Enterobacter sphenomenus with synergistic effect on locust microsporidia and application of enterobacter sphenomenus

Abstract

The invention relates to enterobacter sphenomenus with a synergistic effect on locust microsporidia and application of the enterobacter sphenomenus. Wherein the strain of the enterobacter sphenomenus is 1-31, the enterobacter sphenomenus is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.31405. A new biocontrol resource is provided for locust control.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, in particular to Enterobacter kojira having a synergistic effect on locust microsporidia and application thereof. Background Art

[0002] The East Asian migratory locust belongs to the superfamily Acridoidea of ​​the order Orthoptera of the class Insecta and is one of the most important and typical agricultural pests. Locusts have a large food intake and a wide range of diets, and most green plants can become their food targets. At the same time, locusts have strong migration and reproduction capabilities. Therefore, when a locust plague breaks out, it will not only cause serious losses to agricultural production, but also cause serious threats and panic to society.

[0003] Nosema locustica is a microsporidia that parasitizes locusts. It is non-toxic to humans or other non-target organisms and is highly safe to use. Nosema locustica has become one of the main biological control methods for grassland locusts. However, the effect of Nosema locustica in controlling locusts is slow. It is difficult to quickly apply Nosema locustica to control the spread of locust plagues during a locust outbreak, which has become one of the important reasons limiting the development of Nosema locustica. Summary of the invention

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

[0005] The second aspect of the present invention provides an engineered bacterium obtained by genetically modifying the Kobe Enterobacter strain 1-31 as described in the first aspect of the present invention. Since the engineered bacterium is targeted at the Kobe Enterobacter CGMCC No. 31405 of the present invention, and the means generally adopted are to transfer and / or knock out specific genes and / or DNA fragments, etc., the engineered bacterium is still an Enterobacter. In addition, the engineered bacterium can be used to improve the microsporidia of locusts ( Paranosema locustae ) Engineered bacteria with locust control activity. It can also be an engineered strain that has other pest activity and / or is endowed with other beneficial properties.

[0006] 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.

[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 Kobe Enterobacter strain 1-31 to improve locust microsporidia ( Paranosema locustae) at least one of the genes that have the effect of controlling locusts.

[0008] The third invention provides the Kobe Enterobacter strain 1-31 according to the first invention or the engineered bacteria according to the second invention for improving locust microsporidia ( Paranosema locustae ) for preventing and controlling locusts. Wherein, the locusts are locusts of the superfamily Acridoidea.

[0009] In a specific embodiment, the locust is Locust migratory locust.

[0010] The fourth aspect of the present invention provides a composition comprising the Kobe Enterobacter strain 1-31 as described in the first aspect of the present invention or the engineered bacteria as described in the second aspect of the present invention, and locust microsporidia ( Paranosema locustae ).

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

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

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

[0014] The fifth aspect of the present invention provides the use of the composition of the fourth aspect of the present invention for controlling locusts, wherein the locusts are locusts of the superfamily Acridoidea.

[0015] In a specific embodiment, the locust is Locust migratory locust.

[0016] Beneficial effects of the present invention: The present invention found that Kobe Enterobacter strain 1-31 can improve locust microsporidia ( Paranosema locustae ) has insecticidal activity against locusts, especially migratory locusts, which is conducive to accelerating the control of microsporidia against locusts, controlling the damage caused by locusts to agricultural and animal husbandry production as early as possible, and reducing production losses. It provides a new biological control resource for locust control.

[0017] Strain preservation: The strains screened by the present invention can improve the microsporidia of locusts ( Paranosema locustae ) The strain with insecticidal activity against locusts was named 1-31. The strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee with the deposit number CGMCC No. 31405 and the deposit date of August 1, 2024. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Its systematic classification is Kobe Enterobacter Enterobacter kobei . BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of insecticidal activity analysis of 1-31, Nosema locustae, and a combination of the two are shown.

[0019] Figure 2 Virulence curves, LT 25 ,LT 50 and significance.

[0020] Figure 3 The colony morphology of strain 1-31 is shown.

[0021] Figure 4 Gram staining results of strains 1-31 are shown.

[0022] Figure 5 The phylogenetic tree of strains 1-31 is shown.

[0023] Figure 6 The results of resistance of strains 1-31 to antibiotics are shown. DETAILED DESCRIPTION

[0024] 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.

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

[0026] The locust microsporidia used in the present invention ( Paranosema locustae ) is numbered PL-GM1 and its preservation number is CGMCC No. 19390.

[0027] Example 1: Isolation of strains.

