls short of the concept of green plant protection. This study departs from this approach, selecting Beauveria bassiana strains with high pathogenicity against the oriental fruit fly for mixed application, optimizing the spore ratio to achieve better control effects. Summary of the Invention
[0006] The technical problem this invention aims to solve is to address the shortcomings of existing technologies by providing a highly pathogenic Beauveria bassiana insecticidal composition against the oriental fruit fly, its preparation method, and its application. This Beauveria bassiana insecticidal composition exhibits higher insecticidal activity against the oriental fruit fly than either of the individual Beauveria bassiana species used alone. Co-toxicity testing using the co-toxicity factor method shows that, at the optimal ratio, its toxicity is significantly higher than either single agent. Furthermore, the use of a mixture of two Beauveria bassiana species completely eliminates the need for chemical pesticides, adhering to an environmentally friendly pest control philosophy.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] The present invention first provides an insecticidal composition of Beauveria bassiana, which has high pathogenicity against the oriental fruit fly. The insecticidal composition of Beauveria bassiana is obtained by compounding Beauveria bassiana spore suspension 20251932 and Beauveria bassiana spore suspension 2008658.
[0009] In the above technical solution, the Beauveria bassiana 20251932 is collected by the China Center for Type Culture Collection, with accession number CCTCC No: M 20251932.
[0010] In the above technical solution, the Beauveria bassiana 2008658 is collected by the China Center for Type Culture Collection, with accession number CCTCC CF 2008658.
[0011] In the above technical solution, the Beauveria bassiana spore suspension 20251932 has a concentration of 1×10⁻⁶. 5 ~2×10 6 Spores / ml, preferably 1.928 × 10⁻⁶ 6 Spores / ml.
[0012] In the above technical solution, the Beauveria bassiana spore suspension 2008658 has a concentration of 1×10⁻⁶. 5 ~9×10 6 Spores / ml, preferably 8.711 × 10⁻⁶ 6 Spores / ml.
[0013] In the above technical solution, the volume ratio of the Beauveria bassiana spore suspension 2008658 and the Beauveria bassiana spore suspension 20251932 is 1~10:1~10, preferably 1:10, 1:5, 1:2, 1:1, 2:1, 5:1 and 10:1, and more preferably 1:10, 1:5, 1:2, 1:1 and 2:1.
[0014] The present invention also provides the application of the Beauveria bassiana insecticidal composition, which has high pathogenicity against the oriental fruit fly, in the control of the oriental fruit fly.
[0015] In the above technical solution, when the Beauveria bassiana insecticidal composition is used to control the oriental fruit fly, the oriental fruit fly refers to the adult oriental fruit fly after it has emerged from its molt.
[0016] In the above technical solution, the Beauveria bassiana insecticidal composition is applied by spraying when controlling the oriental fruit fly. Each spray consists of 0.05-0.15 ml of spore suspension, and the spray is repeated 5-15 times. Preferably, each spray consists of 0.1 ml of spore suspension, and the spray is repeated 10 times.
[0017] Compared with existing technologies, it has the following characteristics:
[0018] (1) Synergistic effect. After mixing, the co-toxic factor was greater than 20, which enhanced the pathogenicity against the oriental fruit fly.
[0019] (2) Excellent compatibility: the two strains have the same taxonomic position and similar physiological and ecological characteristics, thus avoiding mutual antagonism.
[0020] (3) Reduce the amount of fungicide used. The spore concentration required to achieve the same control effect is reduced, saving production and application costs.
[0021] (4) Improve the stability of the protective effect. Complementary effects between strains reduce the risk of failure due to a single strain.
[0022] (5) Environmental friendliness and safety. It is a pure biological agent with no chemical residues, safe for non-target organisms, and meets the requirements of organic agriculture and green food production. Attached Figure Description
[0023] Figure 1 The colony (a) and conidial morphology (b) of strain 20251932 are shown.
[0024] Figure 2 The colony (a) and conidial morphology (b) of strain 2008658 are shown.
[0025] Figure 3 To verify the mortality rate of oriental fruit fly larvae after treatment with Beauveria bassiana 20251932 in Example 1;
[0026] Figure 4 To verify the mortality rate of oriental fruit fly larvae after treatment with Beauveria bassiana 2008658 in Example 1;
[0027] Figure 5 To verify the mortality rate of *Bacillus citrinum* pupae treated with *Beauveria bassiana* 20251932 in Example 1;
[0028] Figure 6To verify the mortality rate of *Bacillus citrinum* pupae treated with *Beauveria bassiana* 2008658 in Example 1;
[0029] Figure 7 To verify the daily cumulative mortality rate of adult oriental fruit flies after treatment with Beauveria bassiana 20251932 in Example 1 (immersion method).
