A strain of Bacillus thuringiensis used to control fall armyworm

Abstract

This invention relates to a Bacillus thuringiensis strain for controlling fall armyworm. The strain is IPPO68, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37126. The extracellular polysaccharides produced by this strain exhibit insecticidal activity against fall armyworm.

Description

Technical Field

[0001] This invention relates to the field of microorganisms, and particularly to insecticidal microorganisms. Background Technology

[0002] fall armyworm ( Spodoptera frugiperda The larvae and adults of the fall armyworm can damage a variety of major economic crops, mainly gramineous crops. Based on their preference for host crops, they are divided into corn type and rice type. The fall armyworm is most harmful to corn, and it can cause damage at all stages of corn growth. The most serious damage occurs during the seedling stage and the large trumpet stage (Lin Danmin, Huang Dechao, Shao Tun, Li Ziyuan, Wang Lei, Chen Kewei, Lu Yongyue, 2020; Occurrence and damage patterns of fall armyworm on corn at different growth stages; Journal of Environmental Entomology, 42(06): 1291–1297).

[0003] Screening for highly virulent Bacillus thuringiensis (Bt) Bacillus thuringiensis Bt strain resources can provide an effective way to achieve efficient, green and sustainable control of fall armyworm. Summary of the Invention

[0004] One of the present inventions provides a Bacillus thuringiensis (Bt) Bacillus thuringiensis It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37126.

[0005] The engineered bacteria obtained by genetically modifying the strains of the present invention can be endowed with superior and / or more properties. For example, by combining the characteristics of the strain itself, its insecticidal and / or insect-resistant properties can be increased and / or broadened according to practical applications, or it can also be endowed with antibacterial properties. That is, through genetic modification of the strains of the present invention, they can possess at least one of the above-mentioned properties. Since the engineered strain uses Bacillus thuringiensis of the present invention as the target of modification, that is, specific genes and / or sequences are introduced and / or knocked out there, the genetically modified strain is still Bacillus thuringiensis.

[0006] Therefore, the second aspect of the present invention provides an engineered bacterium obtained by genetically modifying the Bacillus thuringiensis described in the first aspect of the present invention. For example, the genetically modified engineered bacterium can be an engineered strain obtained by introducing a plasmid carrying a functional gene, or it can be an engineered strain obtained by recombining a functional gene into the genome of a wild-type strain.

[0007] Therefore, in one specific embodiment, the engineered bacteria are obtained by transferring a functional gene into Bacillus thuringiensis as described in one of the present invention.

[0008] In one specific embodiment, the functional gene is at least one of the following: a gene for controlling plant pests, a gene for controlling plant pathogenic microorganisms, and a gene that enhances the effect of Bacillus thuringiensis in controlling plant pests.

[0009] Although genetically modified organisms (GMOs) face considerable skepticism from some groups, engineered bacteria obtained through genetic modification of Bacillus thuringiensis are not intended for direct human or animal consumption. Furthermore, before being commercialized, they must first undergo safety evaluations by relevant national authorities to avoid potential safety issues. Their use will then be based on the safety assessments and approval from the relevant national authorities.

[0010] The third invention provides a composition comprising Bacillus thuringiensis as described in the first invention or engineered bacteria as described in the second invention.

[0011] In one specific embodiment, the dosage form of the composition is one of a suspension, powder, and granules.

[0012] In one specific embodiment, the dosage form of the composition is an oil suspension or a wettable powder.

[0013] The fourth invention provides the use of at least one of the following in the control of fall armyworm: Bacillus thuringiensis as described in the first invention, engineered bacteria as described in the second invention, and a composition as described in the third invention.

[0014] Unless otherwise specified, the terminology used in this invention refers to general terms in the prior art.

[0015] Strain Preservation: The microorganism IPPO68 strain screened in this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 37126, on December 19, 2025. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its systematic classification is Bacillus thuringiensis. Bacillus thuringiensis . Attached Figure Description

[0016] Figure 1 The phylogenetic tree of IPPO68 strain 16S is shown. Detailed Implementation

[0017] The present invention will now be described in detail with reference to the embodiments and accompanying drawings. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications and substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0018] Unless otherwise specified, all reagents used in the embodiments of this invention are commercially available.