[0028] Healthy East Asian migratory locusts that had been starved for 12 hours were dissected, the whole intestine was removed, the midgut and hindgut parts were cut, and they were placed in sterilized 1.5 ml centrifuge tubes, respectively, and a small amount of PBS buffer was added for full grinding to obtain midgut suspension and hindgut suspension, respectively. Among them, 3 replicates were set for the midgut and hindgut, and each replicate treated 5 migratory locusts.

[0029] Add 0.3% beef extract, 0.5% peptone and 2% agar powder to distilled water, adjust the pH to 7.2, prepare beef extract peptone solid culture medium, sterilize, and obtain beef extract peptone solid culture medium plate by pouring the plate. Separate and purify the intestinal microorganisms of the East Asian migratory locust on the plate by the method of plate coating and plate streaking of the midgut suspension and hindgut suspension respectively. After culturing at 37 degrees Celsius for 12 hours, pick single colonies with inconsistent size, morphology and color on the plate, separate and culture and purify them by plate streaking multiple times to obtain purified isolated strains. Number the isolated strains.

[0030] Example 2: Analysis of the biological activity of the isolates against Locusta migratoria.

[0031] The isolated strain was picked from the plate and inoculated into a 1.5 ml centrifuge tube containing 500 μl of beef extract peptone liquid medium. The tube was cultured again in a shaker at 37 degrees Celsius for 24 hours, centrifuged, washed, and then diluted to 1×10 with sterile ultrapure water. 4 Colony forming units / mL (cfu / mL), 1×10 5 Colony forming units / ml, 1×10 6 Colony forming units / ml, 1×10 7 The inoculum size was 20 CFU / head, 200 CFU / head, 2000 CFU / head, and 20000 CFU / head locust larvae.

[0032] Healthy 3rd-instar East Asian migratory locust nymphs were selected indoors, packed in plastic boxes with upper and lower diameters of 11.9 cm and 8.5 cm respectively, and starved for 12 hours.

[0033] Counting locust microsporidia using a hemocytometer Paranosema locustae ) mother solution concentration. According to the number of locusts to be treated in the experiment, the corresponding locust microsporidia solution was drawn and centrifuged to obtain locust microsporidia precipitate.

[0034] The locust microsporidia precipitate was resuspended in sterile ultrapure water to prepare a microsporidia suspension, wherein the concentration of locust microsporidia in the microsporidia suspension was 1×10 8 The inoculum size was 200,000 nymphs per locust.

[0035] Four different concentrations of isolate dilutions were used, i.e., the locust microsporidia sediment was resuspended to prepare isolate-microsporidia suspensions. The concentration of locust microsporidia in the isolate-microsporidia suspensions was 1×10 82 μL of isolate dilution, microsporidia suspension, and isolate-microsporidia suspension were evenly applied on corn leaves cut into roughly the same area and scraped, and the corn leaves were fixed with needles and triangular cards to feed 3rd-instar locust nymphs. That is, each locust nymph was inoculated with the same dose of isolate or locust microsporidia. In addition, a sterile ultrapure water negative control group (CK) was set up.

[0036] After 3 hours, the locusts that had eaten all the corn leaves were selected and placed in insect boxes. They were fed with fresh wheat seedlings. Ten successfully inoculated locusts were placed in each insect box. Three insect boxes were used for each treatment, and three biological replicates were set. The death of locusts was observed daily and the mortality rate was calculated, as well as the corrected mortality rate based on the ultrapure water negative control, for a total of 20 days.

[0037] GraphPad software was used to analyze the indoor biological test results of each isolated strain, microsporidia, and the combination of isolated strains and microsporidia, and a survival curve of the East Asian migratory locust nymphs and a bar graph of the mortality rate of the East Asian migratory locust nymphs at different time points were produced.

[0038] Among them, the isolate 1-31 has a more obvious effect on locust microsporidia and is easy to culture. The bar graph of the mortality rate of East Asian migratory locust nymphs after treatment with isolate 1-31, microsporidia, and isolate 1-31 combined with microsporidia is shown in the figure below. Figure 1 As shown in the figure, the inoculum size of isolate 1-31 is 200 colony-forming units / head, and the inoculum size of locust microsporidia is 2×10 5 Pieces / head.

[0039] Figure 1 The results showed that from 0 to 10 days, the lethal effect of isolate 1-31 on the East Asian migratory locust was not significantly different from that of the negative control, that is, it was considered to have basically no toxicity to the East Asian migratory locust, but when it was used in combination with locust microsporidia, it could significantly increase the mortality rate of the East Asian migratory locust, indicating that it has a synergistic effect on locust microsporidia killing the East Asian migratory locust.

