Kobea enterobacter that has a synergistic effect on Nosema locustae and its application

Abstract

The present invention relates to Enterobacter kobei with synergistic effect on Nosema locustae and its application. Among them, the strain of Enterobacter kobei is 1-31, which is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC No. 31405. This provides a new biological control resource for locust control.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and particularly to Enterobacter kobei with synergistic effect on Nosema locustae and its application. Background Art

[0002] Oriental migratory locust belongs to Orthoptera, Acridoidea, and is one of the important and typical agricultural pests. Locusts have a large food intake and a wide range of food habits. Most green plants can be the feeding targets of locusts. At the same time, locusts have strong migratory and reproductive abilities. Therefore, when locust plagues break out, they will not only cause serious losses to agricultural production, but also pose serious threats and panic to society.

[0003] Nosema locustae is a microsporidium that specifically parasitizes locusts. It has no toxic effect on humans or other non-target organisms and has high safety in use. Nosema locustae has become one of the main biological control means for grassland locusts. However, the control effect of Nosema locustae on locusts is slow, and it is difficult to quickly apply Nosema locustae to control the spread of locust plagues during large-scale locust outbreaks, which has become one of the important reasons restricting the development of Nosema locustae. Summary of the Invention

[0004] One aspect of the present invention provides a strain of Enterobacter kobei ( Enterobacter kobei ) strain 1-31, which is deposited in the China General Microbiological Culture Collection Center, and the deposit number is CGMCC No. 31405.

[0005] Another aspect of the present invention provides an engineered bacterium obtained by genetically modifying the Enterobacter kobei strain 1-31 as described in one aspect of the present invention. Among them, since this engineered bacterium targets the Enterobacter kobei CGMCC No. 31405 of the present invention, and the means generally adopted is to transfer into and / or knock out specific genes and / or DNA fragments, etc., therefore, this engineered bacterium is still an Enterobacter. In addition, this engineered bacterium can be an engineered bacterium for improving the activity of Nosema locustae ( Paranosema locustae ) in controlling locusts. It can also be an engineered strain with other pest and disease activities and / or endowed with other beneficial characteristics.

[0006] In a specific embodiment, the engineered bacterium obtained after genetic modification is an engineered strain obtained by transferring a plasmid carrying a functional gene, or an engineered strain obtained by recombining a functional gene into the genome of a wild strain.

[0007] In a specific embodiment, the functional gene is a gene for controlling plant pests, a gene for controlling plant pathogenic microorganisms, and a gene for enhancing the ability of the Enterobacter kobei strain 1-31 to improve Nosema locustae ( Paranosema locustaeAt least one of the genes for controlling the effect of locusts.

[0008] The third aspect of the present invention provides the application of the Enterobacter kobei strain 1-31 described in the first aspect of the present invention or the engineered bacterium described in the second aspect of the present invention in enhancing the control of locusts by Nosema locustae ( Paranosema locustae ). Among them, the locusts are locusts of the superfamily Acridoidea.

[0009] In a specific embodiment, the locusts are Locusta migratoria manilensis.

[0010] The fourth aspect of the present invention provides a composition, which comprises the Enterobacter kobei strain 1-31 described in the first aspect of the invention or the engineered bacterium described in the second aspect of the invention, and Nosema locustae ( Paranosema locustae ).

[0011] In a specific embodiment, the composition further comprises an acceptable carrier.

[0012] In a specific embodiment, the dosage form of the composition is one of suspension, powder and granule.

[0013] In a specific embodiment, the dosage form of the composition is oil suspension or wettable powder.

[0014] The fifth aspect of the present invention provides the application of the composition described in the fourth aspect of the present invention in controlling locusts. Among them, the locusts are locusts of the superfamily Acridoidea.

[0015] In a specific embodiment, the locusts are Locusta migratoria manilensis.

