CN113337448B

CN113337448B - Citrobacter and application thereof in preventing and treating scarab beetles

Info

Publication number: CN113337448B
Application number: CN202110851414.5A
Authority: CN
Inventors: 耿丽丽; 束长龙; 张�杰; 徐文玥; 米良
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2021-07-27
Filing date: 2021-07-27
Publication date: 2022-04-19
Anticipated expiration: 2041-07-27
Also published as: CN113337448A

Abstract

The invention relates to a Citrobacter and application thereof in preventing and treating scarab beetle. The Citrobacter is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 22672.

Description

Citrobacter and application thereof in preventing and treating scarab beetles

Technical Field

The invention relates to a Citrobacter, in particular to a Citrobacter citrobacter which can be used for preventing and treating scarab beetles.

Background

Citrobacter (Citrobacter), also known as Citrobacter, was first introduced in 1932 and belongs to the family Enterobacteriaceae. The citrobacter is about 1.0 μm in diameter and 2.0-6.0 μm in length, is a gram-negative, rod-shaped, nonfilamentous facultative anaerobe which utilizes citrate as a carbon source to ferment glucose and carbohydrates and produce acid and gas. With the development of DNA hybridization technology, Citrobacter species have been developed from 3 original species to 11 species at present, and Citrobacter species are known to grow optimally in an environment at 37 ℃, are commonly found in water, soil, esophagus and intestine of animals and humans, and although some strains may cause urinary tract infection, sepsis and infant meningitis, they are not considered as important pathogens of human diseases, but are considered as conditional pathogens. The citrobacter has the capacity of adsorbing Cd2+ and heavy metal uranium, and the adsorption capacity is influenced by pH, thallus concentration and proton concentration. Citrobacter bacteria are enriched in heavy metals by some enzymes releasing phosphate groups and then synthesizing the phosphate form. Yanliping et al separates a Citrobacter with sulfate reducing function under anaerobic and micro-aerobic conditions, and utilizes a specific gene primer with sulfate reducing function to amplify to a dissimilatory sulfite reductase gene. Citrobacter with alkane degradation function was isolated by Sunpiki et al.

More beneficial functions of Citrobacter are yet to be developed.

Disclosure of Invention

One of the invention provides a Citrobacter sp which is preserved in the common microorganism center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 22672.

The second invention provides an engineering bacterium obtained by genetically modifying the citrobacter according to the first invention. Wherein, the engineering bacterium is the citric acid bacillus CGMCC No.22672 as a target, and the adopted means is generally to transfer and/or knock out specific genes and/or DNA fragments and the like into the engineering bacterium, so the engineering bacterium is still the citric acid bacillus. In addition, the engineering bacteria can be engineering bacteria with improved activity on scarab pests. Engineered strains that have other pesticidal activities and/or are endowed with other beneficial properties may also be used.

In a specific embodiment, the engineered bacterium is obtained by transforming the Citrobacter with a plasmid carrying an anti-pathogenic and/or anti-insect gene.

The third invention provides a composition, which comprises the citric acid bacillus as described in the first invention or the engineering bacteria as described in the second invention, and an acceptable carrier.

The fourth invention provides the application of the citrobacter according to the first invention, the engineering bacteria according to the second invention or the composition according to the third invention in preventing and treating scarab beetle (Scarabaeidae).

In a specific embodiment, the chafer is a gill-chafer (Holotrichia) and/or a star-chafer (Protaetia).

In a specific embodiment, the gill-gold (Holotrichia) is a Holotrichia parallela (Holotrichia parallela) and/or a Holotrichia magna (Holotrichia oblita); the scarab beetle (Protaetia) is a Chrysomya Cineralis (Protaetia brevitarsis).

In a specific embodiment, the chafer (Scarabaeidae) is a larval stage chafer.

In a specific embodiment, the gill-metal turtle (Holotrichia) is a larval stage gill-metal turtle (Holotrichia); the star-flower scarab (Protaetia) is larval-stage star-flower scarab (Protaetia).

