Application of bacillus amyloliquefaciens N-22 in prevention and treatment of aphids
By using Bacillus amyloliquefaciens N-22 fermentation liquid to treat aphids, the problems of pesticide resistance and environmental pollution caused by chemical pesticide control of aphids were solved, achieving a highly efficient and environmentally friendly aphid control effect.
Patent Information
- Application Number
- CN202511418343.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-30
- Publication Date
- 2025-12-16
AI Technical Summary
Existing chemical pesticides are prone to causing pesticide resistance and environmental pollution when controlling aphids, and are also harmful to human and animal health. Finding environmentally friendly biological control methods is an urgent problem to be solved.
Aphids were treated with Bacillus amyloliquefaciens N-22 fermentation broth at a concentration of 10⁸–10¹⁰ CFU/mL. The treatment of aphids with different dilution ratios and treatment times showed a highly effective insecticidal effect.
After treating aphids with fermented bacterial solution, the corrected mortality rate can reach up to 91.89%, which has a better control effect and is environmentally friendly.
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Figure CN121128743A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological control technology for plant pests, and in particular to the application of Bacillus amyloliquefaciens N-22 in the control of aphids. Background Technology
[0002] Aphids are global piercing-sucking pests with a wide host range. The pea aphid, a dominant species in leguminous crops, is particularly destructive. Pea aphids can reduce leguminous crop yields and are highly resistant to pesticides, posing a challenge to control. They can severely damage a variety of crops, causing economic losses, and are now widely distributed throughout the world. Pea aphids can use a variety of plants as hosts, damaging herbaceous leguminous plants including peas, broad beans, alfalfa, sweet clover, red clover, and soybeans. Both nymphs and adults of the pea aphid use their piercing-sucking mouthparts to penetrate the phloem of tender stems, leaves, flowers, and young fruits, sucking the plant's sap. This causes the leaves to curl, and the flowers and buds to turn yellow and eventually fall off. These phenomena indicate that pea aphid damage affects normal plant growth, development, flowering, fruiting, and final yield.
[0003] Currently, the most common method for aphid control is the use of chemical pesticides, with different pesticides used for different crops. Chemical control methods include seed coating or spraying aphids during the plant's growth cycle. However, continuous use of chemical pesticides can lead to aphid resistance, causing aphid outbreaks, and pesticide residues can also affect human and animal health, resulting in food safety issues and environmental degradation.
[0004] Biological control of plant pests using microorganisms has become a popular research field in recent years, attracting much attention because it can completely or partially replace chemical agents, reduce the environmental harm of disease control, and promote the development of sustainable plant protection. Bacillus is one of the most studied genera among biological control agents, possessing unique advantages in biological control: on the one hand, it can form stress-resistant spores, enhancing the bacteria's survival ability under adverse environmental conditions; on the other hand, it can produce secondary metabolites with antagonistic activity, including antifungal proteins, low-molecular-weight volatile compounds, and antibiotics. Summary of the Invention
[0005] In view of this, the present invention provides the application of Bacillus amyloliquefaciens N-22 in the control of aphids.
[0006] Bacillus amyloliquefaciens ( Bacillus amyloliquefaciensThe application of Bacillus amyloliquefaciens N-22 in the control of aphids, the preservation number of Bacillus amyloliquefaciens N-22 is CCTCC NO: M 2020256, and it was deposited at the China Center for Type Culture Collection on July 1, 2020, at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.
[0007] Furthermore, the aphid is the pea aphid.
[0008] A fermentation broth, wherein the fermentation broth is prepared from the above-mentioned Bacillus amyloliquefaciens N-22.
[0009] Furthermore, the concentration of *Bacillus amyloliquefaciens* N-22 spores in the fermentation broth is 10. 8 ~10 10 cfu / mL.
[0010] The above-mentioned fermentation liquid is used in the preparation of aphid control products.
[0011] The beneficial effects of the technical solution provided by this invention are as follows: The *Bacillus amyloliquefaciens* strain used in this invention has advantages such as rapid growth rate, high sporulation rate, and strong antagonism against pests. The fermentation broth prepared using this *Bacillus amyloliquefaciens* can effectively control aphids. After dilution of the N-22 fermentation broth at 100x, 300x, and 500x, aphids were treated, and the corrected mortality rate was statistically analyzed at 24h, 48h, and 72h, respectively. The highest corrected mortality rate reached 80.21%. The fermentation broth was serially diluted (1×10⁻⁶) to... 3 CFU / mL, 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 After treatment with CFU / mL, pea aphids were treated, and corrected mortality rates were calculated at 24h, 48h, 72h, and 96h. The corrected mortality rate of pea aphids reached a maximum of 91.89% with increasing fermentation broth concentration and treatment time. This *Bacillus amyloliquefaciens* strain exhibits better control efficacy than other biocontrol agents and represents a promising aphid control method. Attached Figure Description
[0012] Figure 1 The colony morphology (a), Gram staining results (b), and cell morphology (c) of Bacillus amyloliquefaciens N-22 used in this invention are shown. Figure 2 Line graph showing the corrected mortality rate of aphids after 100-fold dilution of N-22 and H17 fermentation broth; Figure 3Line graph showing the corrected mortality rate of aphids after 300-fold dilution of N-22 and H17 fermentation broth; Figure 4 Line graph showing the corrected mortality rate of aphids after 500-fold dilution of N-22 and H17 fermentation broth; Figure 5 Different gradient concentrations (1×10) 3 CFU / mL, 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 Linear trend of mortality rate of pea aphids after treatment with N-22 fermentation broth (CFU / mL) under different conditions. Detailed Implementation
[0013] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described below with reference to the accompanying drawings.
