Bacillus thuringiensis and application thereof in control of plutella xylostella

Abstract

According to the bacillus thuringiensis and the application thereof in preventing and treating the plutella xylostella, the bacillus thuringiensis with specific activity on the plutella xylostella is screened, the types of insecticidal proteins of the bacillus thuringiensis are identified, and a new microbial resource is provided for biological prevention and treatment of the plutella xylostella. The preservation number of the provided bacillus thuringiensis B-T-3 strain is CCTCC (China Center For Type Culture Collection) No: M20252325. The high-toxicity bacillus thuringiensis obtained by screening has remarkable and excellent insecticidal activity on 2-instar and 3-instar larvae of plutella xylostella. Under the same treatment concentration, the corrected death rate is obviously higher than that of other isolated strains.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for agricultural pests, specifically relating to a Bacillus thuringiensis that is highly toxic to diamondback moth. Background Technology

[0002] Diamondback moth ( Plutella xylostella Diamondback moth (L.) is a major global pest affecting cruciferous vegetables, characterized by high reproductive capacity, short generation cycles, and overlapping generations, causing severe losses to agricultural production. For a long time, chemical pesticides have been the primary means of controlling the diamondback moth. However, the excessive and irrational use of chemical pesticides has led to environmental pollution and pesticide residues, seriously impacting human health. Simultaneously, the diamondback moth has rapidly developed moderate to high levels of resistance to various chemical pesticides, exhibiting a broad resistance spectrum, high resistance levels, and rapid resistance development. This results in a continuous decline or even complete ineffectiveness of conventional chemical control methods, becoming a key bottleneck restricting effective control of the diamondback moth. Existing chemical control technologies are insufficient to meet actual production needs; therefore, the search for and application of efficient and green control technologies are receiving increasing attention.

[0003] Bacillus thuringiensis (Bt) is a Gram-positive bacterium that produces insecticidal proteins. Abundant in nature, it is one of the most widely used microbial insecticides in the world. Bt is widely used in green control systems for the diamondback moth, but the diamondback moth was also one of the first agricultural pests to be found to have developed resistance to Bt. This resistance development has led to problems such as unsatisfactory control effects when using Bt. Furthermore, different Bt strains carry different types and numbers of insecticidal protein genes, resulting in variations in insecticidal activity. Therefore, screening for novel Bt strains with specific insecticidal activity against the diamondback moth and identifying their insecticidal protein genes can continuously enrich Bt strains and insecticidal protein gene resources, providing an effective approach for efficient and green control of the diamondback moth. Summary of the Invention

[0004] This invention provides a Bacillus thuringiensis and its application in the control of diamondback moth. It screens Bacillus thuringiensis (Bt strain) with specific activity against diamondback moth and identifies the types of its insecticidal proteins, providing a new microbial resource for the biological control of diamondback moth.

[0005] The Bacillus thuringiensis BT-3 strain provided by this invention ( Bacillus thuringiensis BT-3 was deposited on October 24, 2025, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC No: M20252325.

[0006] The strain provided by the present application shows high insecticidal activity on Plutella xylostella larvae, and the lethal concentration (LC 50 ) of the strain on Plutella xylostella larvae is 7.92×10 6 cfu / mL.

[0007] The vegetative cells of the provided strain are rod-shaped, single or in chains, the spores are oval, and the parasporal crystals are rhombic.

[0008] The insecticidal crystal proteins of the provided strain are mainly 130 kDa and 70 kDa proteins.

[0009] The insecticidal protein genes contained in the strain include Cry1Aa (the protein sequence is SEQ ID NO: 1), Cry1Ab, Cry1Ac (the protein sequence is SEQ ID NO: 2), Cry1Ia (the protein sequence is SEQ ID NO: 3), Cry2Aa, Cry2Ab (the protein sequence is SEQ ID NO: 4), Mpp46Ab (the protein sequence is SEQ ID NO: 5), Vip3Aa (the protein sequence is SEQ ID NO: 6), Vpb4Ca (the protein sequence is SEQ ID NO: 7), and Spp1Aa (the protein sequence is SEQ ID NO: 8).

[0010] The present application also provides a use of the Bacillus thuringiensis B-T-3 strain, which is used for preventing and treating Plutella xylostella, or used for preparing a product for preventing and treating Plutella xylostella.

