Bacillus thuringiensis L3 and application thereof

Abstract

The invention discloses a bacillus thuringiensis L3 and an application thereof. The preservation number of the bacillus thuringiensis L3 is CCTCC (China Center for Type Culture Collection) NO: M 2025487. The gene sequence of the 16S rDNA of the bacillus thuringiensis L3 is as shown in SEQ ID NO: 1. Under different adversity stress conditions, the bacillus thuringiensis L3 shows a certain stress resistance potential. Research finds that the bacillus thuringiensis L3 has certain acid resistance, high temperature resistance, cholate resistance and drought resistance, and has strong alkali resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a Bacillus thuringiensis L3 and its application. Background Art

[0002] Agricultural pests are the main factors causing losses of agricultural products in the processes of planting, storage, circulation, etc. The control of agricultural pests such as Plutella xylostella often relies on chemical insecticides. However, due to the frequent and unscientific application of chemical pesticides in recent years, resistant pest populations have gradually formed in different regions. At the same time, chemical pesticide residues are likely to pollute the environment and affect the quality of grains, fruits and vegetables. In severe cases, it will threaten human health. The abuse of chemical insecticides will also cause phenomena such as the destruction of natural enemies and the resurgence of secondary pests. At present, the prevention and control technologies for such pests mainly include chemical control, physical control, agricultural technical control, biological control, etc. Each prevention and control technology has its own advantages and disadvantages. Among them, biological control is the most effective and safest method in pest management. At the same time, this method has many advantages such as no environmental pollution, strong targetability, and low cost, so it has become the focus of future insecticidal research and advocacy.

[0003] There are various means of biological control, and tools such as pest natural enemies, biocontrol plants, and biocontrol microorganisms are often selected for control. Actively searching for efficient and low-toxic means of controlling vegetable pests is of great significance. There is an urgent need to introduce and develop biological control agents to rebuild the balance of the farmland ecosystem and achieve sustainable control of agricultural pests such as Plutella xylostella in newly invaded areas. The development and application of biological pesticides are the research hotspots in the current field of biological control. Microbial pesticides have become the focus of attention of scientific research personnel in the current field of pest control because they meet the development requirements of "ecologically reasonable pesticides" and "environmentally friendly pesticides".

[0004] Bacillus thuringiensis, abbreviated as Bt, is the most widely used and important microbial pesticide at present. It shows good activity against pests, has strong target specificity, is harmless to humans, animals and the environment, and has rich strain resources. There are great differences in the types, quantities and expression levels of insecticidal proteins produced by different Bt strains, and they show different insecticidal characteristics. Whether it contains insecticidal genes highly effective against target pests is the most basic condition for screening highly virulent Bt strains. However, the lack of exploited and utilized Bt strain resources, the single variety of commercial Bt preparation strains, and the easy emergence of pest resistance; when Bt is applied, the content of insecticidal proteins, spores and some active substances produced by fermentation culture is closely related to the strength of its insecticidal activity. Therefore, it is necessary to continuously isolate new Bt strains, produce new insecticidal crystal proteins and genes, develop and expand Bt resources. At the same time, it is necessary to explore the stress resistance performance, fermentation optimization culture conditions and formulation creation of Bt strains, which is crucial for optimizing highly active target biocontrol strains, increasing the diversity of toxins, overcoming potential problems related to resistance, improving the growth of strains and insecticidal activity effects, and better applying Bt strains, and has important significance in realizing efficient ecological prevention and control of pests and other pest control aspects. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a Bacillus thuringiensis L3 and its application. The Bacillus thuringiensis L3 of the present invention is isolated from dead insect bodies, and its stress resistance potential, suitable fermentation optimization culture method and the creation research of wettable powder formulation are studied. The Bacillus thuringiensis L3 of the present invention has high insecticidal activity against agricultural pests such as Plutella xylostella.

[0006] To achieve the above purpose, the technical solutions designed by the present invention are as follows:

[0007] The present invention provides a Bacillus thuringiensis L3 with a preservation number of CCTCC NO: M 2025487.

[0008] Furthermore, the gene sequence of the 16S rDNA of the Bacillus thuringiensis L3 is as shown in SEQ ID NO: 1.

[0009] The preservation unit of the above-mentioned Bacillus thuringiensis L3 is the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the postal code is 430072, the preservation number is: CCTCC NO: M 2025487, and the preservation date is: March 17, 2025.

[0010] The present invention also provides an application of the Bacillus thuringiensis L3 in any one of acid resistance, alkali resistance, bile salt resistance, high temperature resistance and drought resistance.

[0011] The present invention also provides an application of the described Bacillus thuringiensis L3 in controlling Plutella xylostella.

[0012] The present invention also provides a Bacillus thuringiensis fermentation bactericide, which contains the described Bacillus thuringiensis L3, and its concentration is 3.235×10 10 ~7.193×10 10 CFU / mL.

[0013] The present invention also provides an application of the described Bacillus thuringiensis fermentation bactericide in preparing a wettable powder of Bacillus thuringiensis L3.

[0014] The present invention also provides a wettable powder of Bacillus thuringiensis L3. The raw materials of the wettable powder of Bacillus thuringiensis L3 include mother powder, dispersant, wetting agent, stabilizer and ultraviolet protectant. Among them, the mass ratio of mother powder, dispersant, wetting agent, stabilizer and ultraviolet protectant is 1∶0.055~0.065∶0.035~0.045∶0.015~0.025∶0.015~0.025;

[0015] The raw materials of the mother powder include a carrier and the described Bacillus thuringiensis fermentation bactericide. Among them, the mass-volume ratio of the carrier and the Bacillus thuringiensis fermentation bactericide is 1∶0.75~0.80 g / mL.

[0016] Further, the carrier is any one of light calcium carbonate, talcum powder, kaolin, diatomite and activated carbon; the dispersant is any one of sodium lignosulfonate, sodium carboxymethyl cellulose and polyvinyl alcohol; the wetting agent is any one of ammonium sulfate, soluble starch and calcium chloride; the ultraviolet protectant is any one of ascorbic acid, dextrin, xanthan gum, humic acid and congo red; the stabilizer is any one of calcium carbonate, potassium dihydrogen phosphate and sodium carboxymethyl cellulose.