[0016] Advantages of the present invention: The present invention discovers that the Enterobacter kobei strain 1-31 can improve the insecticidal activity of Nosema locustae ( Paranosema locustae ) against locusts, especially Locusta migratoria manilensis, in a short period of time. This is beneficial to accelerating the control speed of Nosema locustae against locusts, early controlling the harm caused by locusts to agricultural and livestock production, and reducing production losses. It provides a new biological control resource for controlling locusts.

[0017] Strain preservation: The strain screened in the present invention that can improve the insecticidal activity of Nosema locustae ( Paranosema locustae ) against locusts is named 1-31. This strain is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the preservation number of CGMCC No. 31405, the preservation date of August 1, 2024, and the preservation address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. Its systematic classification is Enterobacter kobei Enterobacter kobei . Description of the drawings

[0018] Figure 1 Shows the results of the insecticidal activity analysis of 1-31, Nosema locustae, and their combination.

[0019] Figure 2 Shows the virulence curves of Nosema locustae and the combination of Nosema locustae and isolate 1-31, LT 25 , LT 50 and significance.

[0020] Figure 3 Shows the colony morphology of strain 1-31.

[0021] Figure 4 Shows the Gram staining results of strain 1-31.

[0022] Figure 5 Shows the phylogenetic tree of strain 1-31.

[0023] Figure 6 Shows the antibiotic resistance results of strain 1-31. Detailed implementation manners

[0024] The above content of the present invention is further described in detail below in the form of preferred implementation cases, but it does not limit the present invention.

[0025] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0026] The strain of Nosema locustae used in the present invention ( Paranosema locustae ) has a strain number of PL-GM1 and a deposit number of CGMCC No. 19390.

[0027] Example 1: Isolation of the strain.

[0028] Dissect healthy Locusta migratoria manilensis that have been starved for 12 hours, take out the whole intestine, cut the middle and hindgut parts, and place them in sterilized 1.5 ml centrifuge tubes respectively. Add a little PBS buffer and grind thoroughly to obtain a middle intestine suspension and a hindgut suspension respectively. Among them, 3 replicates are set for the middle and hindgut respectively, and 5 locusts are treated in each replicate.

[0029] Add 0.3% beef extract, 0.5% peptone and 2% agar powder to distilled water, adjust the pH to 7.2, prepare a solid beef extract peptone medium, sterilize it, and obtain a solid beef extract peptone medium plate by pouring the plate. Separate and purify the intestinal microorganisms of Locusta migratoria manilensis on the plate by spreading and streaking the midgut suspension and hindgut suspension respectively. After culturing at 37 °C for 12 hours, pick single colonies with different sizes, morphologies and colors on the plate, and separate, culture and purify them by streaking on the plate multiple times to obtain purified isolated strains. Number the isolated strains.

[0030] Example 2: Bioactivity analysis of isolates against Locusta migratoria manilensis.

[0031] Pick single colonies of the isolated strains from the plate and inoculate them into 1.5 mL centrifuge tubes containing 500 μL of liquid beef extract peptone medium, and culture them again in a shaker at 37 °C for 24 hours, centrifuge, wash the bacterial cells, and then dilute the bacterial cells with sterile ultrapure water to 1×10 4 colony forming units / mL (cfu / mL), 1×10 5 colony forming units / mL, 1×10 6 colony forming units / mL, 1×10 7 colony forming units / mL to prepare a diluted solution of the isolate. The inoculation amounts are 20 colony forming units / head, 200 colony forming units / head, 2000 colony forming units / head, and 20000 colony forming units / head of nymphs respectively.

[0032] Select healthy 3rd instar nymphs of Locusta migratoria manilensis indoors, divide them into plastic boxes with upper and lower diameters of 11.9 cm and 8.5 cm respectively, and starve them for 12 hours.

[0033] Use a hemocytometer to calculate the concentration of the mother liquor of Nosema locustae ( Paranosema locustae ). According to the number of locusts to be treated in the experiment, suck the corresponding Nosema locustae solution and centrifuge to obtain a precipitate of Nosema locustae.