[0030] Figure 8 To verify the daily cumulative mortality rate of adult oriental fruit fly after treatment with Beauveria bassiana 2008658 in Example 1 (immersion method).
[0031] Figure 9 To verify the daily cumulative mortality rate of adult oriental fruit flies after treatment with Beauveria bassiana 20251932 in Example 1 (spraying method).
[0032] Figure 10 To verify the image of hyphae growing on the body surface of the oriental fruit fly after being infected by Beauveria bassiana 20251932 in Example 1;
[0033] Figure 11 To verify the daily cumulative mortality rate of adult oriental fruit flies after treatment with Beauveria bassiana 2008658 in Example 1 (spraying method).
[0034] Figure 12 To verify the image of hyphae growing on the body surface of the oriental fruit fly after being infected by Beauveria bassiana 2008658 in Example 1;
[0035] Figure 13 To verify the growth of hyphae on the body surface of *Bacillus citrinum* after infection with *Beauveria bassiana* 20251932 and *Beauveria bassiana* 2008658 in Example 2, the following diagrams are provided: a represents group 2008658; b represents group 20251932; c to i represent groups with a 2008658:20251932 ratio of 1:10, 1:5, 1:2, 1:1, 2:1, 5:1 and 10:1, respectively. Detailed Implementation
[0036] The following describes in detail the specific embodiments of the technical solution of the present invention, but the present invention is not limited to the following description:
[0037] The materials and methods used in the following verification embodiments and examples of the present invention are as follows:
[0038] 1. Materials and Methods:
[0039] 1.1. Test insect source:
[0040] The oriental fruit fly was provided by the oriental fruit fly research group of the College of Plant Protection, South China Agricultural University. The insect had been continuously bred indoors for multiple generations. After being transferred to our research group, it was raised in an artificial climate chamber with a temperature of 26±1℃, relative humidity (RH%) of 65±5%, and photoperiod of 14L:10D, using artificial feed. Its life parameters were monitored regularly to prevent population degradation and other phenomena.
[0041] 1.2 Test strains:
[0042] The tested strain 20251932 was provided by Qingdao Agricultural University and is collected by the China Center for Type Culture Collection, with accession number CCTCC No: M 20251932.
[0043] The tested strain 2008658 was purchased from the China Center for Type Culture Collection (CCTCC), accession number CCTCC CF2008658. Before the experiment, Beauveria bassiana was cultured on glucose agar (PDA) plates and placed in a constant temperature incubator (temperature 25 ± 1°C, RH% > 90%, photoperiod 14L:10D).
[0044] The colony and conidial morphology of strains 20251932 and 2008658 are as follows: Figure 1 and Figure 2 As shown.
[0045] 1.3. Experimental culture medium and conditions:
[0046] Potato Dextrose Agar (PDA): 200 g / L potato, 20 g / L glucose, 15-20 g / L agar, 1 L water.
[0047] Yeast Extract Peptone Dextrose (YEPD) medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L; water 1 L
[0048] Note: Glucose solution needs to be sterilized separately.
[0049] Aseptic operating conditions: All instruments and utensils used in the experiment were sterilized in a high-temperature sterilizer (121℃, 20 min), and all inoculation and other operations were carried out in a clean bench.
[0050] 1.4 Preparation of spore suspension:
[0051] Select vigorous sporulating strains growing on PDA plates. Use a perforator to collect fresh mycelial cakes from the edge of the colonies and transfer them to Erlenmeyer flasks containing 100 ml of YEPD medium. Then, incubate the flasks on a shaker at 26°C (200 rpm) for 6-10 days. Filter the culture medium after shaking through a 40 µm cell filter to obtain a pure spore suspension. First, determine the spore concentration using a hemocytometer, then prepare a 1×10⁻⁶ spore suspension. 9 A spore suspension of spores / ml is used as a base, which is then diluted to the required concentration before use.