[0017] Still further, the raw materials of the wettable powder of Bacillus thuringiensis L3 include mother powder, sodium lignosulfonate, soluble starch, dextrin and sodium carboxymethyl cellulose. Among them, the mass ratio of mother powder, sodium lignosulfonate, soluble starch, dextrin and sodium carboxymethyl cellulose is 1∶0.06∶0.04∶0.02∶0.02;

[0018] The raw materials of the mother powder include kaolin and the described Bacillus thuringiensis fermentation bactericide. Among them, the mass-volume ratio of kaolin and the Bacillus thuringiensis fermentation bactericide is 1∶0.76 g / mL.

[0019] The present invention also provides a preparation method of the described wettable powder of Bacillus thuringiensis L3, which includes the following steps:

[0020] S1: Weigh the carrier and the Bacillus thuringiensis fermentation inoculant according to the above mass-volume ratio.

[0021] S2: Mix the carrier and the Bacillus thuringiensis fermentation inoculant evenly, pre-freeze at -80 °C, and then use a freeze dryer to sublime the moisture in it to obtain a mixed bacterial powder. After grinding it into powder, the mother powder is obtained.

[0022] S3: Weigh the mother powder, dispersant, wetting agent, stabilizer and ultraviolet protectant according to the above mass ratio.

[0023] S4: Mix the mother powder, dispersant, wetting agent, stabilizer and ultraviolet protectant evenly to obtain the wettable powder of Bacillus thuringiensis L3.

[0024] Further, in step S2, pre-freeze for 3 - 4 h, set the temperature of the freeze dryer at -60 °C, the vacuum degree at 1 Pa, and freeze-dry for 9 - 12 h.

[0025] The present invention also provides an application of the wettable powder of Bacillus thuringiensis L3 in controlling agricultural pests, and the agricultural pests are any one of Plutella xylostella, Spodoptera litura and Spodoptera exigua.

[0026] Advantages of the present invention:

[0027] 1. The Bacillus thuringiensis L3 of the present invention has high pathogenicity to major agricultural pests such as Plutella xylostella, Spodoptera exigua, Helicoverpa armigera and Spodoptera litura, and can effectively kill pests such as Plutella xylostella, Spodoptera exigua and Helicoverpa armigera.

[0028] 2. The present invention studies the insecticidal protein genes of Bacillus thuringiensis L3, and finds that it may contain a variety of insecticidal protein genes, such as Cry1Db, Cry2Aa and some new genes.

[0029] 3. Under different stress conditions, the Bacillus thuringiensis L3 strain shows certain stress resistance potential. It is found that the Bacillus thuringiensis L3 strain has certain acid tolerance, high temperature tolerance and drought tolerance, and its alkali tolerance ability is relatively strong.

[0030] 4. The present invention studies the culture medium formula suitable for the growth of Bacillus thuringiensis L3 strain and the corresponding fermentation optimization culture conditions, and a large number of bacterial cells, spores and parasporal crystals can be obtained.

[0031] 5. The present invention also creates and provides a wettable powder formula suitable for the Bacillus thuringiensis L3 strain, and after detection, it meets the national standard value.

[0032] 6. In the situation where the wild strains available for existing Bt preparations are relatively scarce, the Bacillus thuringiensis strain L3 of the present invention is another supplement to the wild resources of Bt biocontrol bacteria. In addition, there are currently few patents in the application aspects such as stress resistance performance, fermentation optimization, and formulation creation of Bacillus thuringiensis strains. The present invention can provide a reference for related research and contribute to the research and development and scientific and effective application of microbial pesticides taking Bt as an example. Description of the Drawings

[0033] Figure 1 It is the RFLP map of the primer amplification products of some insecticidal proteins of strain L3;

[0034] In the figure, A and B are the electrophoresis maps of Cry1-type genes, and C and D are the electrophoresis maps of Cry2-type genes;

[0035] Figure 2 It is the morphological observation and microscopic observation map of strain L3;

[0036] In the figure, both A and B are the colony maps grown by streak plating on the plate,

[0037] Both C and D are the single colony maps grown by spread plating on the plate,

[0038] Both E and F are the larger single colony maps with clearer morphology;

[0039] G is the microscopic observation diagram of the culture of undiluted strain L3 on the LA plate,

[0040] H is the microscopic observation diagram of the culture of strain L3 diluted with sterile water on the LA plate, and I is the microscopic observation diagram of the culture of undiluted strain L3 on the LB plate,

[0041] J is the microscopic observation diagram of the culture of strain L3 diluted with sterile water on the LB plate;

[0042] Figure 3 It is the phylogenetic tree construction diagram of strain L3;

[0043] Figure 4 It is the schematic diagram of the biocompatibility of different adjuvants and Bacillus thuringiensis L3;

[0044] In the figure, A is the schematic diagram of the biocompatibility of different carriers,

[0045] B is the schematic diagram of the biocompatibility of different dispersants,

[0046] C is the schematic diagram of the biocompatibility of different wetting agents,

[0047] D is the schematic diagram of the biocompatibility of different stabilizers,

[0048] E is the schematic diagram of the biocompatibility of different UV protectants. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0050] Example 1: Isolation, screening and identification of strain L3

[0051] 1. Isolation of strains

[0052] 1. Collect dead insect bodies from open-air cabbage fields in Chibi City, Xianning City, Hubei Province, carefully peel them off the leaves with tweezers, put them into a 1.5mL centrifuge tube filled with 1mL 75% alcohol and bring them back, and make relevant collection records (sampling number, sampling location, sampling date, etc.).

[0053] 2. In the clean bench, take out the dead insect body, rinse it in sterile water to remove the alcohol, then use tweezers to pick up the insect body and place it on toilet paper to absorb excess water; put the insect body in a glass homogenizer, add sterile water, and move the piston to fully grind; take the supernatant and dilute it 10 times with sterile water to obtain a gradient suspension; select the grinding liquid with a suitable concentration gradient, and apply 100 μL each on the LA plate, 3 plates for each gradient, and culture it at 28°C after evenly applying it, and observe it for use.