In a specific embodiment, the Holotrichia parallela is a larval stage Holotrichia parallela; the Holotrichia parallela (Holotrichia oblite) is a larval-stage Holotrichia parallela (Holotrichia oblite); the Chrysomya megacephala (Protaetia brevitarsis) is larva stage Chrysomya megacephala (Protaetia brevitarsis).

The invention has the beneficial effects that:

the invention discovers that the Citrobacter exists in the bacterial strain with insecticidal activity to scarab beetle for the first time. Not only does this broaden the application of Citrobacter, but also broadens the means for preventing and treating scarab beetle. In addition, the citric acid bacillus has insecticidal activity on scarab for the first time, and belongs to a different genus from known microorganisms such as bacillus thuringiensis and the like on a classification position, so that the insecticidal mechanism is different from that of the insecticidal microorganisms or insecticidal substances in the prior art, and the insecticidal activity is beneficial to pest resistance treatment.

Drawings

FIG. 1 is a 16S rDNA-based phylogenetic tree of the IPPBiotC41 strain.

Strain preservation

The filtered citrobacter is named IPPBiotC41, and the strain is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.22672, the preservation date of 2021, 06 months and 07 days, and the preservation addresses are as follows: the institute of microbiology, national academy of sciences No.3, Xilu No.1, Beijing, Chaoyang, Beijing. Its system is classified as Citrobacter sp.

Detailed Description

The above-described aspects of the invention are explained in more detail below by means of preferred embodiments, but they are not intended to limit the invention.

The reagents in the examples of the present invention were all commercially available unless otherwise specified.

The primer is synthesized by the synthesis part of Beijing division of biological engineering, the sequencing is completed by the open laboratory of the national important scientific engineering of crop gene resources and gene improvement of Chinese agricultural academy of sciences, and biochemical and molecular biological reagents are all commercially available and analytically pure.

TSB medium was purchased from sigma under the accession number T8907-1 KG.

Example 1

Isolation and Classification of strains

1.1 peanut planting in the field and grub inoculation

The method is characterized in that 1 mu of land is selected for planting peanuts in a test field of an experimental base of a Hebei gallery, and five points are selected before sowing to deeply bury a 304 stainless steel net cage in the field so as to limit the grubs inoculated subsequently to spread to the periphery. When sowing, 3 peanut seeds are sown in each hole, and water is poured once every 3 days. Inoculating the third-instar larvae of Holotrichia parallela which are subjected to starvation treatment for 48 hours in the pod bearing period of the peanuts, taking samples 7 days after the Holotrichia parallela larvae are inoculated, and repeatedly collecting peanut rhizosphere soil for 3 points in each net cage.

1.2 Collection and treatment of rhizosphere soil in field

The peanut rhizosphere soil is collected and processed as follows:

taking out the peanut root system completely, shaking off non-rhizosphere soil on the peanut root, only leaving rhizosphere soil with a distance of less than 1mm from the root surface, shearing off the peanut root system with the rhizosphere soil by using sterile scissors, putting the root system into a sterile 50mL centrifuge tube, adding 1 XPBS buffer solution until the whole peanut root system is submerged, soaking and standing for 15min, carrying out vortex oscillation for 15S, taking out the peanut root system, filtering out fibrous roots, impurities and the like in rhizosphere soil suspension by using a 100-mesh sieve, centrifuging for 3200g, 15min, removing part of supernatant, and suspending centrifugal precipitate (rhizosphere soil) again by using the left 5mL of supernatant to obtain rhizosphere soil suspension. The rhizosphere soil suspension was used for isolation of rhizosphere bacteria.

1.3 isolation of the rhizosphere culturable bacteria

Referring to the article by Bai et al (Bai et al, 2015), culturable bacteria in rhizosphere soil suspension were isolated using TSB rhizosphere bacteria isolation medium: diluting the rhizosphere soil suspension by 10 times in a gradient way, and selecting and diluting 10 times⁷、10⁸、10⁹Coating with three gradients, eachThe suspension was repeatedly smeared with 40. mu.L of soil suspension and incubated in a 30 ℃ incubator for 1 day, 1 plate counted as 1 replicate, with 3 replicates per gradient. After one week, dilution to 10 was found³In the process, the amount of colonies growing on each flat plate is large and dense, and a single colony is difficult to pick for secondary culture; diluting to 10⁵When the number of colonies grown on each plate was too small; only diluting to 10⁴At this time, the number of colonies grown on each plate was between 100 and 300, and therefore the dilution gradient was selected to pick colonies. 300 single colonies were picked and subjected to purification culture using a TSB plate, and thus purification culture was performed 3 times to obtain purified isolates.