[0019] LB liquid medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, sterilized at 121 degrees Celsius for 20 min.

[0020] LB solid medium: LB liquid medium with 15 g / L agar, sterilized at 121 degrees Celsius for 20 min.

[0021] Fall armyworm tested ( Spodoptera frugiperda (Provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences)

[0022] Artificial feed formula for fall armyworm: 200 g corn flour, 100 g soybean flour, 90 g yeast powder, 50 g sucrose, 15 g agar, 1.8 g sorbic acid, 1.8 g methylparaben, and 1000 ml water.

[0023] Example 1: Isolation and Morphological Identification of Strains

[0024] LB agar was used to screen and isolate Bacillus from soil samples collected from the Qinghai Plateau region. First, the soil samples were dried at 70°C for 4 hours. The dried soil samples were then added to 50 mL centrifuge tubes, along with 15 mL of sterile water and approximately 10 3 mm glass beads. The mixture was vortexed and thoroughly diluted. The serially diluted samples were then placed in an 80°C water bath for 20 minutes. Under aseptic conditions, 100 μL of each dilution was spread onto LB agar plates and incubated at 30°C for 48 hours. Colonies exhibiting a non-slippery, moist, thick morphology with slightly diffused and irregular outer edges were purified. The purified single colonies were then preserved for subsequent identification and bioactivity analysis.

[0025] Purified single colonies were cultured on LB solid medium at 30°C, and samples were taken at different time points for microscopic observation of colony morphology and crystal characteristics. The results observed at different stages of culture on LB medium are as follows: Vegetative cells: rod-shaped, with blunt ends, approximately 1.0 × 0.5 μm to 1.5 × 0.5 μm in size; existing singly or in chains of two or more. Spores: oval, approximately 1.0 × 0.5 μm to 1.3 × 0.5 μm in size, dormant; exhibiting strong resistance to adverse environments such as high temperature or dryness. Parasporal crystals: spherical, rhomboid, and square, etc. These morphological characteristics are basically consistent with the description of Bacillus thuringiensis in the *Handbook of Systematic Identification of Common Bacteria* (edited by Dong Xiuzhu et al., Science Press, 2001). Therefore, strains with this morphological colony were preliminarily identified as *Bacillus thuringiensis*.

[0026] The isolated strains were numbered.

[0027] Example 2: Screening of insecticidal strains for fall armyworm

[0028] Single clones of each isolated strain were inoculated into test tubes containing 5 mL of LB liquid medium and activated by incubation at 30°C for 12 hours. The activated bacterial solutions were then transferred to LB solid medium and incubated at 30°C for 12 hours. The bacterial cells were collected using a sterile scraper and resuspended uniformly in phosphate-buffered saline (PBS). Plate counting was performed on the suspended cells. The counted cells were then sonicated (amplitude 70%, pulse 3 s, pause 5 s) to release total protein from the cells, yielding the bacterial solutions of each isolated strain.

[0029] Weigh 15 g of artificial feed for fall armyworm and place it in a sterile petri dish. Add bacterial suspensions of each isolated strain separately to achieve a final bioassay concentration of 1×10⁻⁶. 8 The feed was mixed thoroughly by kneading with gloved hands and left at room temperature for 1 to 2 hours to allow excess moisture to evaporate. The feed was then evenly distributed into 24-well plates. Newly hatched larvae that were active, uniform in size, and exhibiting good filamentous growth were then picked up with a brush and placed into each well, one larva per well. The plates were then covered with a top cover containing a plastic cardboard insert and secured with rubber bands to prevent escape. The 24-well plates were placed in a rearing room at (27±1)°C, RH (65±5)%, and a light cycle of 16 L:8 D. Each treatment was repeated three times, with 24 larvae per replicate. Phosphate-buffered saline (PBS) was used as a negative control (CK). Light, humidity, temperature, and the presence of mold or condensation in the feed were checked daily. After 7 days, the number of dead and live larvae was recorded, and the average mortality rate was calculated. The results are shown in Table 1.