[0054] 3. The above cultured plates were placed in a clean bench to complete the separation and purification of bacterial strains. Different types of strains were separated and purified by three-zone lines on the LA plate according to the morphology, diameter, color, viscosity, etc. of the strains; the process was repeated until a single colony spot appeared, and then the strains with the characteristics of Bacillus thuringiensis strains were transferred to LB medium, and cultured on a shaker at 30°C and 200rpm for 72 hours to obtain a single strain culture solution.

[0055] 4. Streak the isolated single strains again on the LA plate and culture them in a 30℃ constant temperature box for 24 hours or more. When a large number of plaques are observed, they can be stored in a 4℃ refrigerator. When reviving, pick a single colony on the plate and transfer it to 5mL LB medium. Culture it in a shaking incubator at 200rpm and 30℃ for 8-10 hours to complete the activation.

[0056] Through the above method, a total of 236 strains were isolated in this example.

[0057] 2. Functional screening of isolated strains

[0058] 1. Preparation of fermentation stock solution and treatment solution: Pipette 1 mL of the above-activated bacterial solution and transfer it to 100 mL of LB medium. Incubate it in a shaker at 200 rpm and 30 °C for 72 h to obtain the fermentation stock solution. Mix the fermentation stock solution and 1‰ Triton X-100 aqueous solution in a volume ratio of 1:1 to obtain the treatment solution for standby.

[0059] 2. Preparation of leaves: Prepare cabbage leaves of the same size, soak them in 100 mL of the treatment solution for 2 min, air-dry them and transfer them to a petri dish or 24-well plate. Use cabbage leaves not treated with the bacterial solution as a blank control to correct the mortality of the insects.

[0060] 3. Preparation of feed: Cut artificial feed of the same size and put it into a sterilized 24-well plate. Add the above treatment solution in a laminar flow hood to allow the bacterial solution to fully contact and be absorbed by the feed, and then air-dry it. Use feed not treated with the bacterial solution as a control to correct the mortality of the insects.

[0061] 4. Activity screening: Gently dip the reared 1st instar larvae of Plutella xylostella, Spodoptera litura, Spodoptera exigua or Helicoverpa armigera with a brush and transfer them to a petri dish or 24-well plate containing cabbage leaves or feed treated with the bacterial solution. Use feed or cabbage leaves not treated with the bacterial solution as a blank control for repeated experiments. Place the petri dish or 24-well plate in a constant temperature insect rearing room at 26 °C, relative humidity of 60 - 80%, and light cycle of 12L:12D. Observe the state of the insects every day, record the number of dead larvae, and calculate the mortality and corrected mortality after 3 days to obtain strains with high insecticidal toxicity for subsequent experiments.

[0062] Corrected mortality = (mortality of treatment group - mortality of control group) / (1 - mortality of control group) × 100%

[0063] In this example, a total of 236 strains were isolated. According to the preliminary identification results, some strains were selected, and the relatively sensitive 1st instar larvae of Plutella xylostella, Spodoptera litura, Spodoptera exigua and Helicoverpa armigera reared indoors were used as target insects for indoor biological activity screening, and the Bacillus strain L3 with high insecticidal activity was screened out.

[0064] The results are shown in Table 1: Strain L3 has the strongest toxic effect on the 1st instar larvae of Plutella xylostella and Spodoptera exigua, and the corrected mortality after 3 days is 100%. It has a relatively strong toxic effect on the 1st instar larvae of Helicoverpa armigera, and the corrected mortality after 3 days is 75%. However, the toxic effect of strain L3 on the 1st instar larvae of Spodoptera litura is relatively weak.

[0065] Table 1 Insecticidal activity of strain L3 against 1st instar larvae of Lepidoptera pests

[0066]

[0067] Note: The data in the table are mean ± standard error.

[0068] III. Identification of the insecticidal protein genotype of strain L3

[0069] Extract the total DNA of strain L3, and use the insecticidal protein primers K5un2 / K3un2, K5un3 / K3un3, S5un2 / S3un2 or II(+) / II(-) to perform PCR amplification on the total DNA respectively. Four PCR products are obtained, among which two are Cry1-type gene PCR products and the other two are Cry2-type gene PCR products. Then use the restriction enzymes PstI / XbaI, EcoRI / PstI, HincII / MspI, DdeI to perform restriction enzyme identification on the four PCR products respectively. Its RFLP pattern is as shown in Figure 1 shown. By comparing with the restriction fragments of the model gene, it can be speculated that Figure 1 the Cry1-type gene in B may be Cry1Db, Figure 1 the Cry2-type gene in C may be Cry2Aa, Figure 1 the Cry1 in A and Figure 1 the Cry2-type genotype gene in D may be a new gene. The nucleotide sequences of the primers K5un2 / K3un2, K5un3 / K3un3, S5un2 / S3un2 and II(+) / II(-) are shown in Table 2 below:

[0070] Table 2 Primer sequences for identification of insecticidal protein genotype

[0071]

[0072] IV. Identification of strain L3

[0073] 1. Observation of cell morphology and culture characteristics

[0074] Spread or streak the activated strain L3 on the LA plate, and incubate it in an incubator at 28 °C for 10 - 12 h. Observe and record the characteristics of the strain community, such as Figure 2 A - Figure 2 F shown. Observe the colony morphology of strain L3 growing on the LA plate. By comparing the colonies grown from streak plating on the plate ( Figure 2 A, Figure 2 B), the single colonies grown from spread plating on the plate ( Figure 2 C, Figure 2 D) and the larger single colonies with clearer morphology grown after spread plating culture on the plate ( Figure 2 E, Figure 2 F), it is determined that the morphological characteristics of this strain are that the colonies are white, round, wax-drop-shaped, slightly raised in the center, with notches at the edges and radiating, the surface is dry and uneven, and it is an aerobic bacterium, which conforms to the typical morphological characteristics of Bacillus thuringiensis.

