Application of Bacillus amyloliquefaciens N-22 in the control of tea lace bug

Abstract

This invention relates to the field of biological control technology for plant pests, and particularly to the application of *Bacillus amyloliquefaciens* in the control of tea lace bugs. The strain is *Bacillus amyloliquefaciens* (…). Bacillus amyloliquefaciens Lactobacillus subtilis N-22, accession number CCTCC NO:M 2020256, was deposited at the China Center for Type Culture Collection (CCTCC) on July 1, 2020. The fermentation broth prepared from Bacillus amyloliquefaciens used in this invention can effectively control tea lace bugs. A 1.0 × 10⁻⁶ culture medium was used. 8 The 96-hour corrected mortality rate of tea lace bug nymphs in CFU / mL fermentation broth reached a maximum of 84.31%. 50 7.0×10 2 CFU / mL.

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 tea lace bugs. Background Technology

[0002] Tea lace bug ( Chinese Stephanitis The tea lace bug (Laminaria japonica), belonging to the family Laminariaceae in the order Hemiptera, is an adult and nymph that congregates on the underside of tea leaves to suck sap. Affected leaves develop dense clusters of small white spots, giving the tea plant a grayish-white appearance from a distance. Black, sticky excrement appears on the underside of the leaves, hindering photosynthesis. In severe cases, it leads to leaf drop, weakened plant vigor, and slow, small, or halted bud development, significantly impacting tea yield and quality. In recent years, the tea lace bug has spread rapidly in tea-producing areas of several provinces in my country, causing widespread crop failure in many tea gardens and becoming one of the major threats to tea production safety. Its astonishingly rapid reproduction rate makes control extremely challenging. Chemical control is the primary method for controlling the tea lace bug and is widely used in tea production. However, the long-term and excessive use of chemical pesticides not only poses serious risks to tea production but may also damage the tea garden's ecological environment, triggering a series of ecological problems.

[0003] Biopesticides, as a convenient, readily available, environmentally friendly, and residue-free method of pest control, are receiving increasing research and application. Using biopesticides to control pests can effectively reduce the use of chemical pesticides. Most microbial pesticides used for pest control focus on entomopathogenic fungi such as Beauveria bassiana and Metarhizium anisopliae, as well as Bacillus thuringiensis (primarily for controlling lepidopteran pests). However, there are no reports of using Bacillus thuringiensis to control the tea lace bug, a piercing-sucking insect. Summary of the Invention

[0004] In view of this, the present invention provides the application of Bacillus amyloliquefaciens N-22 in the control of tea lace bugs.

[0005] Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens The application of N-22 in the control of tea lace bugs, the Bacillus amyloliquefaciens ( 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.

[0006] A fermentation broth, said fermentation broth being prepared from the aforementioned Bacillus amyloliquefaciens N-22.

[0007] Furthermore, the concentration of *Bacillus amyloliquefaciens* N-22 spores in the fermentation broth is 10. 8 ~10 10 cfu / mL.

[0008] The above-mentioned fermentation broth was used in the preparation of a solution for controlling tea lace bugs.

[0009] The beneficial effects of the technical solution provided by the present invention are: (1) The Bacillus amyloliquefaciens strain used in the present invention has the advantages of fast growth rate, large sporulation and strong resistance to pests. The fermentation liquid prepared by using Bacillus amyloliquefaciens can effectively control tea lace bugs.

[0010] (2) This Bacillus amyloliquefaciens has a better control effect than other biocontrol agents and is a promising method for controlling tea lace bugs. Attached Figure Description

[0011] Figure 1 The mortality rate of tea lace bugs under treatment with 10 biological agents; Figure 2 The mortality rate of tea lace bugs under different concentrations of Bacillus amyloliquefaciens N-22 fermentation broth was determined. Detailed Implementation

[0012] 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.