Each isolate was numbered.

Example 2

Biological activity assay

(1) Determination of biological activity of Holotrichia parallela larvae

A monoclonal antibody numbered IPPBiotC41 was inoculated into a test tube containing 5mL of a liquid LB medium (tryptone 10.0g/L, yeast extract 5.0g/L, NaCl 10.0g/L, sterilized at 121 ℃ for 20min), activated at 30 ℃ for 12 hours, the activated bacterial solution was transferred to a solid LB medium (LB liquid medium plus agar 15g/L, dish diameter 15cm), cultured at 30 ℃ for 12 hours, collected with a sterile spatula, uniformly suspended with PBS buffer, and the suspended cells were subjected to plate counting. The counted cells were sonicated (Ampl 70%, pulse 3s, stop 5s) to release total protein from the cells, yielding a disrupted bacterial solution.

Preparing carrot shreds, washing the carrot shreds with clear water, and airing until the surface is free of moisture. Soaking carrot shreds in the crushed bacteria solution for about 20min, taking out, uniformly placing in 6-hole bioassay plates with 4-5 strips per hole, mixing the rest bacteria solution with 60g of soil, and measuring the final concentration of 1 × 10⁸And 1X 10⁹cfu/g. And uniformly subpackaging the mixed soil into 6-hole bioassay plates with corresponding concentrations, inoculating 1 larva of the 2-day old Holotrichia parallela which is hatched initially into each hole, and feeding the larva in an incubator at the temperature of 25 ℃ and under the illumination of L: D ═ 16: 8. Each repeat was 20, 5 repeats. Buffered with PBSThe wash served as a negative control. After 7 days, the number of dead and live insects was investigated, and the average mortality and corrected mortality were calculated.

The results are shown in Table 1.

TABLE 1 measurement of biological Activity of IPPBiotC41 on Holotrichia parallela larvae

Bioassay samples (cfu/g)	Average mortality	Correcting mortality
			1×10⁸	59％	51.65％
1×10⁹	70％	64.6％
			Negative control	15.3％	—

From the results in table 1, IPPBiotC41 was found to have insecticidal activity against larvae of holotrichia parallela.

(2) Determination of biological activity of Holotrichia parallela larvae

The bioassay concentration was set to 1X 10⁶、1×10⁷、1×10⁸And 1X 10⁹cfu/g, replacing the larvae which are hatched for 2 days at the beginning of the Holotrichia parallela with the larvae which are hatched for 2 days at the beginning of the Holotrichia parallela in the biological activity determination object. Otherwise, the same as the section (1) of this embodiment.

The results are shown in Table 2.

TABLE 2 measurement of biological Activity of IPPBiotC41 on Holotrichia parallela larvae

Bioassay samples (cfu/g)	Average mortality	Correcting mortality
			1×10⁶	8.3％	2.9％
1×10⁷	19.4％	14.6％
			1×10⁸	30.6％	26.5％
1×10⁹	63.9％	61.8％
			Negative control	5.6％	—

From the results in table 2, IPPBiotC41 was found to have insecticidal activity against larvae of Holotrichia parallela.

(3) Determination of biological activity of platysternon megacephalum

A monoclonal antibody numbered IPPBiotC41 was inoculated into a test tube containing 5mL of a liquid LB medium (tryptone 10.0g/L, yeast extract 5.0g/L, NaCl 10.0g/L, sterilized at 121 ℃ for 20min), activated at 30 ℃ for 12 hours, the activated bacterial solution was transferred to a solid LB medium (LB liquid medium plus agar 15g/L, dish diameter 15cm), cultured at 30 ℃ for 12 hours, collected with a sterile spatula, uniformly suspended with PBS buffer, and the suspended cells were subjected to plate counting. The counted cells were diluted to 1X 10 with PBS buffer⁹cfu/mL, and ultrasonication (Ampl 70%, pulse 3s, stop 5s) was performed to release total protein from the cells, resulting in a disrupted bacterial solution.