[0030] Table 1

[0031]

[0032] Note: Different lowercase letters after the data in the same column indicate that the differences are significant at the P<0.05 level according to the one-way ANOVA multiple comparison analysis.

[0033] Example 3: Cluster analysis of IPPO68 strains

[0034] The 16S rRNA gene is an evolutionary benchmark for bacteria. For an unknown species, the phylogenetic relationship of its 16S rRNA gene is usually examined first. Therefore, the 16S rRNA gene of IPPO68 was sequenced and a phylogenetic tree was constructed.

[0035] Refer to Song FP, et al. (Identification of cry1I-type genes from Bacillus thuringiensis Genomic DNA was extracted from isolated strains such as IPPO68 using the method described in [J]. Applied and environmental microbiology. 2003, 69(9), 5207-5211. The 16S rDNA sequences of each isolated strain were amplified using the universal primers for bacterial 16S rDNA: 16SF1 (SEQ ID No. 1) and 16SR1 (SEQ ID No. 2). The fragments obtained by PCR amplification were purified using an Axygen gel extraction kit (Axygen Biotechnology (Hangzhou) Co., Ltd.), and then TA cloned into pMD-18T (Takara) and transformed into Escherichia coli (E. coli). Escherichia coli The transformed bacteria were cultured using standard methods to obtain transformants. After verification by PCR, the transformants were sent to Beijing Liuhe Huada Genomics Co., Ltd. for sequencing. The 16S rDNA sequence of strain IPPO68 was 1549 bp (see SEQ ID No. 3). The sequenced sequences were submitted to the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) for homology comparison. The comparison results showed that strains such as IPPO68 are homologous to Bacillus thuringiensis (Bt). Bacillus thuringiensis The sequence NR043403.1 (with a published 1486 bp 16S rDNA fragment) showed 99.9% similarity. A phylogenetic tree was constructed using MEGA 6.0 based on the 16S rDNA sequence; the results are shown below. Figure 1 .according to Figure 1The phylogenetic tree shows that isolates such as IPPO68 are most closely related to Bacillus thuringiensis. Therefore, isolates such as IPPO68 are systematically classified as Bacillus thuringiensis. Bacillus thuringiensis Furthermore, IPPO68 is more closely related to D3, D18, D21, S17, S50, and S191 than to the known strain NR043403.1.

[0036] This strain was deposited on December 19, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37126. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0037] Example 4: Biological activity of crude extracellular polysaccharide of IPPO68 (i.e., extracellular polysaccharide extract)

[0038] Extraction of crude extracellular polysaccharides from IPPO68: 10 μL of Bacillus thuringiensis IPPO68 glycerol bacteria were inoculated into 5 mL of LB liquid medium and activated at 30°C and 220 r / min for 10 h to obtain an activated bacterial solution. The activated bacterial solution was transferred to 100 mL of LB liquid medium at a 1% inoculation rate and cultured in a 500 mL Erlenmeyer flask for 30 h. The cultured bacterial solution was collected, and the pH was adjusted to 8.5 with 1 mol / L NaOH. The solution was centrifuged at 4°C and 7000 r / min for 30 min, and the first supernatant was collected. 1 / 10 volume of 5 wt% trichloroacetic acid aqueous solution was added to the first supernatant, and the protein was denatured at 25°C for 2 h. The solution was then centrifuged at 4°C and 7000 r / min for 30 min, and the second supernatant was collected. The pH was adjusted to 6.5. Three volumes of anhydrous ethanol were added, and the solution was precipitated at 4°C for 12 h. The solution was then centrifuged at 4°C and 7000 r / min for 30 min. Discard the supernatant and freeze-dry the precipitate to obtain crude extracellular polysaccharide powder.