[0011] Another aspect of the present application also provides a product for preventing and treating Plutella xylostella, which contains the Bacillus thuringiensis B-T-3 strain.

[0012] The high-toxicity Bacillus thuringiensis (Bt) strain B-T-3 obtained by screening in the present application shows significantly excellent insecticidal activity on 2nd instar and 3rd instar larvae of Plutella xylostella. Under the same treatment concentration, the corrected mortality of B-T-3 is significantly higher than that of other isolated strains. Further toxicity determination shows that the LC 50 of B-T-3 is 7.92×10 6 cfu / mL, which is about 1.78 times higher than that of the commercial control strain, and the high efficiency prevention and treatment can be realized at a lower dosage, which is beneficial to reduce the frequency of pesticide application and the prevention and treatment cost.

[0013] This strain carries multiple insecticidal protein genes, including Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ia, Cry2Aa, Cry2Ab, and Vip3Aa, forming a multi-toxin synergistic mechanism. It possesses broad-spectrum and highly effective insecticidal properties, helping to delay the development of pest resistance. The strain is morphologically stable and grows rapidly, making it suitable for large-scale fermentation production. As a source of biopesticides, BT-3 is safe for non-target organisms, environmentally friendly, and meets the requirements of green pest control, showing promising application and promotion prospects. Attached Figure Description

[0014] Figure 1 Graph showing the toxicity of strain BT-3 and the control strain against diamondback moth larvae. Figure 2 Vegetative morphology diagram of strain BT-3. Figure 3 Crystal morphology of spores and paraspores of strain BT-3. Figure 4 Growth curve of strain BT-3 Figure 5 Electrophoresis diagram of insecticidal crystal proteins from strain BT-3. Detailed Implementation

[0015] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0016] Example 1: Screening for strains highly toxic to diamondback moth For the 14 separated ( Bacillus thuringiensis , Preliminary screening of the insecticidal activity of Bt strains was conducted. The Bt strains were activated and inoculated onto LB solid medium using the three-zone streak method. After inoculation, the plates were incubated at 28°C. Single colonies were picked using an inoculation loop and transferred to LB liquid medium, then incubated at 28°C and 200 rpm for 72 h in a shaking incubator until spores and paraspore crystals were produced. The bacterial suspension was collected, centrifuged at 8000 rpm for 15 min, the supernatant was discarded, and the cells were resuspended in 10 mL of sterile water. After counting with a hemocytometer, the concentration of all Bt strains was adjusted to 1×10⁻⁶. 8cfu / mL. Well-grown 3rd instar Plutella xylostella larvae were selected as target insects for activity determination. The insecticidal activity determination method used a leaf dipping method. Cabbage leaves were washed with water and dried, fresh and uniform cabbage leaves were selected and cut into similar pieces, dipped in the Bt sample, dried, and placed in a bioassay cup. It was placed in a 28°C incubator, maintained at 70% relative humidity, and the photoperiod was set to 12 hours light: 12 hours dark (12L:12D). The number of dead insects was observed and recorded at 24, 48, 72, and 96 h, and the corrected mortality rate was calculated. The preliminary screening results of insecticidal activity showed that among the 14 Bt strains, B-T-3 had the highest insecticidal activity against 3rd instar Plutella xylostella larvae at a bacterial liquid concentration of 1×10 8 cfu / mL, with a corrected mortality rate of 62.2%.

[0017] Using Wuhan Konno Biological Technology Co., Ltd.'s Invincible Boy (Bacillus thuringiensis) as a control, strains B-T-3 and the control strain were diluted to 4.0×10 8 cfu / mL, 1.0×10 8 cfu / mL, 2.5×10 7 cfu / mL, 6.25×10 6 cfu / mL, 1.56×10 6 cfu / mL, and 3.9×10 5 cfu / mL, respectively, and bioassay was performed according to the above method. Well-grown 2nd instar Plutella xylostella larvae were selected, 15 larvae were treated at each concentration, each treatment was repeated 3 times, and distilled water was used as a blank control. It was placed in a 28°C incubator, maintained at 70% relative humidity, and the photoperiod was set to 12 hours light: 12 hours dark (12L:12D). The number of dead insects was observed and recorded at 24, 48, 72, and 96 h, and the lethal concentration (LC 50 ) was calculated. The bioactivity determination results ( Figure 1 ) showed that the LC 50 of strain B-T-3 against Plutella xylostella larvae was 7.92×10 6 cfu / mL, and the LC 50 of the control strain against Plutella xylostella larvae was 1.41×10 7 cfu / mL, indicating that strain B-T-3 had high insecticidal activity against Plutella xylostella larvae, 1.78 times that of the control strain.