[0034] Resuspend the precipitate of Nosema locustae with sterile ultrapure water to prepare a suspension of Nosema locustae, wherein the concentration of Nosema locustae in the suspension of Nosema locustae is 1×10 8 cells / mL. The inoculation amount is 200000 cells / head of nymphs.

[0035] Use 4 different concentrations of the diluted solution of the isolate, that is, resuspend the precipitate of Nosema locustae to prepare an isolate-Nosema locustae suspension. Among them, the concentration of Nosema locustae in the isolate-Nosema locustae suspension is 1×10 8colony-forming units per milliliter (cfu / mL). Take 2 μL of the isolate dilution, the microsporidia suspension, and the isolate-microsporidia suspension, and evenly apply them on corn leaves that have been cut into approximately the same area and treated by scraping. Fix the corn leaves with a needle and a triangular card, and feed them to 3rd-instar nymphs of Locusta migratoria manilensis. That is, each nymph is inoculated with the same dose of the isolate or Nosema locustae. In addition, a negative control group of sterile ultrapure water (CK) is set up.

[0036] After 3 hours, select the nymphs that have eaten all the corn leaves and put them into a rearing box, and feed them with fresh wheat seedlings. Put 10 successfully inoculated nymphs in each rearing box, use 3 rearing boxes for each treatment, and set up 3 biological replicates. Observe the daily death of the locusts every day, calculate the mortality rate, and calculate the corrected mortality rate based on the negative control of ultrapure water, for a total of 20 days.

[0037] Use GraphPad plotting software to analyze the indoor bioassay results of each isolated strain, microsporidia, and the combination of the isolate and microsporidia, and make a survival curve graph of the nymphs of Locusta migratoria manilensis and a bar graph of the mortality rate of the nymphs of Locusta migratoria manilensis at different time points.

[0038] Among them, the isolate that has a more obvious synergistic effect on Nosema locustae and is easy to culture is 1-31. After treatment with the isolated strain 1-31, microsporidia, and the combination of the isolated strain 1-31 and microsporidia, the bar graph of the mortality rate of the nymphs of Locusta migratoria manilensis is as Figure 1 shown. Among them, the inoculation amount of the isolated strain 1-31 in the figure is 200 colony-forming units per head, and the inoculation amount of Nosema locustae is 2×10 5 per head.

[0039] Figure 1 The results show that from day 0 to day 10, there is no significant difference in the lethal effect of the isolated strain 1-31 on Locusta migratoria manilensis compared with the negative control, that is, it is considered to have basically no toxicity to Locusta migratoria manilensis. However, when it is used in combination with Nosema locustae, it can significantly increase the mortality rate of Locusta migratoria manilensis, indicating that it has a synergistic effect on the killing of Locusta migratoria manilensis by Nosema locustae.

[0040] When the inoculation amount of Nosema locustae is 2×10 5 per head; and in the combination of Nosema locustae and the isolated strain 1-31, the inoculation amount of the isolated strain is 200 colony-forming units per head, and the inoculation amount of Nosema locustae is 2×10 5 per head, use SPSS software to obtain the virulence curve and the correlation R value based on time and the mortality rate of Locusta migratoria manilensis. Among them, x represents the number of days of the experiment, and y represents the mortality rate of Locusta migratoria manilensis changing with time. Calculate LT 25 and LT 50 . The virulence curves, LT of Nosema locustae and the combination of Nosema locustae and the isolated strain 1-3125 and LT 50 and significance are shown in Figure 2 .

[0041] Example 3: Classification and identification of active isolated strains.

[0042] Colony morphology of isolate 1-31: Isolate 1-31 was cultured upright at 37 °C on nutrient agar solid medium for 2 hours, and then inverted for 24 hours. Observe the colony morphology and take pictures, as shown in Figure 3 . According to Figure 3 , it can be seen that the colony of isolate 1-31 is nearly circular in shape, with an irregular edge, showing a serrated shape, slightly raised, slightly concave in the center, white in color, smooth and moist on the surface, and semi-transparent.