[0040] The inoculum size of locust microsporidia was 2 × 10 5 The inoculum size of the isolate in the combination of locust microsporidia and isolate 1-31 was 200 colony-forming units / head, and the inoculum size of locust microsporidia was 2×10 5 In the case of 1000 locusts / head, the toxicity curve and the correlation R value were obtained based on time and locust mortality using SPSS software, where x represents the number of days of the experiment and y represents the mortality of locusts over time. 25 and LT 50 . Virulence curves of locust microsporidia and the combination of locust microsporidia and isolate 1-31, LT25 ,LT 50 And significant Figure 2 .

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

[0042] Colony morphology of 1-31 isolate: 1-31 isolate was cultured upright on beef extract peptone solid medium at 37 degrees Celsius for 2 hours, then inverted for 24 hours. The colony morphology was observed and photographed. Figure 3 .according to Figure 3 It can be seen that the colony shape of the 1-31 isolate is nearly circular, with irregular edges, serrated, slightly raised, slightly concave in the center, white in color, and a smooth, moist, and translucent surface.

[0043] Gram identification of 1-31 isolates: drop a drop of sterile water on the slide, use an inoculation loop to gently pick up the bacterial moss of 1-31 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 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 1-31 isolates - 16S rDNA identification: The total DNA of each isolate was extracted according to the operating procedures of the bacterial DNA genome kit, and 341F (as shown in SEQ ID No. 1) / 806R (as shown in SEQ ID No. 2) was used as primers, and the total DNA of 1-31 isolates 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 Bomadegene for sequencing. Among them, the sequence of the 16S rDNA measured for the 1-31 strain was (as 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 the 1-31 strain was different from Kobe Enterobacter ( Enterobacter kobei ) are closely related. The phylogenetic tree was constructed using MEGA7.0. The phylogenetic tree of strains 1-31 is shown in Figure 5 .according to Figure 5 The phylogenetic tree shows that strain 1-31 is related to Kobe E. Enterobacter kobei ) strains are most closely related.

[0045] In summary, the 1-31 strain was systematically classified as Enterobacter kobeensis Enterobacter kobei .

[0046] The strains 1-31 were deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC No. 31405 and the deposit date 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 Kobe Enterobacter Enterobacter kobei .

[0047] Resistance test of 1-31 isolated strain: Prepare five antibiotics: gentamicin sulfate, working concentration 50 μg / ml; streptomycin sulfate, working concentration 50 μg / ml; kanamycin, working concentration 25 μg / ml; chloramphenicol, working concentration 25 μg / ml; ampicillin, working concentration 50 μg / ml. Prepare resistance plates of beef extract peptone solid culture medium, drop 1-31 bacterial solution in the center of the plate, culture it upright in a 37 degree Celsius incubator for 2 hours, then culture it upside down for 24 hours, observe whether the bacteria can grow, and judge the resistance of the bacteria to different antibiotics. See the results. Figure 6 .according to Figure 6 From the results, it can be seen that it cannot grow on the beef extract peptone solid culture medium plates containing gentamicin sulfate, streptomycin sulfate, kanamycin and chloramphenicol (a, b, c, d respectively), that is, it has no resistance to gentamicin sulfate, streptomycin sulfate, kanamycin and chloramphenicol; it can grow on the beef extract peptone solid culture medium plate containing ampicillin (e), indicating that it is resistant to ampicillin.

Claims

1. A strain of Enterobacter kobeensis ( Enterobacter kobei ) strain 1-31, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No. 31405.

2. An engineered bacterium obtained by genetically modifying the Kobe Enterobacter strain 1-31 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 genes are genes for preventing and controlling plant pests, genes for preventing and controlling plant pathogenic microorganisms, and genes for enhancing the ability of the Kobe Enterobacter strain 1-31 to improve locust microsporidia ( Paranosema locustae ) at least one of the genes that have the effect of controlling locusts.

3. The Kobe Enterobacter strain 1-31 according to claim 1 or the engineered bacteria according to claim 2 is used to improve the locust microsporidia ( Paranosema locustae ) Application in locust control.

4. The use according to claim 3, characterized in that: The locust is the East Asian migratory locust.

5. A composition comprising the Kobe Enterobacter strain 1-31 as claimed in claim 1 or the engineered bacteria as claimed in claim 2, and locust microsporidia ( Paranosema locustae ).

6. The composition according to claim 5, characterized in that The composition also includes an acceptable carrier.

7. The composition according to claim 5, characterized in that The dosage form of the composition is one of a suspension, a powder and a granule.

8. The composition according to claim 5, characterized in that The dosage form of the composition is oil suspension or wettable powder.

9. Use of the composition according to any one of claims 5 to 8 for controlling locusts.

10. The use according to claim 9, characterized in that: The locust is the East Asian migratory locust.

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