[0075] 2. Strain staining and microscopic observation

[0076] Pick an appropriate amount of the L3 strain colonies for staining with carbol fuchsin stain, and observe the strain morphology under a 100× oil immersion microscope; observe the microscopic observation of the L3 strain on the LA plate before and after dilution with sterile water ( Figure 2 G, Figure 2 H), and the microscopic observation of the L3 strain in the LA plate before and after dilution with sterile water ( Figure 2 I, Figure 2 J). The microscopic identification results showed the microscopic observation characteristics of Bacillus thuringiensis, and the strain was preliminarily identified as Bacillus thuringiensis.

[0077] 3. Physiological and biochemical identification

[0078] For the determination of physiological and biochemical indexes, refer to the relevant experimental methods in "Bergey's Manual of Determinative Bacteriology" (8th Edition) and "Microbiology Experiment Manual" to identify the physiological and biochemical characteristics of the L3 strain.

[0079] As can be seen from Table 3, the L3 strain is a Gram-positive bacterium, and its physiological and biochemical characteristics conform to the classification criteria of Bacillus thuringiensis in "Bergey's Manual of Determinative Bacteriology" (8th Edition) and "Manual of Common Bacterial Identification".

[0080] Table 3 Identification results of physiological and biochemical characteristics of the L3 strain

[0081]

[0082] Note: "+" indicates positive, and "-" indicates negative.

[0083] 4. Molecular biology identification

[0084] Pick a single colony of the L3 strain from the LA plate and transfer it to a 20 mL shake flask containing 5 mL of LB medium. After culturing with shaking at 30 °C and 200 rpm for 12 h, take 1 mL and transfer it to a centrifuge tube; use a bacterial genomic DNA extraction kit to extract the total DNA of the L3 strain according to the operating steps. After the extracted total DNA passes the quality inspection, use a small amount of DNA for 16S rDNA molecular identification, and send the PCR product after agarose gel electrophoresis detection to Wuhan Branch of Beijing Tsingke Biotechnology Co., Ltd. for sequencing. Use Basic BLAST on the NCBI website to perform homology comparison on the measured 16S rDNA sequence, select sequences with higher homology for comparison in MEGA 11 and construct a phylogenetic tree, and the results are as Figure 3 shown. The 16S rDNA gene sequence of the L3 strain is shown in SEQ ID NO: 1.

[0085] As can be seen from the phylogenetic tree, strain L3 belongs to the same genetic branch as Bacillus thuringiensis and has a high support degree, indicating that their genetic relationship is very close. Combining the strain activity, morphological characteristics, microscopic observation and physiological and biochemical tests, strain L3 was identified as Bacillus thuringiensis, and was classified and named as Bacillus thuringiensis L3.

[0086] 5. Slant preservation: Streak the single colony of Bacillus thuringiensis L3 on the LA plate to obtain the target strain single colony. Use an inoculation loop to pick the single colony and streak it on the sterilized LA slant. After culturing at about 30°C for 1 day, the streaked colonies grow out. Preserve the slant test tubes in a 4°C refrigerator.

[0087] 6. Glycerol tube preservation: Shake-culture Bacillus thuringiensis L3 in LB medium for 10 - 12 h to obtain the target strain. Take 1 mL of the bacterial liquid and add it to 1 mL of the sterilized 40% glycerol tube, mix well, and preserve it in an -80°C refrigerator.

[0088] At the same time, the glycerol tube of Bacillus thuringiensis L3 was preserved on March 17, 2025. The preservation unit is the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the postal code is 430072, the preservation number is: CCTCC NO: M 2025487, and the preservation date is: March 17, 2025.

[0089] Example 2: Study on the stress resistance of Bacillus thuringiensis L3

[0090] I. Preparation of overnight seed liquid / 3-day seed liquid

[0091] Pick a single colony of Bacillus thuringiensis L3 into 5 mL of LB medium, and culture it in a shaker at 200 rpm and 30°C for 8 - 10 h. Transfer 1 mL of the bacterial liquid to 100 mL of LB medium, and culture it in a shaker at 200 rpm and 30°C overnight (10 - 12 h) or for 3 days to obtain the overnight seed liquid or 3-day seed liquid.

[0092] II. Acid tolerance test

[0093] Adjust the pH value of the LB medium to 2.0, 3.0, 4.0, and 5.0 respectively with dilute hydrochloric acid. Inoculate the overnight seed liquid or 3-day seed liquid at a ratio of 2% respectively, and culture it in a shaker at 30°C and 200 rpm. Use the blank LB medium as a control. Take 0.1 mL of the sample for plate counting at 3 h and 24 h respectively, calculate the survival rate of Bacillus thuringiensis L3, and measure its OD 600 value. The results are shown in Tables 4 and 5 below, indicating that Bacillus thuringiensis L3 has certain acid tolerance.

[0094] Table 4 Results of Acid Resistance Test of Overnight Seed Solution

[0095]

[0096]

[0097] Note: The data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P<0.05 level by Duncan's new multiple range test.

[0098] Table 5 Results of Acid Resistance Test of 3d Seed Solution

[0099]

[0100] Note: The data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P<0.05 level by Duncan's new multiple range test.

[0101] III. Alkaline Resistance Test

[0102] The composition of the LB medium remained unchanged, and the pH was adjusted to 8.0, 9.0, 10.0, 11.0, and 12.0 respectively. The overnight seed solution or 3d seed solution was inoculated into the LB medium with different pH values at an inoculation amount of 2% respectively, and cultured at 30 °C and 200 rpm. The blank LB medium was used as a control. 0.1 mL of the sample was taken at 3 h and 24 h respectively for plate counting, and the survival rate of Bacillus thuringiensis L3 was calculated and its OD 600 value was measured. The results are shown in Tables 6 and 7, indicating that Bacillus thuringiensis L3 has excellent alkaline resistance.

[0103] Table 6 Results of Alkaline Resistance Test of Overnight Seed Solution

[0104]

[0105]

[0106] Note: The data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P<0.05 level by Duncan's new multiple range test.

