A Bacillus thuringiensis granule for the prevention and control of grubs

Abstract

This invention provides a Bacillus thuringiensis granule formulation. The granules comprise a nutrient matrix composed of soybean meal and corn flour, and also contain an adsorbent and a binder. The mass ratio of soybean meal to corn flour is 2:1. This invention uses Bacillus thuringiensis to control grubs, screening for suitable and inexpensive nutrients and adjuvants for Bt proliferation in soil to obtain a stable and highly effective Bt granule product. It solves the problems of storage stability and easy inactivation, effectively increasing the Bt biomass in the soil after application, improving the stability and persistence of the agent, and realizing the resource utilization of agricultural byproducts. This invention is green and environmentally friendly, reduces environmental pollution, is easy to use, harmless to humans and crops, and has a significant effect on controlling grubs, showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biological pest control technology, specifically relating to a Bacillus thuringiensis granule for controlling grubs. Background Technology

[0002] Grubs are the larvae of scarab beetles. Grubs are recognized both domestically and internationally as difficult-to-control soil-dwelling pests. Common species in Chinese agriculture include the large black-breasted scarab beetle, the dark black-breasted scarab beetle, and the green-breasted scarab beetle. Their life cycle is generally one generation per year or two years. Damage is primarily caused by the larvae, which feed on plant roots, leading to plant wilting and death. Grubs particularly favor oil crops such as peanuts, damaging peanut pods and severely impacting yield and quality. Severe grub infestations combined with inadequate control can result in complete crop failure in peanut fields. Many methods exist for controlling grubs, with chemical control being the most prevalent. However, the long-term irrational use of chemical pesticides leads to a series of negative problems, including increased pesticide residues, increased resistance, deterioration of soil physical and chemical properties, and environmental pollution. Biological control, on the other hand, offers numerous advantages such as high selectivity, good safety, minimal impact on the ecosystem, and low likelihood of developing resistance, making it a promising area for application.

[0003] Bacillus thuringiensis (Bt) possesses insecticidal activity against a variety of agricultural and forestry pests and is widely used as a biological insecticide. One of the most important characteristics of Bt strains is that during their spore formation and stable growth phases, they can produce Cry or Cyt proteins, primarily in the form of companion crystals. These protein crystals act as poisons to target pests, while remaining safe and harmless to non-target pests, humans, and animals.

[0004] In 1992, the first Bt strain Buibui with specific insecticidal activity against grubs was discovered abroad. The United States and Japan subsequently applied for related patents and product registrations, demonstrating the huge application potential of Bt in the biological control of grubs.

[0005] In recent years, more than ten Bt strains with insecticidal activity against grubs have been isolated in China. However, the development of Bt formulations and products for the control of grubs in China is still relatively lagging behind. The main problems are the short shelf life of the formulations and the poor stability of the parasitic crystals that play the main insecticidal role. At the same time, Bt is easily diluted by soil and water when applied in the field, which leads to a decrease in the Bt content in the soil, resulting in unstable efficacy, short duration of effect, and unsatisfactory control effect. Summary of the Invention

[0006] This invention addresses the problems of poor storage stability, easy inactivation, and low Bacillus thuringiensis (Bt) content in soil after application of Bt granules. It provides a Bacillus thuringiensis granule for controlling grubs, thereby improving the storage stability and control efficacy of Bt.

[0007] The present invention first provides a Bacillus thuringiensis granule, wherein the granule contains a nutrient matrix composed of soybean meal and corn flour, and also contains an adsorbent and a binder.

[0008] Preferably, the mass ratio of soybean meal to corn flour is 2:1.

[0009] The adsorbent is any one of vermiculite, acid-washed and un-acid-washed biochar, and silica.

[0010] Preferably, the adsorbent is unwashed biochar.

[0011] The binder is one or more of gelatin, microcrystalline cellulose, and sodium alginate.

[0012] Preferably, the binder is sodium alginate.

[0013] Furthermore, the Bacillus thuringiensis granules are prepared by adding 5% sodium alginate and 1% biochar to the nutrient matrix, with a Bt concentration of 2.3 × 10⁻⁶. 9 cfu / g; the mass ratio of soybean meal to corn flour in the nutrient matrix is ​​2:1.