[0013] The inventors have discovered that *Bacillus amyloliquefaciens* exhibits a significant control effect against tea lace bugs, providing new technical support for reducing the use of chemical pesticides and ensuring the quality and safety of tea. This not only ensures the quality and safety of tea but also reduces damage to the tea garden's ecological environment. *Bacillus amyloliquefaciens* N-22, isolated from peach leaves, has advantages such as rapid growth, high sporulation rate, and strong antagonism against pests. Its fermentation broth has a significant effect on controlling tea lace bugs and holds promise for development as a novel tea lace bug control agent.

[0014] In this invention: Test insects: The test insects were tea lace bugs, collected from Wujia Tai Tea Garden in Xuan'en, Enshi, Hubei. Fourth instar nymphs of tea lace bugs with basically the same size and good vitality were selected for the test.

[0015] Test inoculants: 1 billion CFU / mL Bacillus amyloliquefaciens N-22 fermentation broth, 1 billion CFU / mL Bacillus amyloliquefaciens H-12 fermentation broth, 1 billion CFU / mL Bacillus subtilis H-17 fermentation broth, 1 billion CFU / mL *Bacillus thuringiensis* suspension, 1 billion CFU / mL *Bacillus thuringiensis* BTK-P15 suspension, 3 billion CFU / mL *Bacillus thuringiensis* BTT, *Beauveria bassiana* I (8 million spores / g), *Beauveria bassiana* II (100 billion spores / g), *Beauveria bassiana* III (mutant, 1 billion spores / g), and 200 million CFU / mL foliar microbial inoculant (Anhui Guoren Biotechnology Co., Ltd.).

[0016] Bacillus amyloliquefaciens H-12 and Bacillus subtilis H-17 were purchased from Yunnan Agricultural University; Beauveria bassiana was purchased from Hubei Zhongxiang Biotechnology Co., Ltd. Other bacterial agents are stored in our laboratory.

[0017] It should be noted that the Bacillus amyloliquefaciens described in this invention (…) Bacillus amyloliquefaciens The accession number of Bacillus amyloliquefaciens N-22 is CCTCC NO: M 2020256. It 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 fermentation broth preparation method of Bacillus amyloliquefaciens N-22 used in this invention is the same as that application.

[0018] Fermentation of Bacillus amyloliquefaciens 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.

[0019] 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.

[0020] 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.

[0021] <Experimental Example 1> This experiment aimed to investigate the corrected mortality rate of *Bacillus amyloliquefaciens* N-22 fermentation broth at different dilutions after treatment with *Tea lace bug*, and to compare it with the corrected mortality rate of *Tea lace bug* after treatment with nine other biological agents. Experimental steps: 1. The experiment consisted of 10 treatments, as follows: Treatment 1: Bacillus amyloliquefaciens N-22 fermentation broth diluted 100 times; Treatment 2: Bacillus amyloliquefaciens H-12 fermentation broth diluted 100 times; Treatment 3: Bacillus subtilis H-17 fermentation broth diluted 100 times; Treatment 4: Bacillus thuringiensis BTK-P15 diluted 100 times; Treatment 5: Beauveria bassiana with 8 million spores / g diluted 300 times; Treatment 6: Bacillus flavus suspension diluted 300 times; Treatment 7: Acaricide diluted 100 times; Treatment 8: Foliar microbial agent diluted 300 times; Treatment 9: Beauveria bassiana with 100 billion spores / g diluted 1000 times; Treatment 10: Beauveria bassiana mutant strain diluted 100 times.

[0022] Sterile water was used as a control, and each treatment was repeated 3 times.

[0023] Fresh tea tree branches free from pests and diseases, collected from the tea garden, were rinsed clean with water and allowed to air dry naturally. Two tea leaves were then cut and placed in a petri dish. The cut area was wrapped with a moist cotton ball to keep it moist. Twenty tea lace bugs of uniform size were attached with a fine brush and sprayed evenly with the prepared bacterial solution. Sterile water was used as a control. Each treatment was repeated three times.