The three instar platysternon leucocephalus larvae are placed on ice and slightly frozen to be inactive. And sucking a proper amount of the resuspended bacteria liquid by using a syringe, inserting the needle of the syringe into a mouth device to be injected with the insects, and injecting 50 mu L of the mixture. The injected larvae were placed in a dark box containing appropriate amount of feed, 7 per group, 3 replicates. PBS buffer was used as negative control. After culturing for 7d in an incubator at 25 ℃, the number of dead and live insects is investigated, and the mortality is calculated.

The results are shown in Table 3.

TABLE 3 measurement of biological Activity of IPPBiotC41 on Scarabaeus albus larvae

From the results in table 3, IPPBiotC41 was found to have insecticidal activity against larvae of platysternon leucocephala.

(3) Determination of the biological Activity of the California gibsonii

A single clone, designated IPPBiotC41, was inoculated into a medium containing 5mL of liquid LB medium (tryptone 10.0g/L, yeast extract 5.0g/L, NaCl 10.0g/L, 121 ℃ C.)Sterilized for 20min), cultured at 30 ℃ for 12 hours for activation, then the bacterial solution obtained by activation is transferred to a solid LB medium (LB liquid medium plus agar 15g/L, dish diameter 15cm), cultured at 30 ℃ for 12 hours, collected with a sterile spatula, evenly suspended with PBS buffer, and the suspended bacteria are counted on a plate. The counted cells were diluted to 1X 10 with PBS buffer⁹cfu/mL, and ultrasonication (Ampl 70%, pulse 3s, stop 5s) was performed to release total protein from the cells, resulting in a disrupted bacterial solution.

A9 cm dish was filled with 9cm of sterilized qualitative filter paper, and wet-sprayed with sterilized distilled water. Cleaning Chinese cabbage leaves, and air drying. Taking 10mL of the crushed bacterial liquid, adding 100 mu L of 10% detergent into the bacterial liquid, uniformly mixing, soaking cabbage leaves, then airing the cabbage leaves, putting the cabbage leaves into a culture dish, inoculating the preliminarily hatched larvae of the simian diabrotica by using a writing brush, wherein 30 insects are inoculated in each dish, strictly sealing the inoculated insects by 2 layers of toilet paper, and repeating each group for three times. PBS buffer was used as negative control. Culturing in 25 deg.C incubator for 48 hr, investigating dead and live insect number, and calculating mortality.

The results are shown in Table 4.

TABLE 4 measurement of the biological Activity of IPPBiotC41 on Calicium Browns

Raw test sample	Average mortality	Correcting mortality
			IPPBiot41	10.0％	3.6％
Negative control	6.7％	—

From the results in table 4, IPPBiotC41 was found to have no insecticidal activity against the larvae of the great ape beetle.

Example 3

Identification of IPPBiotC41 Strain

Extraction of gram-negative bacteria from reference molecular clones the method described for extraction of IPPBiotC41 genomic DNA. With bacterial 16S rDNA universal primers: 27F (shown as SEQ ID No. 1) and 1492R (shown as SEQ ID No. 2) amplify the 16S rDNA sequence of IPPBiotC41 to obtain a multiple fragment of about 1.4 kb. TA cloning is carried out by purifying the kit, a clone obtained by transforming escherichia coli is subjected to bacterial liquid PCR verification and then is sent to a company Limited in biological engineering (Shanghai) for sequencing, and the obtained sequence is 1404bp (shown as SEQ ID No. 3). The sequences were submitted to the ezbiocoud website for homology comparison and a phylogenetic tree was constructed using MEGA6.0 (figure 1). It can be seen from the phylogenetic tree of FIG. 1 that it is closely related to Citrobacter braakii strain of Citrobacter, and therefore the IPPBiotC41 system of the present invention is classified as Citrobacter sp.

The IPPBiotC41 strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.22672, the preservation date of 2021, 06 months and 07 days, and the preservation addresses are as follows: the institute of microbiology, national academy of sciences No.3, Xilu No.1, Beijing, Chaoyang, Beijing. Its system is classified as Citrobacter sp.