[0052] Verification Example 1: Determination of the pathogenicity of Beauveria bassiana to Orientia citrinum
[0053] 1. Test method:
[0054] 1.1 The pathogenicity of two Beauveria bassiana species to the larvae of the oriental fruit fly was determined by the immersion method:
[0055] Prepared according to the experimental design to 1×10 9 A spore suspension with a concentration of spores / mL was prepared, with sterile water as a control. Each group consisted of 20 larvae, replicated 5 times. Mature larvae of the oriental fruit fly of the same instar were selected and inoculated by immersing them in the spore suspension for 30 seconds. The surface moisture was then blotted dry with filter paper, and the mature larvae were placed in a device containing moist sand to await pupation. The device was placed in a constant temperature incubator (temperature 25±1°C, RH%>60%, photoperiod 14L:10D) for one week. The number of non-pupae, i.e., the number of dead larvae, was recorded.
[0056] 1.2. The pathogenicity of two Beauveria bassiana strains to the pupae of the citrus fruit fly was determined using the immersion method:
[0057] Configured as 1×10 according to the experimental design. 9 Spore concentration / ml, with sterile water as a control. Pupae of the oriental fruit fly (Bactrocera dorsalis) pupated for 48 hours were inoculated into a spore suspension for 1 minute. The pupae were then removed and placed in a 10cm high transparent cup filled with 1cm of moist sand. The cup was sealed with a mesh for ventilation. Each group contained 20 oriental fruit fly pupae, with 5 replicates for both the control and treatment groups. The pupae were then placed in a constant temperature incubator (temperature 25±1℃, RH%>80%, photoperiod 14L:10D).
[0058] Starting from the second day after treatment, the number of oriental fruit fly pupae emerging from each treatment was checked daily. Emerged adults were fed artificial feed and water. After 7 days of observation, the number of dead oriental fruit fly pupae in each group was counted, and the dead pupae were removed and placed in petri dishes containing sterilized moist filter paper. They were then cultured at 25±1℃ to maintain humidity and observe whether mycelium grew on the pupae.
[0059] 1.3. The pathogenicity of two Beauveria bassiana strains to adult fruit fly was determined using the immersion method:
[0060] Spore suspensions with different concentration gradients were used, namely 1×10⁻⁶. 5 1×10 6 1×10 7 1×10 8 and 1×10 9 Spores / mL, with sterile water as a control. Healthy adult oriental fruit flies (Bactrocera dorsalis) three days after emergence were selected as test subjects. Ten healthy three-day-old adults were treated in each group, with five replicates. Adult oriental fruit flies were inoculated by immersing themselves in the spore suspension for 30 seconds and then placed on filter paper to absorb excess water. Ten adults were placed in the apparatus as a replicate and reared with artificial feed and water in an incubator with suitable temperature and humidity. Starting from the second day after treatment, the number of dead adults was recorded daily. Dead adults were removed from the apparatus and placed in a petri dish containing sterilized moist filter paper, and cultured at 25±1℃. The presence of mycelium on the insect body was observed to determine the cause of death, and the mortality rate was calculated.
[0061] 1.4. The pathogenicity of two Beauveria bassiana strains against adult oriental fruit fly was determined using the spray method:
[0062] Considering practical application scenarios, a spray method was used to test the pathogenicity of two Beauveria bassiana strains against adult Bactrocera orientalis. Different concentration gradients of spore suspensions were used, including 1×10⁻⁶ spore suspensions. 5 1×10 6 1×10 7 1×10 8 and 1×10 9 Spores / mL, with sterile water as a control. Healthy adult orientalis flies, 3 days after emergence, were selected as test subjects. Ten healthy 3-day-old adults (female:male = 1:1) were treated in each group, with 5 replicates. The orientalis flies were placed on ice during the experiment. Filter paper with a diameter of 9 cm and an area of 6358.5 mm² was used. 2 Each time, 10*5 groups of fruit flies (50 flies in total) were placed on filter paper. ImageJ software was used to calculate the area of the sprayed liquid that the adult fruit flies could contact. The area of the sprayed liquid was 37.37 mm for females. 2 The male insect measured 33.463 mm. 2Each spray consisted of 0.1 ml of liquid, with 50 adult insects treated at each concentration sprayed a total of 10 times, resulting in 1 ml of suspension. During the experiment, each female insect was exposed to 5.877 μL of liquid, and each male insect was exposed to 5.2626 μL of liquid. Ten adult insects were placed in the apparatus as a replicate and reared in an incubator with artificial feed and water at suitable temperature and humidity. Starting from the second day after treatment, the number of dead adult insects was recorded daily. Dead adults were removed from the apparatus and placed in petri dishes containing sterilized moist filter paper, and cultured at 25±1℃. The presence of mycelium on the insect bodies was observed to determine the cause of death, and the mortality rate was calculated.
