Enterobacter hormaechei and application thereof in luring noctuidae pests
By genetically improving the Enterobacter coli strain BiotT-6121, an engineered strain that can induce fall armyworm was obtained, which solved the problem that the existing technology was difficult to efficiently control fall armywormworm, and achieved effective inducing and controlling the pest.
Patent Information
- Application Number
- CN202411945415.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-04-01
AI Technical Summary
Falligeria poses serious harm to corn and other economic crops, and it is difficult for the existing technology to provide efficient and green prevention and control methods.
By genetic modification of Enterobacter hormaechei strain BiotT-6121, an engineered strain capable of seducing pests from the family Desertaceae family was obtained, and a composition made of this strain was combined with the strain to control Fallia meadow.
This engineering strain has a significant inducing effect on Fallia meadow, providing a new biodefense resource that can effectively prevent and control Fallia meadow.
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Figure CN119736197A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of microorganisms, in particular to an Enterobacterium hallii and a method for using the same as an attractant to control Noctuidae pests. Background Art
[0002] Both larvae and adults of the fall armyworm (Spodoptera frugiperda) can cause serious damage to a variety of major economic crops such as corn. Screening resource microorganisms can provide an effective way for efficient, green and sustainable prevention and control of fall armyworm. Summary of the invention
[0003] One of the present invention provides a strain of Enterobacter holmesii ( Enterobacter hormaechei ) strain BiotT-6121, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.31840.
[0004] The second aspect of the present invention provides an engineered bacterium obtained by genetically improving the Enterobacter holmesii strain BiotT-6121 as described in the first aspect of the present invention. Among them, since the engineered bacterium is targeted at the Enterobacter holmesii CGMCC No.31840 of the present invention, and the means generally adopted are to transfer and / or knock out specific genes and / or DNA fragments therein, the engineered bacterium is still an Enterobacter. In addition, the engineered bacterium can be an engineered bacterium with improved attracting effect on Noctuidae pests (such as fall armyworm). It can also be an engineered strain that is active against pests and diseases and / or is endowed with other beneficial properties.
[0005] In a specific embodiment, the engineered bacteria obtained after genetic improvement are engineered strains obtained by introducing plasmids carrying functional genes, or engineered strains obtained by recombining functional genes into the genome of wild strains.
[0006] In a specific embodiment, the functional gene is at least one of a gene for controlling plant pests, a gene for controlling plant pathogenic microorganisms, and a gene that enhances the effect of the Enterobacter holmii strain BiotT-6121 in attracting plant pests.
[0007] The third aspect of the present invention provides a composition, which includes the Enterobacter hallii strain BiotT-6121 as described in the first aspect of the present invention or the engineered bacteria as described in the second aspect of the present invention.
[0008] In one embodiment, the composition further comprises an acceptable carrier.
[0009] In a specific embodiment, the dosage form of the composition is one of a suspension, a powder and a granule.
[0010] In a specific embodiment, the composition is in the form of an oil suspension or a wettable powder.
[0011] The fourth aspect of the present invention provides the use of at least one of the Enterobacterium hallii strain BiotT-6121 according to the first aspect of the present invention, the engineered bacteria according to the second aspect of the present invention, or the composition according to the third aspect of the present invention for luring Noctuidae pests.
[0012] In one embodiment, the Noctuidae pest is Spodoptera frugiperda ( Spodoptera fruit fly ).
[0013] Beneficial effects of the present invention: The present invention found that the Enterobacter holmenii strain BiotT-6121 has an attracting effect on Noctuidae pests, especially the fall armyworm, which provides a new biological control resource for controlling the fall armyworm.
[0014] Strain preservation: The strain screened by the present invention that has an attractant effect on Noctuidae pests is named BiotT-6121, and the strain is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC No.31840, a deposit date of September 3, 2024, and a deposit address of: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its system classification is Enterobacter hallii Enterobacter hormaechei . BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 The phylogenetic tree of the BiotT-6121 strain is shown.
[0016] Figure 2 The results of the resistance of BiotT-6121 strain to antibiotics are shown. Among them, (a) is a negative control plate without any resistance; (b) is a kanamycin resistance plate; (c) is an ampicillin resistance plate; (d) is a streptomycin sulfate resistance plate; (e) is an erythromycin resistance plate; (f) is a rifampicin resistance plate. DETAILED DESCRIPTION
[0017] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation to the present invention.
[0018] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.
[0019] 10% TSB liquid culture medium: casein hydrolysate 17.0 g, soy peptone 3.0 g, NaCl 5.0 g, K2HPO4 2.5 g, glucose 2.5 g, dilute to 1.0 L with distilled water, sterilize at 121 degrees Celsius for 15 min.