[0039] Quantification of IPPO68 extracellular polysaccharides: First, a glucose standard curve was prepared as follows: 0.1 g of glucose was dissolved in 250 mL of sterile distilled water and stirred until homogeneous to obtain a glucose stock solution. Then, glucose standard solutions of different concentration gradients were prepared using the glucose stock solution. 0.1 mL of 98% concentrated sulfuric acid and 0.5 mL of 6wt% phenol aqueous solution were added to each glucose standard solution, and sterile distilled water was added to bring the total volume to 0.8 mL. The mixture was slowly mixed, cooled, and allowed to stand at room temperature for 20 min. 200 μL of the solution was then transferred to an ELISA plate and the absorbance was measured at 490 nm. Sterile distilled water was used as a blank control instead of the sample. Each treatment group was repeated in triplicate, and a standard curve was plotted. Then, quantification was performed using the phenol-sulfuric acid method as follows: Crude extracellular polysaccharide powder was dissolved in sterile distilled water and diluted to a concentration that met the OD values ​​of the crude extracellular polysaccharide aqueous solution. 490nm The absorbance was between 0.2 and 1.0. 0.1 mL of concentrated sulfuric acid and 0.5 mL of 6wt% phenol aqueous solution were added, followed by the addition of sterile distilled water to a final volume of 0.8 mL. The mixture was thoroughly mixed, cooled, and allowed to stand at room temperature for 20 min. 200 μL of the solution was then transferred to an ELISA plate, and the absorbance was measured at 490 nm. The content of extracellular polysaccharides in the crude extracellular polysaccharide powder was calculated based on the plotted standard curve. The concentration of extracellular polysaccharides in the crude extracellular polysaccharide aqueous solution was 100 mg / mL.

[0040] Biological activity assay of IPPO68 crude extracellular polysaccharide: 15 g of artificial feed for fall armyworm was weighed and placed in a sterile petri dish. A crude extracellular polysaccharide aqueous solution with a concentration of 100 mg / ml was added to make the extracellular polysaccharide content in the artificial feed 10 mg / g. The feed was repeatedly kneaded and mixed with gloved hands and left at room temperature for 1 to 2 hours to allow excess moisture to evaporate. All feed was evenly distributed into 24-well plates. Then, newly hatched larvae that were active, uniform in size, and had a stringy texture were picked up with a brush and transferred to the 24-well plates, one larva per well. After the larvae were transferred, the plates were covered with a top cover containing a blown cardboard insert and secured with a rubber band to prevent larvae from escaping. The 24-well plates were placed in a rearing room at a temperature of (27±1) degrees Celsius, RH of (65±5)%, and a photoperiod of 16 L:8 D. Each treatment was repeated three times, with 24 larvae per replicate. Sterile distilled water was used as a negative control. Daily checks were conducted on light, humidity, temperature, and whether the feed was moldy or showing signs of water vapor condensation. After 7 days, the number of dead and live insects was investigated, and the average mortality rate was calculated. The result was 61.11%.

[0041] Example 5: Biological activity of IPPO68 insecticidal protein

[0042] Insecticidal gene identification: Upstream primer CryF (SEQ ID No. 4) and downstream primer CryR (SEQ ID No. 5) were designed for PCR amplification using the IPPO68 strain genome as a template. The obtained gene, after sequencing, was... cry1Ca15 The nucleic acid sequence of (Genbank No. QBO24619) is completely identical, meaning that the insecticidal protein gene of strain IPPO68 is... cry1Ca15 .Will cry1Ca15 The pSTK-cry1Ca15 recombinant expression vector was obtained by linking the pSTK-cry1Ca15 to the expression vector pSTK-cry1Ca15. The pSTK-cry1Ca15 was then transformed into the Bt amorphous mutant strain HD73- to obtain HD73 / pSTK-cry1Ca15 for expressing the Cry1Ca15 protein.