[0014] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.
[0015] Potato Dextrose Agar (PDA): 200 g peeled potatoes, 20 g glucose, 14 g agar, 1000 mL distilled water, sterilized at 121°C for 20 min.
[0016] Luria Broth (LB): 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 1000 mL distilled water, sterilized at 121°C for 20 min.
[0017] Fermentation medium and fermentation conditions: 10 g / L potato flour, 15 g / L soybean flour, fermentation time 30 h, liquid volume 50%, inoculum size 4%.
[0018] Gram staining kit: manufactured by Beijing Solarbio Biotechnology Co., Ltd.
[0019] Test insects: Aphids were wingless aphids obtained by continuous rearing and breeding in the laboratory using broad bean seedlings. Third-instar nymphs were selected as test insects for the live testing.
[0020] Test bacterial agents: N-22 (Bacillus amyloliquefaciens), H17 (Bacillus amyloliquefaciens).
[0021] It should be noted that the Bacillus amyloliquefaciens described in this invention (…) Bacillus amyloliquefaciens Bacillus amyloliquefaciens N-22, with accession number CCTCC M 2020256, has been disclosed in the invention patent application number 2021113593382, entitled "A strain of Bacillus amyloliquefaciens and its application in the prevention and control of tea cake disease." The method for preparing the fermentation broth of Bacillus amyloliquefaciens N-22 used in this invention is the same as that application. Bacillus amyloliquefaciens H17 is preserved in our laboratory.
[0022] I. Fermentation of strain N-22 Activation of bacteria: Streak the preserved bacteria on a PDA plate and incubate in an incubator for 24-48 hours. After the bacteria have grown, check the purity of the bacteria and pick a single colony in the workbench and transfer it to a 1.5 mL centrifuge tube containing 300 mL of LB medium. Incubate at 30°C and 200 rpm for 48 hours to obtain the activated bacterial solution. Store at 4°C for later use.
[0023] Bacterial culture: The shake culture method was used. 10 mL of the activated bacterial solution was taken into a 100 mL Erlenmeyer flask containing 20 mL of LB medium and shaken at 200 rpm for 48 h in a constant temperature shaker at 30°C to obtain the shake culture solution, which was stored at 4°C for later use.
[0024] Fermentation of the bacteria: Take 600 mL of the culture medium and transfer it to a 100 mL Erlenmeyer flask containing 20 mL of LB medium (or a 250 mL Erlenmeyer flask containing 40 mL of LB medium). Incubate at 30°C with shaking at 200 rpm for 12-16 hours to obtain the seed culture. Take 600 mL of the seed culture and transfer it to a 100 mL Erlenmeyer flask containing 20 mL of LB medium. Incubate at 30°C with shaking at 200 rpm for 30 hours to obtain the fermentation broth. The concentration of viable cells per unit volume of the fermentation broth was measured to be 10. 8 ~10 10 cfu / mL.
[0025] The colony morphology, Gram staining results, and cell morphology of Bacillus amyloliquefaciens N-22 used in this invention are as follows: Figure 1 As shown.
[0026] <Experimental Example 1> This experiment aims to investigate the corrected mortality rate of aphids treated with Bacillus amyloliquefaciens N-22 fermentation broth at different dilutions, and to compare it with the corrected mortality rate of aphids treated with H17. Experimental steps: Select healthy broad bean plants infested with aphids and cut off approximately 7cm sections. Use a paintbrush to remove any dead or weak aphids from the cut sections, ensuring there are 20-25 aphids on each plant. Record the exact total number of aphids.
[0027] Using tweezers, hold the cut end of the plant and spray approximately 0.5 mL of bacterial solution onto the aphids using a spray bottle, ensuring all aphids are sprayed. Perform three replicates for each concentration. Use sterile water as a control group.
[0028] After spraying, wrap the cut part of the plant with a damp cotton ball to keep it moist, and place the plant in a 9cm diameter petri dish. Place the lid of the petri dish on top of the dish, making sure there is a gap between the lid and the dish, and wrap the lid with plastic wrap to secure it.