[0107] Table 7 Results of Alkaline Resistance Test of 3d Seed Solution

[0108]

[0109] Note: The data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P<0.05 level by Duncan's new multiple range test.

[0110] IV. High Temperature Resistance Test

[0111] Prepare overnight seed solution or 3-day seed solution. Take 3 mL of overnight seed solution or 3-day seed solution and place them in a water bath at 70 °C, 80 °C, and 90 °C respectively. Samples are taken at the time points of 3 min, 10 min, and 20 min for plate counting. The bacterial solution without high-temperature treatment is used as a control to calculate the survival rate of Bacillus thuringiensis L3. The results are shown in Tables 8, 9, and 10, indicating that Bacillus thuringiensis L3 has certain high-temperature tolerance.

[0112] Table 8 Results of the high-temperature tolerance test of Bacillus thuringiensis L3 at 70 °C

[0113]

[0114] Note: The data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P < 0.05 level by Duncan's new multiple range test.

[0115] Table 9 Results of the high-temperature tolerance test of Bacillus thuringiensis L3 at 80 °C

[0116]

[0117] Note: The data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P < 0.05 level by Duncan's new multiple range test.

[0118] Table 10 Results of the high-temperature tolerance test of Bacillus thuringiensis L3 at 90 °C

[0119]

[0120] Note: The data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P < 0.05 level by Duncan's new multiple range test.

[0121] V. Drought tolerance test

[0122] 1. Prepare aqueous solutions of PEG-6000 with different concentrations (0%, 10%, 20%, 30%, and 40%), filter and sterilize them for later use;

[0123] 2. Add aqueous solutions of PEG-6000 with different concentrations to LB medium at a ratio of 1:1 to artificially simulate mild, moderate, severe, and extremely severe drought conditions (5%, 10%, 15%, and 20% PEG-6000, corresponding to osmotic potential levels of -0.3, -0.8, -1.6, and -2.4 mPa respectively);

[0124] 3. Inoculate the pre-prepared bacterial suspension (overnight seed solution or 3-day seed solution) stored at 4°C in the refrigerator into the above-mentioned culture media at an inoculation amount of 2% respectively. Using pure LB medium as a control, incubate at 30°C and 200 r·min -1 Samples are taken respectively when shaking culture for 3 h and 24 h, and OD 600 value is measured. Using the magnitude of OD 600 value (turbidity) and the decline rate (the ratio of adjacent osmotic potential OD 600 ), evaluate its growth and reproduction status, and conduct colony counting to calculate the survival rate of the strain. Thus, analyze and obtain the drought tolerance of Bacillus thuringiensis L3. Among them, the decline rate (-A / -B) = [-A average OD value - (-B average OD value)] / -B average OD value, where A and B are two adjacent osmotic levels respectively. The greater the decline rate, the smaller the bacterial growth potential. The results are shown in Table 11 and Table 12, indicating that Bacillus thuringiensis L3 has a certain drought tolerance.

[0125] Table 11 Results of the 3-hour drought tolerance test of Bacillus thuringiensis L3

[0126]

[0127] Note: The data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P < 0.05 level tested by Duncan's new multiple range method.

[0128] Table 12 Results of the 24-hour drought tolerance test of Bacillus thuringiensis L3

[0129]

[0130]

[0131] Note: The data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P < 0.05 level tested by Duncan's new multiple range method.

[0132] VI. Bile salt tolerance test

[0133] 1. Weigh porcine bile salt and prepare an aqueous solution of 0.1 g / mL, and filter and sterilize it;

[0134] 2. Add the porcine bile salt aqueous solution to the LB medium that has been sterilized and cooled to about 50°C to prepare LB media containing 0.1%, 0.3%, and 0.5% porcine bile salt respectively;

[0135] 3. Inoculate the pre-prepared bacterial suspension (overnight seed solution or 3-day seed solution) stored at 4°C in the refrigerator into the above-mentioned LB media containing porcine bile salt at an inoculation amount of 2% respectively. Using pure LB medium as a control, incubate at 30°C and 200 r·min -1Samples were taken at 3 h and 24 h of shaker culture to measure the OD 600 value and perform colony counting, and calculate the survival rate of the strain. From this, the bile salt tolerance of Bacillus thuringiensis L3 was analyzed. The results are shown in Tables 13 and 14, indicating that Bacillus thuringiensis L3 has poor bile salt tolerance.

[0136] Table 13 Results of the 3-h bile salt tolerance test of Bacillus thuringiensis L3

[0137]

[0138]

[0139] Note: The data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P<0.05 level by Duncan's new multiple range test.

[0140] Table 14 Results of the 24-h bile salt tolerance test of Bacillus thuringiensis L3

[0141]

[0142] Note: The data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P<0.05 level by Duncan's new multiple range test.

[0143] Example 3: Field efficacy test study of Bacillus thuringiensis L3

[0144] Taking Plutella xylostella as the control object, a field efficacy test of Bacillus thuringiensis L3 (4.265×10 8 CFU / mL) was carried out in a cabbage field, and the average control effect of the field efficacy was compared with several other pesticide insecticides. The results are shown in Table 15, and it was found that Bacillus thuringiensis L3 could exert a good field control effect.

[0145] Table 15 Field test results of several agents against Plutella xylostella

[0146]

[0147]

[0148] Example 4: Research on the creation of wettable powder of Bacillus thuringiensis L3

[0149] I. Preparation of fermentation inoculum

[0150] 1. Prepare an optimized liquid fermentation medium, and its composition is carbon source (yeast powder)∶nitrogen source (tryptone)∶inorganic salt (magnesium sulfate) = 0.25∶1∶1.5.

[0151] 2. Inoculate Bacillus thuringiensis strain L3 onto a 250 mL Erlenmeyer flask containing 100 mL of LB medium, and culture it on a shaker at 30 °C and 200 rpm for 12 h to obtain the seed liquid. Inoculate the seed liquid into a 500 mL Erlenmeyer flask containing 70 mL of optimized fermentation liquid medium at an inoculation amount of 3% by volume, and culture it on a shaker at 36 °C and 250 rpm for 72 h to obtain the Bacillus thuringiensis fermentation inoculant (the bacterial content is 7.193×10 10 CFU / mL). The fermentation inoculant must have a high bacterial content, the spore rate should be ≥90%, and a large number of parasporal crystals should be produced.