[0024] All the above-mentioned culture dishes were placed in an artificial climate chamber with a temperature of 23±2℃, a relative humidity of 70%±10%, and a photoperiod of 14L:10D. The number of dead insects was checked and recorded at 24h, 48h, 72h, and 96h after the spraying treatment. The insects were considered dead if they did not move when lightly touched with a brush.

[0025] 2. Data Processing Experimental data were statistically processed using SPSS 27.0 software to analyze the significant differences in control effects among treatment groups. Probability values ​​were converted based on corrected mortality rates, and linear regression analysis was performed on the dose to derive the toxicity regression equation and the median lethal concentration (LC50). 50 ) and other parameters.

[0026] The formula is as follows: Mortality rate (%) = (Number of live worms before treatment - Number of live worms after treatment / Number of live worms before treatment) × 100% Corrected mortality rate (%) = (Treatment mortality rate - Control mortality rate / 1 - Control mortality rate) × 100% Experimental results: The bioactivity of 10 biological agents against the tea lace bug was determined in vitro, and the results are shown in Figure 1 and Table 1. Figure 1 shows that the mortality rate of tea lace bugs under each agent treatment gradually increased with the extension of treatment time, exhibiting an upward trend. The control group (CK) maintained a consistently low mortality rate. At 96 hours post-treatment, the mortality rate of tea lace bugs treated with foliar microbial agents was significantly lower than that of the other agents, and the mortality rate of tea lace bugs treated with Bacillus thuringiensis BTK-P15 was also slightly lower than that of the other agents. This result indicates that, compared to other agents, foliar microbial agents and Bacillus thuringiensis BTK-P15 have less toxic effects on tea lace bugs. From 48 to 72 hours post-treatment, the mortality rates of tea lace bugs treated with *Beauveria bassiana* (100 billion spores / g), *Beauveria bassiana* (100 billion spores / g), and *Beauveria bassiana* mutant strains showed significantly different changes compared to the control group. The mortality rate of tea lace bugs varied significantly between 72 and 96 hours after treatment with Bacillus amyloliquefaciens N-22, Bacillus amyloliquefaciens H-12, Bacillus subtilis H-17, and Beauveria bassiana with 8 million spores / g.

[0027] Table 1. Corrected mortality rate of tea lace bugs at different time points after treatment with different inoculants.

[0028] The experimental results (Table 1) show that among these 10 biological agents, Bacillus amyloliquefaciens N-22, Bacillus amyloliquefaciens H-12, Bacillus subtilis H-17, Bacillus flavus, Beauveria bassiana with 8 million spores / g, and the Beauveria bassiana mutant strain all have good toxic effects on tea lace bugs.

[0029] 24 hours post-treatment, the corrected mortality rate of tea lace bugs treated with four agents—Bacillus amyloliquefaciens H-12, Bacillus subtilis H-17, Bacillus thuringiensis BTK-P15, and *Bacillus fumigatus*—exceeded 10%. 48 hours post-treatment, the corrected mortality rate of tea lace bugs treated with Bacillus subtilis H-17, Bacillus thuringiensis BTK-P15, and *Beauveria bassiana* at 100 billion spores / g was 27.78%, the corrected mortality rate of tea lace bugs treated with Bacillus amyloliquefaciens H-12 was 24.07%, and the corrected mortality rates of the other agents were all below 20.00%.

[0030] 72 hours after application, *Betula flavum* showed significantly higher toxicity against tea lace bugs than other treatments, with a corrected mortality rate of 67.30%. *Beauveria bassiana* with 100 billion spores / g also exhibited higher toxicity than other treatments, with a corrected mortality rate of 55.77%, second only to *Betula flavum* in toxicity.

[0031] The corrected mortality rate of tea lace bugs was lowest under foliar microbial inoculant treatment, at only 19.23%. Among the remaining agents, the corrected mortality rate of tea lace bugs was higher than 40.00% under treatment with Beauveria bassiana mutant strains, acaricides, and Bacillus thuringiensis BTK-P15.