[0063] 2. Data statistical analysis:
[0064] Experimental data were analyzed using SPSS 27.0 statistical software. One-way ANOVA was used to evaluate the corrected mortality rates of different Beauveria bassiana treatments on each developmental stage of Oryza sativa. Duncan's multiple range test was used for multiple comparisons between treatments. One-way ANOVA was selected when analyzing three or more groups of data. P < 0.05 was used as the significance criterion. Graphpad Prism 9 was used for plotting.
[0065] Mortality rate (%) = (Number of dead insects / Number of tested insects) × 100%
[0066] Corrected mortality rate (%) = (treatment group mortality rate - control group mortality rate) / (1 - control group mortality rate) × 100%
[0067] 3. Results and Analysis:
[0068] 3.1 Results of Beauveria bassiana infection of oriental fruit fly larvae:
[0069] The mortality rate of oriental fruit fly larvae after treatment with Beauveria bassiana is as follows: Figure 3 and Figure 4 As shown, the larvae of the oriental fruit fly were exposed to 1×10⁻⁶ Beauveria bassiana 20251932. 9 Treatment with spore suspensions at a concentration of spores / mL resulted in a mortality rate of less than 10%. At this concentration, Beauveria bassiana 2008658 caused a mortality rate exceeding 20% in larvae, but this was not significantly different from the control group. Analysis showed that infection of oriental fruit fly larvae with Beauveria bassiana 20251932 and 2008658 via immersion had no significant impact on the transition from larval to pupal stage.
[0070] 3.2 Results of Beauveria bassiana infection of oriental fruit fly pupae:
[0071] The mortality rate of oriental fruit fly pupae after treatment with Beauveria bassiana is as follows: Figure 5 and Figure 6 As shown, the citrus fruit fly pupa underwent 1×10 9Treatment with spore suspensions at a concentration of spores / mL resulted in a mortality rate of less than 30% after pupa infection, with no significant difference compared to the control group. Analysis showed that infection of *Bacillus citrinum* pupae with *Beauveria bassiana* 20251932 and 2008658 via immersion had no significant impact on their emergence process.
[0072] 3.3 Results of Beauveria bassiana infection of adult oriental fruit fly:
[0073] (1) Results of Beauveria bassiana 20251932 infection of adult oriental fruit fly:
[0074] Adult citrus fruit flies were exposed to a concentration of 1×10⁻⁶. 5 1×10 6 1×10 7 1×10 8 and 1×10 9 After treatment with a suspension of Beauveria bassiana spores (spores / ml), the daily cumulative mortality rate of adult fruit flies (Bactrocera dorsalis) was as follows: Figure 7 As shown in the figure, starting from the fourth day, at 1×10 7 1×10 8 and 1×10 9 A significantly higher number of adult fruit flies died when treated with spore concentrations per ml.
[0075] Table 1 shows the corrected cumulative mortality rate of adult oriental fruit flies at different concentrations on day 8 post-infection. As shown in Table 1, the corrected mortality rate of adult oriental fruit flies reached over 90% after treatment with three relatively high concentrations of spore suspension. The highest corrected mortality rate was achieved with a concentration of 1×10⁻⁶ spore suspension. 8 spores / ml and 1×10 9 Treatment with a spore suspension at spore density of 1 / ml resulted in a 100% mortality rate. The median lethal concentration (LC50) of *Beauveria bassiana* 20251932 against adult *Bactrocera dorsalis* was determined by immersion treatment. 50 1.10122×10 5 Spores / ml. At three relatively high concentrations, Beauveria bassiana 20251932 showed good pathogenicity against adult oriental fruit flies using the immersion method.
[0076] Table 1. Corrected cumulative mortality rate of adult oriental fruit fly (Bactrocera dorsalis) on day 8 after treatment with the 20251932 immersion method.