[0014] The application of Bacillus thuringiensis granules provided by this invention in the control of grubs.

[0015] This invention utilizes Bacillus thuringiensis (Bt) to control grubs. It screens suitable and inexpensive nutrients and adjuvants for Bt proliferation in soil, resulting in a stable and highly effective Bt granule product. This addresses the issues of storage stability and easy inactivation, effectively increasing the Bt biomass in the soil after application, improving the stability and persistence of the agent, and achieving resource utilization of agricultural byproducts. This invention is environmentally friendly, reduces pollution, is easy to use, harmless to humans and crops, and has a significant effect on controlling grubs, showing broad application prospects. Attached Figure Description

[0016] Figure 1 Here is a picture of the actual Bt granules;

[0017] Figure 2 The graph shows the changes in viable bacterial counts of Bt granules after storage at 26℃ and 4℃.

[0018] Figure 3The graph shows the changes in the insecticidal crystal protein content of Bt granules after storage at 26℃ and 4℃.

[0019] Figure 4 The graph shows the changes in viable bacterial counts of Bt granules after storage at 54℃ and 45℃.

[0020] Figure 5 The graph shows the changes in the insecticidal crystal protein content of Bt granules after storage at 54℃ and 45℃.

[0021] Figure 6 This is a graph showing the changes in the number of viable bacteria in soil using Bt granules;

[0022] Figure 7 Corrected mortality rate of grubs for indoor bioassay of Bt granules. Detailed Implementation

[0023] This invention uses the Bt larvae-killing strain B-Y7-1 as material. By screening suitable nutrients and adjuvants for Bt proliferation in soil, a stable and efficient Bt granule product is obtained, solving the problems of poor storage stability, easy inactivation, and low Bt content in soil. This is of great significance for improving the stability and duration of efficacy of the agent.

[0024] The present invention will be further illustrated by the following specific embodiments, but these are not intended to limit the invention.

[0025] Example 1: Screening of Granule Formulations

[0026] 1. Activate the preserved Bt strain B-Y7-1, and inoculate it on LB agar using the three-zone streak method. After inoculation, place the plate at 28°C for incubation. Pick a single colony and continue inoculating it on LB agar. After incubation for 72 hours, the bacterial culture can be collected and prepared.

[0027] 2. Screening Bt nutrient matrix formulations

[0028] 1) Different proportions and combinations of soybean meal, peanut meal, and corn flour were used as nutrient substrates. Cultures were performed in 250mL Erlenmeyer flasks with a total flask volume of 60g. The inoculum concentration was 10% of the nutrient substrate, and the bacterial concentration was 1×10⁻⁶. 8 The concentration of cfu / mL was maintained at 20%–25% of the total volume, and each treatment was repeated three times. Samples were taken for four consecutive days, 10g each time, and added to an Erlenmeyer flask containing glass beads. 100mL of sterile water was added, and the mixture was allowed to stand for 20 minutes. Then, it was shaken at 200 rpm for 30 minutes to obtain the mother culture suspension. This suspension was diluted 10 times, and 0.1mL of the suspension was added to LB agar plates using appropriate gradients for plating. The viable cell count was calculated, and a suitable nutrient substrate formulation was selected.

[0029] Calculation formula:

[0030]

[0031] When using pure soybean meal, peanut meal, and corn flour as the nutrient substrate, Bt grew rapidly in the first two days. The viable bacterial concentration in soybean meal and corn flour reached its highest value on day 2, significantly increasing compared to the initial bacterial count, and by day 4 it was several times the initial bacterial count. When soybean meal and peanut meal were mixed in different ratios, the Bt bacterial count in the soybean meal:peanut meal (6:0) mixture continuously increased from day 2, reaching its highest value on day 4, exceeding all other combinations. When soybean meal and corn flour were mixed in different ratios, the Bt bacterial count in the soybean meal:corn flour (2:1) mixture continuously increased, reaching its highest value on day 4, exceeding that of pure soybean meal and all other combinations. After screening, the optimal nutrient substrate formula was determined to be soybean meal:corn flour (2:1).