Sequence listing

<110> institute of plant protection of Chinese academy of agricultural sciences

<120> Citrobacter and application thereof in preventing and treating scarab beetles

<130> LHA2160376

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

agagtttgat cctggctcag 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

cggttacctt gttacgactt 20

<210> 3

<211> 1404

<212> DNA

<213> Citrobacter sp

<400> 3

gcaagtcgaa cggtaacagg aagcagcttg ctgctttgct gacgagtggc ggacgggtga 60

gtaatgtctg ggaaactgcc cgatggaggg ggataactac tggaaacggt agctaatacc 120

gcataacgtc gcaagaccaa agagggggac cttcgggcct cttgccatcg gatgtgccca 180

gatgggatta gcttgttggt gaggtaacgg ctcaccaagg cgacgatccc tagctggtct 240

gagaggatga ccagccacac tggaactgag acacggtcca gactcctacg ggaggcagca 300

gtggggaata ttgcacaatg ggcgcaagcc tgatgcagcc atgccgcgtg tatgaagaag 360

gccttcgggt tgtaaagtac tttcagcggg gaggaagggg ttaaggttaa taaccttatt 420

cattgacgtt acccgcagaa gaagcaccgg ctaactccgt gccagcagcc gcggtaatac 480

ggagggtgca agcgttaatc ggaattactg ggcgtaaagc gcacgcaggc ggtctgtcaa 540

gtcggatgtg aaatccccgg gctcaacctg ggaactgcat tcgaaactgg caggcttgag 600

tctcgtagag gggggtagaa ttccaggtgt agcggtgaaa tgcgtagaga tctggaggaa 660

taccggtggc gaaggcggcc ccctggacga agactgacgc tcaggtgcga aagcgtgggg 720

agcaaacagg attagatacc ctggtagtcc acgccgtaaa cgatgtctat ttggaggttg 780

tgcccttgag gcgtggcttc cggagctaac gcgttaaata gaccgcctgg ggagtacggc 840

cgcaaggtta aaactcaaat gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 900

taattcgatg caacgcgaag aaccttacct ggtcttgaca tccacagaac ttggcagaga 960

tgccttggtg ccttcgggaa ctgtgagaca ggtgctgcat ggctgtcgtc agctcgtgtt 1020

gtgaaatgtt gggttaagtc ccgcaacgag cgcaaccctt atcctttgtt gccagcggtc 1080

cggccgggaa ctcaaaggag actgccagtg ataaactgga ggaaggtggg gatgacgtca 1140

agtcatcatg gcccttacga ccagggctac acacgtgcta caatggcata tacaaagaga 1200

agcgacctcg cgagagcaag cggacctcat aaagtatgtc gtagtccgga ttggagtctg 1260

caactcgact ccatgaagtc ggaatcgcta gtaatcgtgg atcagaatgc cacggtgaat 1320

acgttcccgg gccttgtaca caccgcccgt cacaccatgg gagtgggttg caaaagaagt 1380

aggtagctta accttcggga gggc 1404

Claims

1. A Citrobacter sp is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 22672.

2. An engineered bacterium obtained by genetically modifying the Citrobacter according to claim 1.

3. The engineered bacterium of claim 2, wherein the engineered bacterium is obtained by transforming the Citrobacter bacterium with a plasmid carrying a disease-resistant gene and/or a pest-resistant gene.

4. A composition comprising the citrobacter according to claim 1 or the engineered bacterium according to claim 2 or 3, and an acceptable carrier.

5. Use of the Citrobacter according to claim 1, the engineered bacterium according to claim 2 or 3, or the composition according to claim 4 for controlling Tortoise (Scarabaeidae), which is a larval stage Tortoise.

6. Use according to claim 5, wherein the chafer is a gill-tortoise (Holotrichia) and/or a platysternon megacephalum (Protaetia).

7. The use according to claim 6, wherein the Holotrichia parallela (Holotrichia parallela) is Holotrichia parallela (Holotrichia parallela) and/or Holotrichia parallela (Holotrichia oblita); the scarab beetle (Protaetia) is a Chrysomya Cineralis (Protaetia brevitarsis).