[0152] II. Biocompatibility experiment

[0153] Add the carrier, dispersant, wetting agent, stabilizer, or ultraviolet protectant into the LA medium separately at a ratio of 5% by mass, 1.5 mg / mL, 0.25 mg / mL, 1.0 mg / mL, or 50 μg / mL, respectively. After autoclaving, make it into a plate. Pipette 4 mL of the Bacillus thuringiensis fermentation inoculant (the bacterial content is 7.193×10 10 CFU / mL) cultured in the optimized fermentation liquid medium and dilute it 10 7 times. Take 0.1 mL of the diluted solution and spread it on the LA plates containing different carriers, dispersants, wetting agents, stabilizers, or ultraviolet protectants. Use the LA plate as a control, with 3 replicates for each group. Place the plates in an incubator at 37 °C and incubate them statically for 12 h, record the number of colonies on each plate, and preliminarily screen out the dominant additives.

[0154] The results are as Figure 4 shown. Taking the viable bacteria content as the detection index, compared with the blank control group, corresponding different additives can be selected for the subsequent research on the creation of wettable powders.

[0155] III. Screening of carriers

[0156] Accurately weigh 5 g of the carrier into a beaker, quantitatively add the Bacillus thuringiensis fermentation inoculant into the beaker, and continuously stir to make the carrier fully adsorb. When the powder shows a state of just sticking to the cup wall, not flowing, and having a suitable viscosity, stop adding the Bacillus thuringiensis fermentation inoculant, record the average adsorption amount, and dry it. Compare the material costs of each carrier. Measure the wetting time and suspension rate.

[0157] As can be seen from Table 16, light calcium carbonate has good wetting performance and suspension rate and the lowest cost, but its adsorption ability is poor; talc powder has a relatively low cost, but its adsorption ability and wettability are the worst and the suspension is not good; kaolin has good adsorption ability and wettability, the best suspension, and a moderate cost: diatomite has strong adsorption ability and the best wetting performance, but the suspension is poor and the cost is relatively high; activated carbon has strong adsorption ability and suspension, but weak wettability and the highest cost.

[0158] Combined with the biocompatibility of each carrier, light calcium carbonate, kaolin and diatomite were initially selected as the carriers for the preparation of wettable powders.

[0159] Table 16 Influence of carriers on the preparation

[0160]

[0161] Note: The data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P<0.05 level tested by Duncan's new multiple range method.

[0162] IV. Preparation of mother powder of wettable powder

[0163] Weigh 30 g of the carrier, add Bacillus thuringiensis fermentation inoculant according to the adsorption capacity of the carrier in Table 16 and stir until the powder shows a state where it can form a mass but does not flow and has a moderate viscosity. Then pre-freeze the item in a -80 °C low-temperature refrigerator. After 3 h, use a freeze dryer to sublime the moisture in the sample. For freeze drying, set the temperature of the freeze dryer at -60 °C and the vacuum degree at 1 Pa, and freeze dry for 9 - 12 h to obtain the bacterial powder mixed with the carrier. Grind the dried powder into powder to prepare the mother powder of the wettable powder for standby, which is used to screen wetting agents and dispersants.

[0164] After the above steps, the mother powders prepared using different carriers (light calcium carbonate, kaolin and diatomite) were obtained.

[0165] V. Screening of the types of the best wetting agent and dispersant

[0166] On the basis of determining the carrier and preparing the mother powder, take 1 g of the mother powder and add different wetting agents or dispersants at a ratio of 10% of the mother powder mass (the addition amount is 0.1 g), fully pulverize and mix evenly, and measure and compare their wettability and suspension rate, so as to select the best wetting agent or dispersant.

[0167] The results are shown in Table 17 and Table 18. Considering comprehensively factors such as biocompatibility, adsorption capacity, wetting time, suspension rate, cost, etc., and combining the experimental results, kaolin was selected as the carrier, soluble starch as the wetting agent, and sodium lignosulfonate as the dispersant for subsequent experiments.

[0168] Table 17 Influence of wetting agents on the preparation

[0169]

[0170] Note: The data in the two columns of wetting time and suspension rate are mean ± standard error, and different letters after the data indicate significant differences at the P<0.05 level tested by Duncan's new multiple range method.

[0171] Table 18 Influence of Dispersant on the Preparation

[0172]

[0173] Note: The data in the columns of wetting time and suspension rate are mean ± standard error. Different letters after the data indicate significant differences at the P < 0.05 level tested by Duncan's new multiple range method.

[0174] VI. Screening of the Ratio of Wetting Agent and Dispersant

[0175] On the basis of determining kaolin as the carrier to prepare the mother powder, 1 g of the mother powder was taken, and different ratios of soluble starch and sodium lignosulfonate were added (the total amount of soluble starch and sodium lignosulfonate was 10% of the mass of the mother powder, i.e., 0.1 g). After fully pulverizing and mixing evenly, their wettability and suspension rate were measured and compared, and thus the optimal ratio of the wetting agent and the dispersant was selected. As can be seen from Table 19, the wetting time of each test group was relatively short, meeting the national standard. At this time, by comparing the suspension rate, the ratio with the highest suspension rate was selected as the optimal ratio. Therefore, the optimal ratio of the wetting agent soluble starch and the dispersant sodium lignosulfonate was 4:6.

[0176] Table 19 Influence of the Ratio of Wetting Agent and Dispersant on the Properties of the Preparation

[0177]

[0178] Note: The data in the columns of wetting time and suspension rate are mean ± standard error. Different letters after the data indicate significant differences at the P < 0.05 level tested by Duncan's new multiple range method.