[0077] Concentration (spores / ml) Number of samples (heads) Average number of deaths (heads) Corrected mortality rate % <![CDATA[1×10 5 ]]> 10 5.25±2.21736 47.22±12.32b <![CDATA[1×10 6 ]]> 10 8.5±0.6455 83.34±7.17ab <![CDATA[1×10 7 ]]> 10 9.4±0.4 93.34±4.44a <![CDATA[1×10 8 ]]> 10 10 100a <![CDATA[1×10 9 ]]> 10 10 100a CK 10 1±0.5773 -
[0078] Note: The mean number of deaths and corrected mortality rates in the table are the mean ± standard error. Counts with the same letter in the same column indicate no significant difference (Duncan multiple comparisons, P < 0.05).
[0079] (2) Results of Beauveria bassiana 2008658 infection of adult fruit fly citrus fruit fly
[0080] Adult citrus fruit flies were exposed to a concentration of 1×10⁻⁶. 5 1×10 6 1×10 7 1×10 8 and 1×10 9 After treatment with a suspension of Beauveria bassiana 2008658 spores per ml, the daily cumulative mortality rate of adult fruit flies was as follows: Figure 8 As shown in Table 2, the corrected cumulative mortality rates of adult *Bacillus cereus* at different concentrations on day 8 after infection with *Beauveria bassiana* 2008658 are corrected. Table 2 shows that after treatment with three higher concentrations of spore suspension, the corrected mortality rates of adult *Bacillus cereus* were 90.77±2.31%, 97.69±2.31%, and 100%, respectively. The median lethal concentration (LC50) of *Beauveria bassiana* 2008658 in adult *Bacillus cereus* under immersion treatment is also shown. 50 It is 1.29956×10 5 Spores / ml. At higher concentrations, Beauveria bassiana, via immersion, showed good pathogenicity against adult oriental fruit flies.
[0081] Table 2. Corrected cumulative mortality rate of adult oriental fruit flies on day 8 after treatment with the 2008658 immersion method.
[0082] Concentration (spores / ml) Number of samples (heads) Average number of deaths (heads) Corrected mortality rate % <![CDATA[1×10 5 ]]> 10 5.5±0.49 48.08±11.04b <![CDATA[1×10 6 ]]> 10 6.0±1.15 53.85±6.66b <![CDATA[1×10 7 ]]> 10 9.2±0.20 90.77±2.31a <![CDATA[1×10 8 ]]> 10 9.8±0.20 97.69±2.31a <![CDATA[1×10 9 ]]> 10 10 100a CK 10 1.33±0.67 -
[0083] Note: The mean number of deaths and corrected mortality rates in the table are the mean ± standard error. Counts with the same letter in the same column indicate no significant difference (Duncan multiple comparisons, P < 0.05).
[0084] 3.4 Results of local infection of adult oriental fruit fly with Beauveria bassiana using the Beauveria bassiana spray method:
[0085] (1) Results of local infection of adult oriental fruit fly with Beauveria bassiana 20251932 by spray method:
[0086] Adult citrus fruit flies were exposed to a concentration of 1×10⁻⁶. 5 1×10 6 1×10 7 1×10 8 and 1×10 9 After treatment with a suspension of Beauveria bassiana spores (spores / ml), the daily cumulative mortality rate of adult fruit flies (Bactrocera dorsalis) was as follows: Figure 9 As shown in the figure. It can be seen from the figure that on the fourth day after treatment with adult oriental fruit flies, at a concentration of 1×10⁻⁶... 9Treatment with spore concentrations per ml resulted in a dramatic increase in the mortality rate of adult oriental fruit flies, reaching nearly 50%. After infection and death, mycelium grew on the bodies of the adults in about 5 days, such as... Figure 10 As shown in the image, the eyes of the oriental fruit fly are slightly sunken, and the mycelium growing there covers the entire oriental fruit fly, mainly concentrated on the head and thorax, while the abdomen is sunken and has some mycelium growing there.
[0087] Table 3 shows the corrected cumulative mortality rate of adult oriental fruit flies at different concentrations on day 8 after infection with Beauveria bassiana 20251932. As shown in Table 3, after treatment with two higher concentrations of spore suspension, the corrected mortality rate of adult oriental fruit flies reached over 90%. Furthermore, compared with a concentration of 1×10⁻⁶... 7 Compared to a spore suspension with spores / ml, the concentration is 1×10 8 Treatment with a spore suspension at spores / ml significantly increased the corrected cumulative mortality rate of adult *Bacteroides citrinum* on day 8. The median lethal concentration (LC50) of *Beauveria bassiana* 20251932 in adult *Bacteroides citrinum* under spray treatment was also determined. 50 1.928 × 10 6 Spores / ml.