[0018] Example 2: Morphology of Bt strain vegetative cells, spores, and parasporal crystals After activation, the strain B-T-3 was inoculated on LB plate medium and cultured in a 28 ℃ incubator. At different culture times, the bacterial cells were fixed on clean glass slides, and carbol fuchsin dye was added dropwise for 40 sec-1 min. Then the dye was washed with water and observed under a microscope to observe the morphology of the bacterial cells and crystals. At the same time, the bacterial samples at different stages were transferred to 1.5 mL centrifuge tubes, and 2.5% glutaraldehyde was added for fixation. After fixation, the samples were washed 2-3 times with PBS buffer. Then the samples were dehydrated with different concentrations (50%, 70%, 90%, and 100%) of ethanol, each for 15-20 min, to gradually remove the water from the samples. The dehydrated samples were added dropwise to small pieces of glass slides and dried in a freeze dryer for 3-5 h. The dried samples were fixed on a metal sample stage with conductive tape, and a 5-10 nm thick metal film was deposited on the sample surface by sputter coating. The samples were observed and imaged under a scanning electron microscope.

[0019] By observation, the vegetative cells of strain B-T-3 were rod-shaped with blunt ends, Figure 2 about 3.5 x 1.2 μm in size, and existed singly or in chains of two or more. The spores were oval-shaped, about 1.7 x 0.8 μm in size, and the parasporal crystals were rhombic, about 1.8 x 0.8 μm in size. Figure 3

[0020] Example 3: Determination of the growth curve of the Bt strain A single colony was picked from the streaked plate and inoculated into 5 mL of LB liquid medium, which was incubated overnight at 28 ℃ and 200 rpm. The culture was inoculated into 50 mL of LB liquid medium at a 1% inoculation amount, and 100 μL of the culture was taken every 2 h and added to a 96-well plate. The absorbance of the taken culture at OD 600 was measured using a microplate reader, with un-inoculated LB liquid medium as a blank control. The OD 600 values were calculated and a growth curve graph was plotted.

[0021] The growth of strain B-T-3 in LB liquid medium is shown in Figure 4 At 0-6 h, the strain grew slowly and did not proliferate significantly. At 6-18 h, the strain entered the logarithmic growth phase and began to grow rapidly, with a rapid increase in OD 600 value. At 18-24 h, the strain entered the stationary growth phase, and as the nutrients were consumed, the growth of the strain gradually stabilized, with the OD 600 value tending to be stable. After 24 h, the strain entered the decline phase, and the OD 600 value began to decrease slowly.

[0022] ​Example 4: Insecticidal crystal proteins of Bt strain and identification of their genes Strain B-T-3 was inoculated on LB plate, about 100 mg of the colony was scraped after spores and crystals were separated, suspended in 100 μL of sterile water, 25 μL of 0.5 mol / L NaOH solution was added, reacted at room temperature for 5 min, 125 μL of 2x sample buffer was added, boiled for 10 min, centrifuged at 10000 r / min for 5 min, and the supernatant was taken for SDS-PAGE. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R250, and the electrophoretogram was analyzed by BandScan software. The results are as follows Figure 5 The molecular weight of the insecticidal crystal proteins of strain B-T-3 is mainly 130 kDa and 70 kDa.

[0023] A single colony of strain B-T-3 was transferred to LB liquid medium with a inoculating loop, and was placed in a shaking incubator at 28°C and 200 r / min until the OD 600 =1.0. The bacterial liquid was centrifuged at 8000 r / min and 4°C for 10 min, the supernatant was discarded, and the bacterial body was collected. The bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit, and the quality and concentration of the bacterial genomic DNA were evaluated by 1% agarose gel electrophoresis and Qubit® 2.0 system. Libraries for Oxford Nanopore Technologies (ONT) and Illumina sequencing were constructed, and whole genome sequencing was performed using Nanopore PromethION and Illumina HiSeq platforms. Nanopore and Illumina data were assembled and merged using Unicycler v0.4.9 software.