[0179] VII. Screening of the Dosage of Wetting Agent and Dispersant

[0180] On the basis of determining kaolin as the carrier, preparing the mother powder and determining the optimal ratio of the wetting agent and the dispersant, 1 g of the mother powder was taken, and soluble starch and sodium lignosulfonate were added according to different total dosages (the ratio of soluble starch and sodium lignosulfonate was 4:6). After fully pulverizing and mixing evenly, their wettability and suspension rate were measured and compared. As can be seen from Table 20, when the total dosage of the wetting agent and the dispersant was 10% of the mass of the mother powder, the wetting time of the experimental group was the shortest and the suspension rate was the highest. Therefore, the optimal total dosage of the wetting agent soluble starch and the dispersant sodium lignosulfonate was 10% of the mass of the mother powder.

[0181] Table 20 Influence of the Total Dosage of Wetting Agent and Dispersant on the Properties of the Preparation

[0182]

[0183]

[0184] Note: The data in the two columns of wetting time and suspension rate are mean ± standard error. Different letters after the data indicate significant differences at the P<0.05 level by Duncan's new multiple range test.

[0185] VIII. Screening of UV protectants

[0186] Take 1 g of the mother powder prepared with kaolin as the carrier. Add the wetting agent soluble starch and the dispersant sodium lignosulfonate (with a ratio of 4:6) to the mother powder prepared with kaolin at a total dosage of 10% of the mother powder mass, and stir and grind evenly in a mortar. Then add the UV protectant to the prepared sample at an addition amount of 2% of the mother powder mass to make a wettable powder (the addition amount of soluble starch is 0.04 g, the addition amount of sodium lignosulfonate is 0.06 g, and the addition amount of the UV protectant is 0.02 g). Mix the wettable powder and sterile distilled water evenly at a ratio of 1:9 g / mL, and place it 40 cm away from the UV lamp (254 nm, 20 W) for irradiation for 0, 12, and 24 h. After irradiation, dilute it step by step with sterile distilled water, take 0.1 mL and coat it on the LB plate, and statically culture it in an incubator at 37°C for 12 h, and calculate the spore content and survival rate. Use the sample without the protectant as the control, and calculate the average value of the data results. Each treatment is repeated 3 times. As can be seen from Table 21, dextrin is the optimal UV protectant.

[0187] Table 21 Effects of UV protectants on the wettable powder L3 of L3

[0188]

[0189] Note: The data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P<0.05 level by Duncan's new multiple range test.

[0190] IX. Screening of stabilizers

[0191] 1. Weigh 1 g of the mother powder prepared with kaolin as the carrier. Add soluble starch and sodium lignosulfonate (with a ratio of 4:6) to the mother powder prepared with kaolin at a total dosage of 10% of the mother powder mass, and stir and grind evenly in a mortar. Then add dextrin to the prepared sample at an addition amount of 2% of the mother powder mass to make a wettable powder, which is the L3 preparation (the addition amount of soluble starch is 0.04 g, the addition amount of sodium lignosulfonate is 0.06 g, and the addition amount of dextrin is 0.02 g).

[0192] 2. Mix three stabilizers, namely calcium carbonate, dipotassium hydrogen phosphate, or sodium carboxymethyl cellulose, with the L3 preparation at an addition amount of 2% of the mother powder mass (the addition amount of the stabilizer is 0.02 g) respectively and mix them evenly. Dry and process them into wettable powders. Use the one without stabilizer as the control. Heat store them at 54 ± 2 °C and cold store them at 4 ± 2 °C for 14 d respectively. Measure the spore content, spore decomposition rate, suspension rate after storage, and wetting time before and after storage. As can be seen from Table 22 and Table 23, the best stabilizer screened out is sodium carboxymethyl cellulose.

[0193] Table 22 Influence of Stabilizers on L3 Preparation under Heat Storage Conditions

[0194]

[0195] Note: The data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P < 0.05 level tested by Duncan's new multiple range method.

[0196] Table 23 Influence of Stabilizers on L3 Preparation under Cold Storage Conditions

[0197]

[0198]

[0199] Note: The data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P < 0.05 level tested by Duncan's new multiple range method.

[0200] X. Determination of Optimal Contents of UV Protectants and Stabilizers

[0201] Weigh 1 g of the mother powder prepared with kaolin as the carrier. Add soluble starch and sodium lignosulfonate (with a ratio of 4:6) to the mother powder prepared with kaolin at a total usage amount of 10% of the mother powder mass. On this basis, add different content combinations of dextrin and sodium carboxymethyl cellulose. Use the wetting time and suspension rate as evaluation indicators, and screen out the optimal contents of the UV protectant and stabilizer through an orthogonal experiment (Table 24). As can be seen from Table 25, considering the wetting time and suspension rate comprehensively, the optimized ratio contents of the UV protectant and stabilizer are 2.0% of the mother powder mass and 2.0% of the mother powder mass respectively. At this time, the wetting time is relatively short at 30.38 s, and the suspension rate is relatively high at 81.08%.

[0202] Table 24 Orthogonal Experiment on Optimal Optimization Ratio of UV Protectant and Stabilizer

[0203]

[0204] Table 25 Results of Orthogonal Experiment on UV Protectant and Stabilizer

[0205]

[0206]

[0207] Note: k1, k2, k3 and k1’, k2’, k3’ respectively correspond to the mean values of the wetting time and suspension rate at levels A or B1-3. The ranges R1 and R2 respectively correspond to the ranges of the mean values of the wetting time and suspension rate at levels A or B1-3. The data in the two columns of wetting time and suspension rate are mean ± standard error. Different letters indicate the significant difference levels among the data in the same column after being tested by Duncan's new multiple range method.

[0208] Example 4: Preparation and determination of Bacillus thuringiensis L3 wettable powder

[0209] The raw materials of Bacillus thuringiensis L3 wettable powder include mother powder, sodium lignosulfonate, soluble starch, dextrin and sodium carboxymethylcellulose. Among them, the mass ratio of mother powder, sodium lignosulfonate, soluble starch, dextrin and sodium carboxymethylcellulose is 1:0.06:0.04:0.02:0.02; and the raw materials of the mother powder include kaolin and Bacillus thuringiensis fermentation inoculum, and the mass-volume ratio of kaolin and Bacillus thuringiensis fermentation inoculum is 1:0.76 g / mL.