[0020] 10% TSB solid medium: Add 1.4% agar powder to the prepared 10% TSB liquid medium and sterilize at 121 degrees Celsius for 15 min. Example 1: Isolation of strains with attraction activity against fall armyworm
[0021] Sterile gauze with fall armyworm egg masses was placed in a sterile bag, cut into a suitable size with sterile scissors, and spread flat on a 9-cm culture dish. The egg masses were scraped into a weighed centrifuge tube with a sterile blue gun tip and weighed to complete the egg collection. The entire experiment was performed in a clean bench.
[0022] In the clean bench, 0.1 g of eggs were added to 1 mL of sterile water and vortexed for 5 min to obtain the bacterial solution on the surface of eggs; the bacterial solution on the surface of eggs was diluted 10 times with 10% TSB liquid culture medium; the bacterial solution on the surface of eggs diluted 10 times was further diluted in 7 gradients of 3×, 9×, 27×, 81×, 243×, 729×, and 2187×, and each dilution was added to a 96-deep-well bacterial culture plate, 600 μL per well, and sealed with a sterile sealing film after the operation. The last well of each deep-well bacterial culture plate was used as a negative control with an equal amount of blank culture medium; the 96-deep-well plate was cultured at room temperature in the dark for 3 days, the bacterial growth was observed, and the number of turbid wells was counted; when about 30% of the wells in the 96-deep-well plate were turbid, the concentration was considered to be the optimal dilution concentration, and the well plate culture solution obtained in each well of the 96-deep-well plate at this time was used for subsequent operations.
[0023] The results showed that when the egg surface bacteria solution was diluted 2187 times, 35-well plate culture solution was obtained from 95 wells. Based on this, the optimal dilution concentration of the egg surface bacteria solution in 10% TSB medium was determined to be 2187×.
[0024] Next, in the clean bench, 0.1 g of eggs were added to 1 mL of sterile water and vortexed for 5 min to obtain the bacterial solution on the surface of the eggs; the bacterial solution on the surface of the eggs was diluted 10 times with 10% TSB liquid culture medium; the bacterial solution on the surface of the eggs diluted 10 times was diluted again based on the optimal dilution concentration determined above to obtain each dilution, and each dilution was dispensed into 96 deep-well bacterial culture plates, and 600 microliters were injected into each well. The last well of each deep-well bacterial culture plate was negatively controlled with an equal amount of blank culture medium. A total of 15 plates. After culturing for 1 week under room temperature and dark conditions, the well plate culture solution of each bacteria was obtained, and the turbidity of the 96 deep-well bacterial culture plates was counted. Excluding contamination during culture and operation, a total of 14 plates were finally obtained. The well plate culture solution of each well plate was numbered.
[0025] Screening of bacteria with attractive activity for fall armyworm larvae: The 14 96-deep-well plates obtained above were mixed according to the wells in each column of each plate (except the negative control), and a total of 168 mixed culture solutions were obtained.
[0026] 50 μL of each of the 168 mixed culture solutions was transferred to a test tube containing 5 mL of 10% TSB liquid culture medium, and cultured overnight at 30 degrees Celsius and 220 rpm in a constant temperature shaker to obtain a test tube culture solution; then the test tube culture solution was taken and inoculated into a 250 mL conical flask containing 50 mL of 10% TSB liquid culture medium at an inoculation rate of 1%, and a 10% TSB liquid culture medium without mixed culture solution was used as a negative control, and cultured at 30 degrees Celsius and 220 rpm in a constant temperature shaker for 48 h to obtain each bacterial culture solution to be tested; 24 actively crawling fourth-instar fall armyworm larvae were selected, placed in a 24-well plate, starved for 30 minutes, and then placed in the animal placement room of the behavior measurement device (CN2017206618327). 5 mL of the bacterial culture to be tested was evenly dripped into a 9 cm culture dish covered with filter paper as the treatment group; 5 mL of 10% TSB liquid culture medium cultured at the same temperature and time was evenly dripped into another 9 cm culture dish covered with filter paper as the negative control group; then the treatment group and the negative control group were placed in the first odor source chamber and the second odor source chamber, respectively. Turn on the air compressor, adjust the glass rotor flowmeter to stabilize the air flow rate to 0.2 L / min, and treat in darkness for 2 h. Count and compare the number of fall armyworms entering the first odor source chamber (treatment group) and the second odor source chamber (negative control group) in the device.