[0029] The treated petri dishes were placed in a light incubator at 25±0.5℃, RH 60±5%, and a light duration (L:D) of 14h:10h. The number of dead insects was checked and recorded after 24h, 48h, and 72h of treatment. The corrected mortality rate was calculated, and the experimental data were analyzed. The formula for calculating the corrected mortality rate is as follows: Mortality rate (%) = (Number of dead insects / Total number of insects) x 100%; Corrected mortality rate (%) = (treatment mortality rate - control mortality rate) / (1 - control mortality rate) x 100%.
[0030] The number of live insects in each treatment was counted at 24h, 48h and 72h after application of the pesticide, and three replicates were set up for each treatment.
[0031] Table 1. Medications and dilution ratios.
[0032] Table 2 Results of 100X dilution treatment on aphids
[0033] Table 3. Results of 300X dilution treatment on aphids
[0034] Table 4. Results of 500X dilution treatment on aphids
[0035] Experimental results: As shown in Tables 2, 3, and 4, and Figure 2-4As shown, the corrected mortality rates at 100-fold dilution were higher than those at 300-fold and 500-fold dilution. Under the conditions of 100-fold dilution and 72h treatment, the corrected mortality rate of aphids in N-22 fermentation broth reached a maximum of 80.21%, higher than the highest corrected mortality rate of H17 fermentation broth. Therefore, N-22 fermentation broth has a better insecticidal effect on aphids.
[0036] <Experimental Example 2> This experiment aimed to investigate the corrected mortality rate and toxic activity (LC50) of Bacillus amyloliquefaciens N-22 fermentation broth at different dilution ratios against pea aphids. 50 ) Experimental steps: Select healthy broad bean plants infested with aphids and cut off approximately 7cm sections. Use a paintbrush to remove any dead or weak aphids from the cut sections, ensuring there are 20-25 aphids on each plant. Record the exact total number of aphids.
[0037] Using tweezers, hold the cut end of the plant and spray approximately 0.5 mL of bacterial solution onto the aphids using a spray bottle, ensuring all aphids are sprayed. Perform three replicates for each concentration. Use sterile water as a control group.
[0038] After spraying, wrap the cut part of the plant with a damp cotton ball to keep it moist, and place the plant in a 9cm diameter petri dish. Place the lid of the petri dish on top of the dish, making sure there is a gap between the lid and the dish, and wrap the lid with plastic wrap to secure it.
[0039] The treated petri dishes were placed in a light incubator at 25±0.5℃, RH 60±5%, and a light duration (L:D) of 14h:10h. The number of dead insects was checked and recorded after 24h, 48h, 72h, and 96h of treatment. The corrected mortality rate was calculated, and the experimental data were analyzed. The formula for calculating the corrected mortality rate is as follows: Mortality rate (%) = (Number of dead insects / Total number of insects) x 100%; Corrected mortality rate (%) = (treatment mortality rate - control mortality rate) / (1 - control mortality rate) x 100%.
[0040] Experimental results: As shown in Table 5 and Figure 5 As shown, with the increase of N-22 fermentation broth concentration, the corrected mortality rate of pea aphids increased from 24.49% to 97.96% after 96 hours of bacterial treatment, indicating that the toxic effect of Bacillus amyloliquefaciens on pea aphids increases with increasing bacterial concentration. When the bacterial concentration was 1×10⁻⁶... 8At a concentration of CFU / mL, the corrected mortality rate of pea aphids was the highest at 97.96%, higher than other concentrations, indicating the best killing effect.
[0041] As shown in Table 6, when the disinfection treatment time is 72 hours, LC 50 The value is 4.2 × 10 6 CFU / mL; when the toxicity treatment time is 96h, LC 50 The value is 2.7 × 10 5 CFU / mL. In the experiment, the LC50 of N-22's toxicity against pea aphids was found to be... 50 The value decreases as the processing time increases. When the poisoning processing time is 96 hours, the LC value decreases. 50 The lowest value is 2.7 × 10. 5 CFU / mL.
[0042] Table 5. Results of different concentration treatments on pea aphids
[0043] Table 6. Toxicity of different concentrations against pea aphids
[0044] The above embodiments merely illustrate several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of this patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.
Claims
1. The application of Bacillus amyloliquefaciens N-22 in the control of aphids, characterized in that, The Bacillus amyloliquefaciens N-22, with accession number CCTCC NO: M2020256, was deposited on July 1, 2020, at the China Center for Type Culture Collection, located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.
2. The application of Bacillus amyloliquefaciens N-22 according to claim 1 in the control of aphids, characterized in that, The aphid in question is the pea aphid.
3. A fermentation broth, characterized in that, The fermentation broth was prepared from Bacillus amyloliquefaciens N-22 as described in claim 1.
4. The fermentation broth according to claim 3, characterized in that, The concentration of Bacillus amyloliquefaciens N-22 spores in the fermentation broth was 10. 8 ~10 10 cfu / mL.
5. The application of the fermentation liquid according to claim 3 or 4 in the preparation of aphid control products.