[0024] The results of genome sequencing showed that the genome size of strain B-T-3 was 5,675,381 bp, and the GC content was 35.29%. Strain B-T-3 contained 11 plasmids, including B-T-3 P1 (411,714 bp), B-T-3 P2 (317,339 bp), B-T-3 P3 (82,531 bp), B-T-3 P4 (80,699 bp), B-T-3 P5 (69,355 bp), B-T-3 P6 (46,634 bp), B-T-3 P7 (14,889 bp), B-T-3 P8 (8,513 bp), B-T-3 P9 (8,279 bp), B-T-3 P10 (7,635 bp), and B-T-3 P11 (2,062 bp).

[0025] The insecticidal protein gene sequences were downloaded from the Bacterial Insecticidal Protein Resource Center (https: / / www.bpprc-db.org), and the insecticidal protein genes of the strain B-T-3 were identified using the Needle and Blast tools. The results show that the strain B-T-3 carries a variety of insecticidal protein genes, including Cry (insecticidal crystal protein), Mpp (beta-pore-forming insecticidal protein), Vip (vegetative insecticidal protein), Vpb (insecticidal protein associated with binary toxin binding component) and Spp (globular hemolysin-like insecticidal protein) and other insecticidal protein types, containing 10 specific insecticidal protein types as follows: Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ia, Cry2Aa, Cry2Ab, Mpp46Ab, Vip3Aa, Vpb4Ca1 and Spp1Aa, wherein the Spp1Aa gene is located on the chromosome, and the rest of the insecticidal genes are located on the plasmid. The specific information of the insecticidal protein genes is shown in Table 1.

[0026] As can be seen from the results, the strain B-T-3 contains genes such as Cry1Ac and Cry2Ab with high insecticidal activity against Plutella xylostella, and also carries Cry1Ab, Cry1Aa, Cry2Aa, Vip3Aa genes with moderate insecticidal activity against Plutella xylostella and multiple genes with potential insecticidal activity. Due to the differences in insecticidal activity, cross resistance and action mechanism of different insecticidal proteins, the strain expressing multiple insecticidal protein genes or the combined application of different insecticidal proteins can significantly improve the control effect on Plutella xylostella, effectively delay the evolution of Plutella xylostella resistance, and has good application potential and prospect.

[0027] Table 1: Insecticidal protein gene type table of B-T-3 strain

[0028] In summary, the high virulence Bt strain B-T-3 screened by the application shows significantly excellent insecticidal activity against the 3rd and 2nd instar larvae of Plutella xylostella. Plutella xylostella At the same concentration (1x10 8 cfu / mL), the preliminary screening corrected mortality rate reaches 62.2%, which is higher than that of the other 13 isolated strains; further virulence determination shows that the LC 50 is 7.92x10 6 cfu / mL, which is about 1 / 1.78 of the LC 50 (1.41x10 7 cfu / mL) of the control strain (Bacillus thuringiensis of Wuhan Kenno Biological Technology Co., Ltd.), and the virulence is increased by about 1.78 times. The strain has higher activity and killing efficiency against Plutella xylostella, can significantly reduce the control dosage, and improve the field control effect.

Claims

1. A Bacillus thuringiensis, characterized in that, The preservation number of the Bacillus thuringiensis is CCTCC No:M20252325.

2. The Bacillus thuringiensis of claim 1, wherein, The Bacillus thuringiensis has a lethal concentration of 7.92 x 10 6 cfu / mL to the Plutella xylostella larvae.

3. The Bacillus thuringiensis of claim 1, wherein The strain of the Bacillus thuringiensis is bacillary in vegetative form, single or in chain arrangement, oval in spore form, and rhombic in parasporal crystal form.

4. The Bacillus thuringiensis of claim 1, wherein, The insecticidal crystal protein of the Bacillus thuringiensis comprises 130 kDa and 70 kDa proteins.

5. The Bacillus thuringiensis of claim 1, wherein The Bacillus thuringiensis comprises an insecticidal gene with an amino acid sequence as any one of SEQ ID NO:1-8.

6. The Bacillus thuringiensis of claim 1 is applied to control Plutella xylostella.

7. The Bacillus thuringiensis of claim 1 is applied to prepare a preparation for controlling Plutella xylostella.

8. An article for controlling Plutella xylostella, characterized by, The preparation comprises the Bacillus thuringiensis of claim 1.

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