[0088] Table 3. Corrected cumulative mortality rate of adult oriental fruit fly (Bactrocera dorsalis) on day 8 after spraying treatment with the 20251932 method.
[0089] Concentration (spores / ml) Number of samples (heads) Average number of deaths (heads) Corrected mortality rate % <![CDATA[1×10 5 ]]> 10 1.5±0.65 12.07±6.68c <![CDATA[1×10 6 ]]> 10 4.8±0.58 46.21±6.03b <![CDATA[1×10 7 ]]> 10 5.5±0.96 53.45±9.90b <![CDATA[1×10 8 ]]> 10 9.2±0.58 91.72±6.03a <![CDATA[1×10 9 ]]> 10 10 100a CK 10 0.2±0.2 -
[0090] (2) Results of local infection of adult fruit fly with Beauveria bassiana 2008658 by spray method
[0091] Adult citrus fruit flies were exposed to a concentration of 1×10⁻⁶. 5 1×10 6 1×10 7 1×10 8 and 1×10 9 After treatment with a suspension of Beauveria bassiana 2008658 spores per ml, the daily cumulative mortality rate of adult fruit flies was as follows: Figure 11 As shown. Among them, at a concentration of 1×10 8 At a concentration of spores / ml, infected adults began to show significant mortality from day 5, and the mortality rate of the oriental fruit fly was higher than 1×10⁻⁶ at this concentration. 9 Death rate at spore / ml concentration. Seven days after infection with Beauveria bassiana 2008658, the entire body surface of the oriental fruit fly was covered by mycelium growth, as shown in the figure. Figure 12As shown in Table 4, the corrected cumulative mortality rates of adult *Bacterium citrinum* at different concentrations on day 8 after infection with *Beauveria bassiana* 2008658 are corrected to 90.00% and 93.33%, respectively. Table 4 shows that after treatment with two higher concentrations of spore suspension, the corrected mortality rates of adult *Bacterium citrinum* were 90.00% and 93.33%, respectively. The median lethal concentration (LC50) of *Beauveria bassiana* 2008658 for adult *Bacterium citrinum* under spray treatment is also shown. 50 It is 8.711 × 10 6 Spores / ml. At higher concentrations, Beauveria bassiana, when used in a spraying method for local infection of the oriental fruit fly, can produce good pathogenicity.
[0092] Table 4. Corrected cumulative mortality rate of adult oriental fruit fly (Bactrocera dorsalis) on day 8 after treatment with the 2008-658 spray method.
[0093] Concentration (spores / ml) Number of samples (heads) Average number of deaths (heads) Corrected mortality rate % <![CDATA[1×10 5 ]]> 10 1.8±0.58 18.00±5.83c <![CDATA[1×10 6 ]]> 10 2.8±0.37 28.00±3.74c <![CDATA[1×10 7 ]]> 10 5.5±0.50 55.00±5.00b <![CDATA[1×10 8 ]]> 10 9.0±0.58 90.00±5.77a <![CDATA[1×10 9 ]]> 10 9.3±0.33 93.33±3.33a CK 10 0 -
[0094] Verification Example 2: Pathogenicity determination of Beauveria bassiana mixed with adult fruit fly citrus spp.
[0095] 1. Test method:
[0096] The synergistic effect of mixing two Beauveria bassiana strains was determined using the co-toxicity factor method. Spore suspensions of the two Beauveria bassiana strains were prepared to a concentration of LC50. 50 The concentration, i.e., the concentration of Beauveria bassiana 2008658, is 8.711 × 10⁻⁶. 6 The concentration of Beauveria bassiana 20251932 was prepared to be 1.928 × 10⁶ spores / ml, and then mixed separately at volume ratios of 1:10, 1:5, 1:2, 1:1, 2:1, 5:1 and 10:1, and the concentrations were then measured in LC50. 50 Corrected mortality rates of adult oriental fruit flies treated with single agents and mixtures were determined. The formula for calculating the co-toxicant is as follows:
[0097] Theoretical mortality rate = Mortality rate of single-dose A × Percentage of single-dose A in the mixture + Mortality rate of single-dose B × Percentage of single-dose B in the mixture
[0098] Co-toxicity factor = [(actual mortality rate - theoretical mortality rate) / theoretical mortality rate] × 100.
[0099] Cotoxicity factor < -20 indicates antagonistic effect, between -20 and 20 indicates additive effect, and cotoxicity factor > 20 indicates synergistic effect.