[0032] At 96 hours post-treatment, *Bacillus amyloliquefaciens* N-22 and *Bacillus fumigatus* showed the most significant toxic effects. The corrected mortality rate of tea lace bugs under *Bacillus amyloliquefaciens* N-22 treatment reached 74.51%, and that under *Bacillus fumigatus* treatment reached 72.55%, both exceeding 70.00%. The corrected mortality rates of the other agents also increased, with six exceeding 60.00%. The corrected mortality rate of tea lace bugs under *Bacillus amyloliquefaciens* H-12 and *Beauveria bassiana* mutant treatments was 64.71%, while the corrected mortality rate under *Bacillus subtilis* H-17 and *Beauveria bassiana* treatments (8 million spores / g) reached 62.75%. The corrected mortality rate of tea lace bugs treated with foliar microbial agents and Bacillus thuringiensis BTK-P15 was less than 50%, with the corrected mortality rate of foliar microbial agents being only 23.55%, significantly lower than that of other agents. This indicates that the two agents were not very effective in killing tea lace bugs.

[0033] The corrected mortality rates of tea lace bugs under each pesticide treatment were as follows: Bacillus amyloliquefaciens N-22 fermentation broth 74.51% > Bacillus flavus suspension 72.55% > Bacillus amyloliquefaciens H-12 fermentation broth 64.71% = Beauveria bassiana mutant strain 64.71% > Bacillus subtilis H-17 fermentation broth 62.75% = 8 million spores / g Beauveria bassiana 62.75% > acaricide 60.78% = 100 billion spores / g Beauveria bassiana 60.78% > Bacillus thuringiensis BTK-P15 suspension 45.10% > foliar microbial agent 23.53%.

[0034] <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 on tea lace bugs after treatment. 50 ).

[0035] Experimental steps: Five concentration gradients were set up for the N22 fermentation broth experiment, with a concentration gradient of 1.0 × 10⁻⁶. 8 CFU / mL, 1.0×10 6 CFU / mL, 1.0×10 4 CFU / mL, 1.0×10 2 CFU / mL, 1.0×10 1 Five concentration gradients of CFU / mL were used, with sterile water as a control, and each concentration was replicated in triplicate.

[0036] Fresh tea tree branches free from pests and diseases, collected from the tea garden, were rinsed clean with water and allowed to air dry naturally. Two tea leaves were then cut and placed in a petri dish. The cut area was wrapped with a moist cotton ball to keep it moist. Twenty tea lace bugs of uniform size were attached with a fine brush and sprayed evenly with the prepared bacterial solution. Sterile water was used as a control. Each treatment was repeated three times.

[0037] All the above-mentioned culture dishes were placed in an artificial climate chamber with a temperature of 23±2℃, a relative humidity of 70%±10%, and a photoperiod of 14L:10D. The number of dead insects was checked and recorded at 24h, 48h, 72h, and 96h after the spraying treatment. The insects were considered dead if they did not move when lightly touched with a brush.

[0038] Experimental data were statistically processed using SPSS 27.0 software to analyze the significant differences in control effects among treatment groups. Probability values ​​were converted based on corrected mortality rates, and linear regression analysis was performed on the dose to derive the toxicity regression equation and the median lethal concentration (LC50). 50 ) and other parameters.

[0039] The formula is as follows: Mortality rate (%) = (Number of live worms before treatment - Number of live worms after treatment / Number of live worms before treatment) × 100% Corrected mortality rate (%) = (Treatment mortality rate - Control mortality rate / 1 - Control mortality rate) × 100% Experimental results: Tea lace bugs were selected and subjected to a 1.0×10⁻⁶ test. 1 CFU / mL, 1×10 2 CFU / mL, 1×10 4 CFU / mL, 1×10 6 CFU / mL, 1×10 8 The toxicity test of Bacillus amyloliquefaciens N-22 concentration gradient with CFU / mL was shown in Figure 2 and Table 2.