[0032] 2) Vermiculite, acid-washed straw biochar, un-acid-washed straw biochar, and silica were added to LB solid medium at a ratio of 1%. Known concentrations of bacterial suspension were spread onto agar plates, with a blank control included. After 24 hours, the average size of bacterial plaques on the medium with un-acid-washed biochar was 21.99 mm. 2 The concentration was significantly higher than that of other adsorbent treatments (excluding the addition of acid-washed biochar) and the blank control (13.32 mm). 2 The average size of the bacterial plaques on the biochar-added medium was 61.41 mm after 48 hours of application. 2 The concentration was significantly higher than that of other adsorbent treatments (excluding the addition of acid-washed biochar) and the blank control (46.64 mm). 2 ).

[0033] Weigh out 0.8g of adsorbent material and 8mL of 1×10⁻⁶ solution respectively. 8 A CFU / mL Bt bacterial suspension was prepared into a composite bacterial suspension and added to 80g of nutrient substrate. A blank control was set up. The viable bacterial count was counted at 1-4 days and 7 days, and other procedures were the same as in section (1) of Example 1. The dynamic indicators of Bt growth were statistically analyzed to determine the effect of different adsorbents on Bt compatibility. There was no significant difference in the bacterial count of Bt in the mixture of different adsorbents and nutrient substrates. At day 7, the Bt bacterial count with added unwashed biochar was higher than that of other groups and several times higher than the initial bacterial count. This indicates that biochar as an adsorbent has no negative effect on Bt growth and has a certain growth-promoting effect. Considering cost and process, unwashed biochar was selected as the adsorbent material.

[0034] 3) Gelatin, microcrystalline cellulose and sodium alginate were selected as binder materials and added to the nutrient matrix at an addition amount of 3%. A blank control was set up. Other aspects were the same as in section (1) of Example 1. Samples were taken for 4 consecutive days and the number of effective viable bacteria was counted.

[0035] The number of Bt bacteria in the nutrient matrix with added sodium alginate continued to increase and was consistently higher than that with other binders. The number of Bt bacteria reached its highest value at 4 days, showing a significant increase compared to the initial number. Sodium alginate was thus selected as the binder material.

[0036] 4) Based on the single-factor experiments, effective variables were selected, and an orthogonal experimental design (Tables 1 and 2) was adopted to investigate the effects of total water content (A), sodium alginate addition (B), and biochar addition (C) on Bt proliferation. The optimized orthogonal experimental screening results (Table 3) show that the actual optimal result is A2B2C2, and the theoretical optimal result is A3B2C2. The relevant indicators of the theoretical and actual optimal formulations were tested (Table 4), and no significant difference was found between the two.

[0037] The final granule formulation was determined to be as follows: nutrient carrier mass ratio (soybean meal:corn flour = 2:1), sodium alginate addition of 5%, biochar addition of 1%, and a concentration of 2.3 × 10⁻⁶ at a 10% addition ratio. 10 The bacterial culture had a concentration of CFU / mL and a total water content of 77%. Granulation was performed to obtain a concentration of 2.3 × 10⁻⁶ CFU / mL. 9 Bt granules with cfu / g.

[0038] Table 1: Factor Level Table

[0039]

[0040] Table 2: Optimized Orthogonal Array

[0041]

[0042] Table 3: Screening Results of Orthogonal Experiments

[0043]

[0044] Table 4: Test Table of Relevant Indicators for Theoretical Optimal Formulation and Actual Optimal Formulation

[0045]

[0046] Example 2: Preparation and index detection of granules

[0047] 1. According to the selected formula, first mix the nutrient carrier and binder, then autoclave them. Simultaneously, prepare a composite bacterial suspension of biochar and bacterial solution. Add the composite bacterial suspension to the mixture, then add water to achieve a total moisture content of 77%. Knead the mixture into a dough, then place it in a granulator for extrusion granulation. After extrusion, dry the granules to obtain the finished product. Figure 1 ).

[0048] (1) During granulation, the machine operates at a frequency of 10 Hz and the particle size is 0.8 mm.