[0100] Healthy adult oriental fruit flies (Bactrocera dorsalis) three days after emergence were selected as test subjects. Ten healthy three-day-old adults (female:male = 1:1) were treated in each group, with five replicates. The oriental fruit flies were placed on ice during the experiment. Filter paper with a diameter of 9 cm and an area of 6358.5 mm² was used.2 Each time, 10*5 groups of fruit flies (50 flies in total) were placed on filter paper. ImageJ software was used to calculate the area of the sprayed liquid that the adult fruit flies could contact. The area of the sprayed liquid was 37.37 mm for females. 2 The male insect measured 33.463 mm. 2 Each spray consisted of 0.1 ml of liquid, with 50 adult insects treated at each concentration sprayed a total of 10 times, resulting in 1 ml of suspension. During the experiment, each female insect was exposed to 5.877 μL of liquid, and each male insect was exposed to 5.2626 μL of liquid. Ten adult insects were placed in the apparatus as a replicate and reared in an incubator with artificial feed and water at a suitable temperature and humidity. Starting from the second day after treatment, the number of dead adult insects was recorded daily. Dead adults were removed from the apparatus and placed in petri dishes containing sterilized moist filter paper, and cultured at 25±1℃. The presence of mycelium on the insect bodies was observed to determine the cause of death, and the mortality rate was calculated.
[0101] 2. Results and Analysis:
[0102] After two types of Beauveria bassiana infect adult fruit flies, a large number of hyphae and spores are produced on the body surface of the adults about 7 days after their death. Figure 13 As shown in the image, the infected insect's body surface is covered by mycelium, mainly concentrated in the thorax and abdomen, with a small amount of mycelium growing in other areas. The mycelium germinating from the insect's surface contains morphological characteristics of two strains, the main difference being that the mycelial structure is more developed in certain areas, and powdery spores are visible among the mycelium. In contrast,... Figure 13 In group (g), where the ratio of 2008658:20251932 is 2:1, the colony morphology formed on the surface of the infected insects is significantly closer to the typical characteristics of strain 2008658. In this group, a large number of powdery spores are visible covering the surface of the insects, a morphology consistent with the germination and growth characteristics of strain 2008658, demonstrating the competitive advantage of this strain under this ratio.
[0103] The virulence (after 8 days) of two Beauveria bassiana strains against adult oriental fruit flies after infection is shown in Table 5. The results showed that the corrected mortality rates of Beauveria bassiana 2008658 and 20251932 after 8 days of single-agent treatment against oriental fruit flies were 46.67% and 50.00%, respectively. The mixed fungi showed synergistic effects at volume ratios of 1:10, 1:5, 1:2, 1:1, and 2:1; antagonistic effects at 5:1; and additive effects at 10:1. This indicates that reducing the proportion of strain 20251932 within a certain range when using a mixture of these two fungi helps to obtain better insecticidal effects, with the best insecticidal effect observed when the proportion of strain 20251932 is around 10%.
[0104] Table 5. Pathogenicity of two different combinations of Beauveria bassiana to adult fruit fly (Bactrocera dorsalis)
[0105] Volume ratio A : B Percentage of single-dose A (%) Percentage of single-dose B (%) Actual mortality rate (%) Theoretical mortality rate Co-toxic factors interaction 2008658 100.00 0.00 46.67 - - - 20251932 0.00 100.00 50.00 - - - 1:10 31.14 68.86 67.50 49.07 37.57 Increase efficiency 1:5 47.53 52.47 65.00 48.42 34.25 Increase efficiency 1:2 69.28 30.72 65.00 47.69 36.30 Increase efficiency 1:1 81.88 18.12 72.50 47.27 53.37 Increase efficiency 2:1 90.05 9.95 80.00 47.00 70.22 Increase efficiency 5:1 95.75 4.25 35.00 46.81 -25.23 Antagonism 10:1 97.83 2.17 37.50 46.74 -19.77 Add
[0106] A: Volume occupied by Beauveria bassiana 2008658 in the mixture; B: Volume occupied by Beauveria bassiana 20251932 in the mixture.
[0107] As can be seen from the above verification examples, the combined effect of Beauveria bassiana 20251932 and 2008658 in this invention is better than that of single agents after strict compounding ratio. The invention will be described below with reference to specific examples.