[0040] Table 2 Corrected mortality rates of tea lace bugs treated with different concentrations of Bacillus amyloliquefaciens N-22 fermentation broth

[0041] As shown in Figure 2, with the concentration of Bacillus amyloliquefaciens N-22 increasing from 1×10⁻⁶, the effect of the treatment on the growth rate of the bacteria decreased. 1 CFU / mL was gradually increased to 1×10 8 With increasing CFU / mL concentrations, the mortality rate of tea lace bugs gradually increased. At all experimental concentrations, the mortality rate of tea lace bugs also gradually increased with prolonged treatment time. Specifically, at 1×10⁻⁶ CFU / mL... 8 The CFU / mL treatment group maintained a significantly higher mortality rate than other groups at every time point, particularly between 48 and 72 hours post-drug administration, at 1×10⁻⁶. 2 CFU / mL, 1×10 4 CFU / mL, 1×10 6 CFU / mL and 1.0x10 8 The mortality rate increased significantly with CFU / mL, especially at a concentration of 1×10⁻⁶. 6 The mortality rate increased most significantly from 48 hours after treatment, when the CFU / mL level was highest.

[0042] Table 2 shows that the toxic activity of *Bacillus amyloliquefaciens* N-22 fermentation broth against tea lace bugs gradually increased with increasing concentration. At 96 hours post-treatment, a concentration of 1.0 x 10⁻⁶... 8 The highest corrected mortality rate (84.31%) for the tea lace bug was observed at a concentration of CFU / mL, indicating the best toxicity; 1.0×10 1 The toxic effect was weakest at a CFU / mL concentration, with a corrected mortality rate of only 27.45% for the tea lace bug. From 24 to 72 hours post-treatment, the corrected mortality rate of the tea lace bug under the treatment generally increased with increasing N-22 fermentation broth concentration. At 24 hours post-treatment, at a concentration of 1.0 × 10⁻⁶ CFU / mL... 8The toxicity of the agent at a concentration of CFU / mL was significantly higher than that of the other four treatments, with a corrected mortality rate of 40.48%. The corrected mortality rates of the remaining groups were around 10% or less. (1.0×10⁻⁶) 8 The CFU / mL concentration was significantly different from the other four treatment groups (p<0.05). 48 h post-treatment, 1.0×10⁻⁶ 8 The corrected mortality rate of tea lace bugs treated with CFU / mL was significantly higher than that of other treatments, reaching 58.76%. At 72 hours post-treatment, the corrected mortality rate increased in all treatments, with the highest increase observed at 1.0 × 10⁻⁶ CFU / mL. 6 The toxic activity of the agent was significantly improved at a concentration of CFU / mL, 1.0×10⁻⁶. 8 CFU / mL treatment showed good persistence of action.

[0043] Based on the corrected mortality rate of *Tea lace bug* under different concentration gradients of *Bacillus amyloliquefaciens* N-22 poisoning experiments, the virulence regression equation, chi-square value, and LC-12 values ​​at different treatment times were calculated. 50 The values ​​and significance were determined, and the data are shown in Table 3.

[0044]

[0045] Table 3 shows that when the processing time is 72 hours, LC 50 The value is 6.0 × 10 5 CFU / mL; when the treatment time is 96 h, LC 50 The value is 7.0 × 10 2 CFU / mL. The toxicity regression equations at 72h and 96h post-drug administration were y=-1.177+0.204x and y=-0.548+0.193x, respectively. Based on the results, the LC50 of *Bacillus amyloliquefaciens* N-22 against *Tea lace bug* was found to be... 50 The value decreases with increasing processing time. When the disinfection processing time is 96 hours, the LC value decreases. 50 The lowest value is 7.0 × 10. 2 CFU / mL.

[0046] 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. Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens The application of N-22 in the control of tea lace bugs is characterized by, The Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens N-22, with accession number CCTCCNO: M 2020256, 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 a fermentation broth in the preparation of a treatment for controlling tea lace bugs, characterized in that, The fermentation broth is prepared from Bacillus amyloliquefaciens N-22 as described in claim 1, and the concentration of Bacillus amyloliquefaciens N-22 spores in the fermentation broth is 10. 8 ~10 10 cfu / mL.

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