[0210] The preparation method of the above-mentioned Bacillus thuringiensis L3 wettable powder is as follows:

[0211] 1. Weigh 30 g of kaolin as the carrier, add Bacillus thuringiensis fermentation inoculum according to the adsorption capacity of the carrier and stir. The adsorption capacity of kaolin is 0.76 L / kg, that is, the addition amount of Bacillus thuringiensis fermentation inoculum in 1 g of kaolin is 0.76 mL. When the powder shows a state where it can form a mass but does not flow and has a moderate viscosity, pre-freeze the item in a -80°C low-temperature refrigerator. After 3 h, use a freeze dryer to sublime the water in the sample, freeze-dry. The temperature of the freeze dryer is set at -60°C, the vacuum degree is 1 Pa, and freeze-dry for 9-12 h to obtain the bacterial powder mixed with the carrier. Grind the dried powder into powder to prepare the mother powder of the wettable powder for standby.

[0212] 2. Weigh the mother powder prepared with kaolin as the carrier, soluble starch, sodium lignosulfonate, dextrin and sodium carboxymethylcellulose, and mix them evenly to obtain Bacillus thuringiensis L3 wettable powder. Among them, the mass ratio of the mother powder prepared with kaolin as the carrier, soluble starch, sodium lignosulfonate, dextrin and sodium carboxymethylcellulose is 1:0.04:0.06:0.02:0.02.

[0213] 3. According to the detection methods specified in national standards GB / T 20287—2006, GB / T 20287—2006, GB / T 20287—2006, GB / T 30361-2013, GB / T 5451-2001, GB / T 16150-1995, GB 20287-2006, GB / T 5451-2001, GB / T 14825-2006, the effective viable count of Bacillus thuringiensis L3 wettable powder was determined to be 3.2×10 10 CFU / g, the number of mold and miscellaneous bacteria was 0 / g, the miscellaneous bacteria rate was 0.31%, the dry weight loss was 0.3%, the water content was 0.84%, the fineness was 0.31%, the pH value was 7.95, the wetting time was 34.99 s, and the suspension rate was 77.41%, all of which met the national standard values (see Table 26).

[0214] Table 26 Results of the quality determination of the wettable powder finished product

[0215]

[0216] Note: The data in the measured value column are the mean ± standard error.

[0217] Other parts not described in detail are all prior art. Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus thuringiensis L3, with the preservation number of CCTCC NO: M 2025487.

2. Use of the Bacillus thuringiensis L3 described in claim 1 in any one of acid resistance, alkali resistance, bile salt resistance, high temperature resistance and drought resistance.

3. Use of the Bacillus thuringiensis L3 described in claim 1 in controlling Plutella xylostella.

4. A Bacillus thuringiensis fermentation inoculant, characterized in that: The fermentation bacterium agent contains Bacillus thuringiensis L3 described in claim 1, and its concentration is 3.235×10 10 ~7.193×10 10 CFU / mL.

5. Use of the Bacillus thuringiensis fermentation inoculant described in claim 4 in preparing the wettable powder of Bacillus thuringiensis L3.

6. A wettable powder of Bacillus thuringiensis L3, characterized in that: The raw materials of the wettable powder of Bacillus thuringiensis L3 include mother powder, dispersant, wetting agent, stabilizer and ultraviolet protector. Among them, the mass ratio of mother powder, dispersant, wetting agent, stabilizer and ultraviolet protector is 1∶0.055 - 0.065∶0.035 - 0.045∶0.015 - 0.025∶0.015 - 0.025; The raw materials of the mother powder include carrier and the Bacillus thuringiensis fermentation inoculant described in claim 4. Among them, the mass - volume ratio of carrier and Bacillus thuringiensis fermentation inoculant is 1∶0.75 - 0.80 g / mL.

7. The Bacillus thuringiensis L3 wettable powder according to claim 6, characterized in that: The carrier is any one of light calcium carbonate, talcum powder, kaolin, diatomaceous earth and activated carbon; the dispersant is any one of sodium lignosulfonate, sodium carboxymethyl cellulose and polyvinyl alcohol; the wetting agent is any one of ammonium sulfate, soluble starch and calcium chloride; the ultraviolet protector is any one of ascorbic acid, dextrin, xanthan gum, humic acid and congo red; the stabilizer is any one of calcium carbonate, potassium dihydrogen phosphate and sodium carboxymethyl cellulose.

8. The Bacillus thuringiensis L3 wettable powder according to claim 7, wherein: The raw materials of the wettable powder of Bacillus thuringiensis L3 include mother powder, sodium lignosulfonate, soluble starch, dextrin and sodium carboxymethyl cellulose. Among them, the mass ratio of mother powder, sodium lignosulfonate, soluble starch, dextrin and sodium carboxymethyl cellulose is 1∶0.06∶0.04∶0.02∶0.02; The raw materials of the mother powder include kaolin and Bacillus thuringiensis fermentation inoculant. Among them, the mass - volume ratio of kaolin and Bacillus thuringiensis fermentation inoculant is 1∶0.76 g / mL.

9. A preparation method of the Bacillus thuringiensis L3 wettable powder according to any one of claims 6 to 8, characterized in that: Including the following steps: S1: Weigh the carrier and the Bacillus thuringiensis fermentation inoculant described in claim 4 according to the above mass - volume ratio; S2: Mix the carrier and the Bacillus thuringiensis fermentation inoculant, pre - freeze at - 80°C, and then use a freeze - dryer to sublime the water therein to obtain a mixed bacterial powder, which is ground into powder to obtain the mother powder; S3: Weigh the mother powder, dispersant, wetting agent, stabilizer and ultraviolet protector according to the above mass ratio; S4: Mix the mother powder, dispersant, wetting agent, stabilizer and ultraviolet protector evenly to obtain the wettable powder of Bacillus thuringiensis L3.

10. Use of the Bacillus thuringiensis L3 wettable powder according to any one of claims 6 to 8 in controlling agricultural pests, characterized in that: The agricultural pest is any one of Plutella xylostella, Spodoptera litura and Spodoptera exigua.

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