[0108] Example 1:
[0109] A Beauveria bassiana insecticidal composition with high pathogenicity against the oriental fruit fly, wherein the Beauveria bassiana insecticidal composition is obtained by compounding Beauveria bassiana spore suspension 20251932 and Beauveria bassiana spore suspension 2008658.
[0110] The aforementioned Beauveria bassiana 20251932 is deposited at the China Center for Type Culture Collection, with accession number CCTCC M20251932.
[0111] The aforementioned Beauveria bassiana 2008658 is deposited at the China Center for Type Culture Collection, with accession number CCTCC CF2008658.
[0112] The Beauveria bassiana spore suspension 20251932 had a concentration of 1.928 × 10⁻⁶. 6 Spores / ml.
[0113] The Beauveria bassiana spore suspension 2008658 had a concentration of 8.711 × 10⁻⁶. 6 Spores / ml.
[0114] The volume ratio of the Beauveria bassiana spore suspension 2008658 and Beauveria bassiana spore suspension 20251932 is 1:10.
[0115] Example 2:
[0116] A Beauveria bassiana insecticidal composition with high pathogenicity against the oriental fruit fly is basically the same as that in Example 1, except that the volume ratio of the Beauveria bassiana spore suspension 2008658 and the Beauveria bassiana spore suspension 20251932 is 1:5.
[0117] Example 3:
[0118] A Beauveria bassiana insecticidal composition with high pathogenicity against the oriental fruit fly is basically the same as that in Example 1, except that the volume ratio of the Beauveria bassiana spore suspension 2008658 and the Beauveria bassiana spore suspension 20251932 is 1:2.
[0119] Example 4:
[0120] A Beauveria bassiana insecticidal composition with high pathogenicity against the oriental fruit fly is basically the same as that in Example 1, except that the volume ratio of the Beauveria bassiana spore suspension 2008658 and Beauveria bassiana spore suspension 20251932 is 1:1.
[0121] Example 5:
[0122] A Beauveria bassiana insecticidal composition with high pathogenicity against the oriental fruit fly is basically the same as that in Example 1, except that the volume ratio of the Beauveria bassiana spore suspension 2008658 and Beauveria bassiana spore suspension 20251932 is 2:1.
[0123] The above examples are merely illustrative of the technical concept and features of the present invention and should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the essence of the present invention should be included within the scope of protection of the present invention.
Claims
1. An insecticidal composition of Beauveria bassiana with high pathogenicity against the oriental fruit fly, wherein the insecticidal composition of Beauveria bassiana is obtained by compounding Beauveria bassiana spore suspension 20251932 and Beauveria bassiana spore suspension 2008658; wherein Beauveria bassiana 20251932 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M20251932; wherein Beauveria bassiana 2008658 is deposited at the China Center for Type Culture Collection (CCTCCCF) with accession number CCTCCF 2008658; The Beauveria bassiana spore suspension 20251932 has a concentration of 1×10⁻⁶. 5 ~2×10 6 Spores / ml; the Beauveria bassiana spore suspension of 2008658 had a concentration of 1×10⁻⁶. 5 ~9×10 6 spores / ml; The volume ratios of the Beauveria bassiana spore suspension 2008658 and Beauveria bassiana spore suspension 20251932 are 1:10, 1:5, 1:2, 1:1 and 2:
1.
2. The highly pathogenic Beauveria bassiana insecticidal composition according to claim 1, characterized in that, The Beauveria bassiana spore suspension 20251932 had a concentration of 1.928 × 10⁻⁶. 6 Spores / ml; the concentration of the Beauveria bassiana spore suspension 2008658 was 8.711 × 10⁻⁶. 6 Spores / ml.
3. The application of the highly pathogenic Beauveria bassiana insecticidal composition according to any one of claims 1-2 in the control of Bactrocera dorsalis, characterized in that, The oriental fruit fly mentioned refers to the adult oriental fruit fly after it has emerged from its molted body.
4. The application according to claim 3, characterized in that, The aforementioned Beauveria bassiana insecticidal composition is applied by spraying when controlling the citrus fruit fly. Each spray consists of 0.05-0.15 ml of spore suspension, and the total number of sprays is 5-15.
5. The application according to claim 4, characterized in that, The aforementioned Beauveria bassiana insecticidal composition is applied by spraying when controlling the oriental fruit fly. Each spray consists of 0.1 ml of spore suspension, and the total number of sprays is 10.