[0027] The mixed culture fluid with attraction activity was traced back to the single-hole well plate culture fluid and screened one by one using the behavior measurement device again. The experiment was repeated three times for each well plate culture fluid. Among them, the well plate culture fluid with the strongest attraction effect on the fall armyworm larvae was separated and purified to obtain a pure cultured monoclonal strain, and the attraction activity of the monoclonal strain was further screened and confirmed using the behavior measurement device. Finally, the strain numbered BiotT-6121 was obtained, which can attract an average of 11.33 fall armyworm larvae, while the negative control group can only attract an average of 5 fall armyworms, with an attraction index of 0.26 ( t =19, P <0.05). Among them, attraction index = (number of insects captured in the treatment group - number of insects captured in the negative control group) / total number of insects tested. Example 2: Classification and Identification of Active Isolated Strains 1. Gram Identification of BiotT-6121 Isolates
[0028] Drop a drop of sterile water on the slide, use an inoculation loop to gently pick up the bacterial moss of BiotT-6121 and put it into the sterile water, gently stir to make the bacterial solution uniform, stain and decolorize according to the Gram staining solution operation method, and then observe under an oil microscope. The staining results of BiotT-6121 are all red, indicating that it is a Gram-negative bacterium. 2. Molecular identification of BiotT-6121 isolates—16S rDNA identification
[0029] According to the operation steps of the bacterial DNA genome kit, the total DNA of each isolate was extracted, and 27F (as shown in SEQ ID No. 1) and 1492R (as shown in SEQ ID No. 2) were used as primer pairs, and the total DNA of the BiotT-6121 isolate was used as a template to perform PCR amplification of 16S rDNA. After the amplified PCR product was detected by gel electrophoresis, the amplified product was sent to Bomedgene for sequencing. Among them, the sequence of 16S rDNA measured by the BiotT-6121 strain is (as shown in SEQ ID No. 3).
[0030] The 16S rDNA sequences were compared and analyzed at NCBI. The results showed that the BiotT-6121 strain was similar to Enterobacter hallii ( Enterobacter hormaechei ) are closely related. The phylogenetic tree was constructed using MEGA7.0. The phylogenetic tree of the BiotT-6121 strain is shown in Figure 1 .according to Figure 1 From the phylogenetic tree, we can see that the BiotT-6121 strain is closely related to Enterobacter hallii ( Enterobacter hormaechei ) strains are most closely related.
[0031] In summary, the strain BiotT-6121 was systematically classified as Enterobacter hallii ( Enterobacter hormaechei ).
[0032] The strain BiotT-6121 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration with the deposit number CGMCC No. 31840 and the deposit date September 3, 2024. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its system classification is Enterobacter hallii ( Enterobacter wall ). 3. Resistance detection of BiotT-6121 isolated strains
[0033] Kanamycin, working concentration: 100 μg / mL; ampicillin, working concentration: 100 μg / mL; streptomycin sulfate, working concentration: 25 μg / mL; erythromycin, working concentration: 60 μg / mL; rifampicin, working concentration: 50 μg / mL.
[0034] Prepare a 10% TSB solid medium resistance plate, streak it on the plate, use the medium without resistance as a negative control, and culture it in a 30 degree Celsius incubator for 12 hours to observe whether the bacteria can grow and determine the resistance of the bacteria to different antibiotics. Figure 2 .
[0035] according to Figure 2 From the results, it can be seen that the strain cannot grow on the solid culture medium plates containing kanamycin, ampicillin, streptomycin sulfate and rifampicin (b, c, d and f respectively), which means that it has no resistance to the four antibiotics; but it can grow on the solid culture medium plate containing erythromycin (e), indicating that it has a certain resistance to erythromycin.
Claims
1. A strain of Enterobacter holmesii ( Enterobacter hormaechei ) strain BiotT-6121, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No. 31840.
2. An engineered bacterium obtained by genetically modifying the Enterobacter holmesii strain BiotT-6121 as claimed in claim 1; The engineered bacteria obtained after genetic improvement are engineered strains obtained by introducing plasmids carrying functional genes, or engineered strains obtained by recombining functional genes into the genome of wild strains; The functional gene is at least one of a gene for controlling plant pests, a gene for controlling plant pathogenic microorganisms, and a gene for enhancing the effect of the Hallii Enterobacter strain in attracting plant pests.
3. A composition comprising the Enterobacter hallii strain BiotT-6121 as claimed in claim 1 or the engineered bacteria as claimed in claim 2.
4. The composition according to claim 3, characterized in that The composition also includes an acceptable carrier.
5. The composition according to claim 3, characterized in that The dosage form of the composition is one of a suspension, a powder and a granule.
6. The composition according to claim 3, characterized in that The dosage form of the composition is oil suspension or wettable powder.
7. Use of the Enterobacterium hallii strain BiotT-6121 according to claim 1, the engineered bacteria according to claim 2, or the composition according to any one of claims 3 to 6 for luring at least one of Noctuidae pests.
8. The use according to claim 7, characterized in that The Noctuidae pest is the fall armyworm ( Spodoptera frugiperda ).