[0043] Gram staining identification of isolate 1-31: Drop a drop of sterile water on a glass slide, gently pick the bacterial colony of 1-31 with an inoculation loop and put it into the sterile water, gently stir to make the bacterial solution uniform, and perform staining and decolorization according to the operation method of Gram staining solution, and then observe under an oil immersion microscope. The staining results of 1-31 are all red, indicating that it is a Gram-negative bacterium. The staining results are shown in Figure 4 .

[0044] Molecular identification of isolate 1-31 - 16S rDNA identification: Extract the total DNA of each isolate according to the operation steps of the bacterial DNA genome kit. Using 341F (shown in SEQ ID No. 1) / 806R (shown in SEQ ID No. 2) as primers and the total DNA of isolate 1-31 as a template, perform PCR amplification of 16S rDNA. After the amplified PCR product is detected by gel electrophoresis, the amplified product is sent to Bomeideji Company for sequencing. Among them, the sequence of 16S rDNA measured by strain 1-31 (shown in SEQ ID No. 3). The sequence results of the measured 16S rDNA were compared and analyzed on NCBI, and the results showed that strain 1-31 had the closest genetic relationship with Enterobacter kobei ( Enterobacter kobei ). Use MEGA7.0 to construct a phylogenetic tree. Among them, the phylogenetic tree of strain 1-31 is shown in Figure 5 . According to Figure 5 's phylogenetic tree, it can be seen that strain 1-31 has the closest genetic relationship with Enterobacter kobei ( Enterobacter kobei ) strain.

[0045] In summary, the systematic classification of strain 1-31 is Enterobacter kobei Enterobacter kobei .

[0046] The 1-31 strain was deposited with the China General Microbiological Culture Collection Center. The deposit number is CGMCC No. 31405. The deposit date is August 1, 2024. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its systematic classification is Enterobacter kobei Enterobacter kobei .

[0047] Resistance detection of the 1-31 isolated strain: Five antibiotics were prepared: Gentamicin sulfate with a working concentration of 50 μg / mL; Streptomycin sulfate with a working concentration of 50 μg / mL; Kanamycin with a working concentration of 25 μg / mL; Chloramphenicol with a working concentration of 25 μg / mL; Ampicillin with a working concentration of 50 μg / mL. Resistance plates of nutrient agar medium were made. 1-31 bacterial solution was dropped in the center of the plate and incubated upright in an incubator at 37 °C for 2 hours, and then incubated upside down for 24 hours. Observe whether the bacteria can grow to judge the resistance of the bacteria to different antibiotics. The results are shown in Figure 6 . According to Figure 6 the results, it was found that it could not grow on the nutrient agar medium plates containing Gentamicin sulfate, Streptomycin sulfate, Kanamycin, and Chloramphenicol (a, b, c, d in sequence), that is, it was not resistant to Gentamicin sulfate, Streptomycin sulfate, Kanamycin, and Chloramphenicol; it could grow on the nutrient agar medium plate containing Ampicillin (e), indicating that it was resistant to Ampicillin.

Claims

1. Kobe Enterobacter strain 1-31 was used to enhance the ability of locust microsporidia ( Paranosema locustae ) for use in controlling locusts. The Kobe Enterobacter strain 1-31 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number being CGMCC No. 31405.

2. The use according to claim 1, characterized in that: The locust is the East Asian migratory locust.

3. A composition comprising the Kobe Enterobacter strain 1-31 used in the application according to claim 1 or 2, and locust microsporidia ( Paranosema locustae ).

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 composition according to any one of claims 3 to 6 for controlling locusts.

8. The use according to claim 7, characterized in that: The locust is the East Asian migratory locust.

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