[0049] (2) After the granules are extruded into granules, they are placed in an oven at 40°C and dried for 48 hours.

[0050] 2. The following indicators were tested according to the requirements of GB 20287-2006 and GB / T 19136-2021: effective viable count, mold and other microbial count, miscellaneous microbial rate, moisture content, pH, and thermal storage stability:

[0051] (1) Samples were serially diluted and plate counts were performed. LB solid medium was used to determine the number of viable bacteria, and Martin medium was used to determine the number of molds and other microorganisms. Plates with a dilution of 20 to 300 colonies were used as the counting standard, and the number of viable bacteria and other microorganisms were counted separately.

[0052] (2) Select an aluminum box, dry it, and weigh it. Place the sample in the container, weigh it, and record the weight. Place the container with the sample in a drying oven at 105℃±2℃ and dry it for 4 to 6 hours. After drying, weigh it and calculate the moisture content.

[0053] Calculation formula

[0054]

[0055] (3) Take 1g of sample and put it into 100mL of sterile water, stir vigorously for 1min, and let stand for 1min. Turn on the pH meter, calibrate it with the standard solution, and after calibration, insert the electrode into the sample solution to measure the pH value. Repeat each sample three times and calculate the average value of the three times.

[0056] (4) The effective viable count, mold and other microbial count, miscellaneous microbial rate, moisture content, and pH of the granules are shown in Table 5. According to GB20287-2006, the effective viable count should be ≥1×10⁻⁶. 8 cfu / g, bacterial count ≤3×10 6 CFU / g, bacterial count ≤30.0%, moisture content ≤20.0%, pH between 5.5 and 8.5. All indicators of this granule meet the requirements.

[0057] Table 5: Results of Tests for Relevant Indicators of Bt Granules

[0058]

[0059] (5) Stability test at room temperature: The granule sample was sealed in a glass bottle and stored in constant temperature incubators at 26℃±2℃ and 4℃±2℃ for 18 weeks, respectively. Samples were taken once a week, and the effective viable bacteria count and protein content were determined within 24 hours. The method for determining the effective viable bacteria count is as follows: Take 1g of sample, add it to a glass beaded Erlenmeyer flask, add 100mL of sterile water, let it stand for 20min, and then shake it on a shaker at 200r / min for 30min to obtain the mother liquor bacterial suspension. Dilute it by a factor of 10, select an appropriate gradient for plating, and calculate the viable bacteria concentration. The calculation formula is the same as section (1) of 2 in Example 1. The protein content determination method is as follows: Take 1.5 mL of the mother culture bacterial suspension, centrifuge, resuspend the precipitate in 100 μL of sterile water, add 25 μL of 0.5 mol / L NaOH, react at room temperature for 5 min, add 125 μL of 2× loading buffer, boil in a water bath for 10 min, centrifuge, take 15 μL of supernatant for SDS-PAGE, stain the gel with Coomassie Brilliant Blue R250, process the protein gel image using the Image Lab system, and calculate the protein content in the sample.

[0060] Results under different storage conditions are shown in the figure. Figure 2 , Figure 3 At 26℃ and 4℃, the bacterial content in the granules remained stable for the first 10 weeks, without significant decrease or increase. After 10 weeks, the bacterial count began to increase, reaching a maximum of 9.0 × 10⁻⁶. 9 The cfu / g count showed no significant decrease compared to the initial bacterial count by week 18. Insecticidal crystalline proteins were still detectable by week 18 at both 26℃ and 4℃, with no significant decrease in content. Long-term storage at 26℃ and 4℃ showed no significant changes in bacterial count and protein content, offering the advantage of saving storage costs. This indicates that Bt can survive well in granules. This granule formulation can slow down the degradation rate of insecticidal crystalline proteins under natural conditions, reducing the impact of external conditions on the insecticidal components and protecting them. Analysis of the experimental results of Bt granules under different storage conditions clarified the storage conditions and storage time for this product, providing a theoretical basis for its application.