[0043] Preparation of HD73 / pSTK-cry1Ca15 protein spore mixture: 400 μL of activated HD73 / pSTK-cry1Ca15 was evenly spread on 1 / 2 LB solid medium and cultured at a constant temperature of 30°C until more than 50% of the cells were lysed. All cells were scraped into a 50 mL centrifuge tube, and washed twice with an appropriate amount of pre-cooled ultrapure water. The tube was centrifuged at 8000 r / min for 10 min each time. The supernatant was discarded, and the precipitate was dissolved in 4 mL of pre-cooled 50 mM Na2CO3 aqueous solution (pH 11.5). The mixture was repeatedly pipetted and mixed to obtain the HD73 / pSTK-cry1Ca15 protein spore mixture. Take a small amount of HD73 / pSTK-cry1Ca15 protein spore mixture, add 1 / 5 volume of pre-cooled 0.5M NaOH solution, react at room temperature for 5 min, then add 5× loading buffer, mix well, boil for 5 min, centrifuge at 12000 r / min for 3 min, and take 10 μL of supernatant for SDS-PAGE electrophoresis analysis. The electrophoresis method is as described by Sambrook and Russell (2002). Protein maps were quantified using Image J2x software for 130 kDa protein bands.

[0044] Preparation of IPPO68 protein spore mixture: The HD73 / pSTK-cry1Ca15 strain in the preparation of HD73 / pSTK-cry1Ca15 protein spore mixture was replaced with IPPO68 strain. The rest of the preparation was the same as that of HD73 / pSTK-cry1Ca15 protein spore mixture. Finally, IPPO68 protein spore mixture was obtained.

[0045] Biological activity assay of HD73 / pSTK-cry1Ca15 protein spore mixture: 15 g of artificial feed for fall armyworm was weighed and placed in a sterile petri dish. HD73 / pSTK-cry1Ca15 protein spore mixture was added to make the Cry1Ca15 protein content in the artificial feed 5 μg / g and 10 μg / g, respectively. The feed was repeatedly kneaded and mixed with gloved hands and left at room temperature for 1 to 2 hours to allow excess moisture to evaporate. All feed was evenly distributed into 24-well plates. Then, newly hatched larvae that were active and of uniform size and had a stringy texture were picked up with a brush and transferred to the 24-well plates, one larva per well. After the larvae were transferred, the plates were covered with a top cover with a built-in blown cardboard and secured with rubber bands to prevent the larvae from escaping. The 24-well plates were placed in a rearing room at a temperature of (27±1) degrees Celsius, RH of (65±5)%, and a photoperiod of 16 L:8 D. Each treatment was repeated three times, with 24 insects per replicate. A 50 mM Na₂CO₃ aqueous solution (pH 11.5) was used as a negative control. Light, humidity, temperature, and feed for mold and condensation were checked daily. After 7 days, the number of dead and live insects was assessed, and the corrected mortality rate was calculated based on the negative control. Results are shown in Table 2.

[0046] Biological activity assay of IPPO68 protein spore mixture: The HD73 / pSTK-cry1Ca15 protein spore mixture was replaced with the IPPO68 protein spore mixture in the biological activity assay of the HD73 / pSTK-cry1Ca15 protein spore mixture. All other parameters were the same as those for the HD73 / pSTK-cry1Ca15 protein spore mixture assay. The corrected mortality rate at 7 days was then obtained. The results are shown in Table 2.

[0047] Table 2

[0048]

[0049] Note: Different lowercase letters after the data in the same row indicate that the differences are significant at the P<0.05 level according to the one-way ANOVA multiple comparison analysis.

Claims

1. A strain of Bacillus thuringiensis (Bt) Bacillus thuringiensis The strain IPPO68 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37126.

2. A composition comprising the Bacillus thuringiensis strain IPPO68 as described in claim 1.

3. The composition according to claim 2, characterized in that, The composition is in the form of a suspension, powder, or granules.

4. The composition according to claim 2, characterized in that, The composition is in the form of an oil suspension or a wettable powder.

5. The use of the Bacillus thuringiensis strain IPPO68 as described in claim 1 or the composition as described in any one of claims 2 to 4 for the control of fall armyworm.

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