[0061] (6) Thermal storage stability test: The samples were sealed in glass bottles and stored for 6 weeks in constant temperature incubators at 54℃±2℃ and 45℃±2℃ respectively. Samples were taken every two weeks, and the effective viable bacteria count and protein content were determined within 24 hours, using the same method as above. The results of thermal storage stability are shown in […]. Figure 4 , Figure 5 The number of viable bacteria in Bt granules stored at 45℃±2℃ and 54℃±2℃ for 2 weeks decreased from the initial 2.30×10⁻⁶. 9 CFU / g decreased to 1.74 × 10⁻⁶. 9 cfu / g and 1.13×10 9The cfu / g count showed no significant decreasing trend from week 2 to week 6, and the viable bacteria count in the granules still met national standards after week 6. Protein content decreased with prolonged storage at 45℃±2℃ and 54℃±2℃, showing a slight decrease in week 2, and a reduction of approximately 1 / 2 to 2 / 3 in both storage conditions by week 6, indicating incomplete degradation. The effective viable bacteria count and protein content in the granules stored at 45℃ were higher than those stored at 54℃. Analysis of the storage test results under high-temperature conditions demonstrates that the formulation maintains good stability during storage and application under harsh conditions such as hot summer weather in practical applications.

[0062] Example 3: Changes in bacterial biomass of Bt granules in soil

[0063] Soil was collected from outdoors, dried until the moisture content was constant, and then divided into small boxes of 20g each. 1g of granules was added to each box, and the mixture was thoroughly mixed with the soil. Sterile water was then sprayed onto the soil at a moisture content of 15%. Samples were taken on days 3, 9, and 15, with three replicates. Plate samples were prepared according to section (1) of Example 1, and the Bt bacteria count in the soil was detected. The results are shown in […]. Figure 6 After 3 days of application of Bt granules to the soil, the Bt bacterial count in the soil significantly increased compared to the initial count, and showed a gradual increasing trend over time, reaching 3.43 × 10⁻⁶ after 15 days. 8 cfu / g, approximately 1.15 × 10⁻⁶ cfu / g, is equivalent to the initial bacterial count in the soil. 8 The concentration was 3 times higher than that of CFU / g. This indicates that Bt can proliferate in the soil after granule application, resulting in a significant increase in the Bt content in the soil.

[0064] Example 4: Determination of the control effect of Bt granules on grubs

[0065] Three treatments were set up: a blank control, Bt granules, and Bt bacterial solution. Twelve-well culture plates were used, and twelve healthy 2-5 day old larvae of the North China black-browed scarab beetle (one larva per well) were added to each well, and the plates were starved for 6 hours. In the blank control group, peanuts soaked in sterile water for 3 hours were placed in each well and covered with sterilized fine soil. In the Bt granule group, 0.02g of granules and soaked peanuts were added to each well, and the plates were covered with sterilized fine soil. In the Bt bacterial solution group, Bt bacterial solution was mixed with soil to achieve a Bt bacterial content of 9 × 10⁻⁶. 6 The soil bacterial content was adjusted to be consistent with that of the granular treatment group, and peanuts were placed in the holes. Corrected larval mortality rates were calculated on days 7, 14, and 21 after treatment. Results of the indoor bioassays are shown below. Figure 7It can be seen that after treatment with Bt granules and Bt bacterial solution, the corrected mortality rate of grubs increased continuously over time. On days 7, 14 and 21, the corrected mortality rate of grubs treated with Bt granules was significantly higher than that of grubs treated with Bt bacterial solution mixed with soil, indicating that granules have a good protective effect against Bt and can significantly improve the control effect of Bt.

Claims

1. A Bacillus thuringiensis granule, characterized in that, The granules are prepared by adding 5% sodium alginate, 1% unwashed biochar, and Bacillus thuringiensis at a concentration of 2.3 × 10⁻⁶ to a nutrient matrix. 9 cfu / g; the mass ratio of soybean meal to corn flour in the nutrient matrix is ​​2:

1.

2. The application of the Bacillus thuringiensis granules as described in claim 1 in the control of grubs.

3. The use of the Bacillus thuringiensis granules according to claim 1 in the preparation of products for the prevention and control of grubs.

4. A product for controlling grubs, characterized in that, The product contains the Bacillus thuringiensis granules as described in claim 1.

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