Xenorhabdus nematophila and application thereof in decomposing ascr#18

Abstract

The application discloses a Xenorhabdus nematophila strain and application thereof in decomposition of ascr#18. The strain is named Xenorhabdus nematophila A11, and the preservation number is GDMCC No: 63492. The Xenorhabdus nematophila A11 is found to have the effect of decomposing ascarid ascr#18 for the first time. The research provides a reference for improving the ascarid ascr#18 level of plant parasitic nematodes.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a nematode-pathogenic bacillus and its application in the decomposition of ASTR#18. Background Technology

[0002] Ascarosides are ubiquitous signaling molecules in nematodes, playing a crucial role in regulating various biological behaviors. The southern root-knot nematode *Meloidogyne incognita* contains abundant ascr#18. Studies have shown that ascr#18 can induce enhanced resistance in plants to viruses, bacteria, fungi, and nematodes. Furthermore, *Caenorhabditis elegans* can metabolize exogenous ascr#10 and ascr#18 into ascr#9. Ascr#9 significantly promotes the dispersal of *M. incognita*.

[0003] The symbiotic bacterium *Xenorhabdus nematophila*, belonging to the phylum *Proteobacteria*, is a Gram-negative bacterium that supports *St. carpocapsae*. It can inhibit the hatching of *S. southern root-knot nematode* eggs and promote the spread of the nematode. Although there are numerous research reports on its role in controlling *S. southern root-knot nematode*, there are no reports on its effect on ascaridin degradation.

[0004] This invention is the first to discover that the nematopathogenic bacillus Xenorhabdus nematophila has the ability to decompose ascr#18. Summary of the Invention

[0005] The purpose of this invention is to provide a nematophila-pathogenic bacillus strain capable of decomposing ascaroside ASCRIZ#18. The nematophila-pathogenic bacillus (Xenorhabdus nematophila) A11 provided by this invention was deposited on May 23, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China (Guangdong Academy of Sciences Institute of Microbiology), Guangdong Province, China, accession number: GDMCC No: 63492.

[0006] A second object of the present invention is to provide a microbial agent comprising Xenorhabdus nematophila A11.

[0007] Preferably, the microbial agent further comprises excipients that can prolong the activity time of the strain, or other excipients acceptable to microbial agents.

[0008] A third objective of this invention is to provide the application of Xenorhabdus nematophila A11 or its agent in the decomposition of ascaroside ascr#18; the structural formula of ascr#18 is shown below:

[0009]

[0010] Furthermore, it provides the application of Xenorhabdus nematophila A11 or its agent in the preparation of products that decompose ascaroside ascr#18.

[0011] The fourth object of the present invention is to provide a method for decomposing ascaroside ascr#18, which involves using Xenorhabdus nematophila A11 to decompose ascaroside ascr#18.

[0012] The method includes the step of mixing a cyclophosphamide (Xenorhabdus nematophila) A11 bacterial solution with ascaroside ascr#18.

[0013] Preferably, the bacterial culture is obtained by mixing and culturing Xenorhabdus nematophila A11 with LB liquid medium.

[0014] A fifth objective of this invention is to provide the application of the above-described method in the decomposition of ascaroside ascr#18.

[0015] The present invention has the following beneficial effects:

[0016] This invention is the first to discover that *Xenorhabdus nematophila* A11 has the ability to decompose ascaroside ASTR18. This research provides a reference for improving the production level of ascaroside ASTR18 by plant-parasitic nematodes.

[0017] Xenorhabdus nematophila A11 was deposited on May 23, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China. The accession number is GDMCC No. 63492. Attached Figure Description

[0018] Figure 1 The result is the alignment of the 16S rDNA sequence of strain A11 with NCBI. Detailed Implementation

[0019] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0020] The ascaroside ascr#18 in the following examples can be obtained through artificial synthesis or commercial purchase.

[0021] Example 1

[0022] 1. Synthesis of ascaroside standard:

[0023] ascr#18 was synthesized in WuXi AppTec with a purity greater than 95%, and its specific structural formula is as follows:

[0024]

[0025] 2. Microbial identification:

[0026] (1) Strain A11 was derived from laboratory preservation;

[0027] (2) Remove strain A11 from -80℃, activate it on LB plates, and after 3 days, pick a single colony from the plate for PCR identification. The identification primers are as follows:

[0028] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0029] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3';

[0030] (3) The PCR product was sent to a biotechnology company for testing. The 16S rDNA sequence is shown in SEQ ID NO.1. The obtained sequence was compared with NCBI, and the results are shown below. Figure 1 This confirmed that strain A11 was X. nematophila.

[0031] Therefore, strain A11 of the present invention belongs to Xenorhabdus nematophila and is named Xenorhabdus nematophila A11. It was deposited on May 23, 2023 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 63492.

[0032] The 16S rDNA sequencing results are shown in SEQ ID NO.1, specifically:

[0033] GGGGGGGGGGCCTAACACATGCAAGTCGGACGGTAACAGGAAACAGCTTGCTGTTTTGCTGACAG

[0034] TGGCGGACGGGTGAGTAATGTCTGGGGATCTGCCCGATGGAGGGGGATAACCACTGGAAACGGTG

[0035] GCTAATACCGCATGACCTCTTGGGAGTAAAGTGGGGGACCTTCGGGCCTCACGCCATCGGATGAA

[0036] CCCAGATGGGATTAGCTGGTAGGCGGGGTAATGGCCCACCTAGGCGACGATCCCTAGCTGGTCTG

[0037] AGAGGATGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGG

[0038] GAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGT

[0039] TGTAAAGTACTTTCAGCGGGGAGGAAGGCGTAAGTCTGAACAGGGCTTACGATTGACGTTACCCG

[0040] CAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATC

[0041] GGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCAATTAAGTTGGATGTGAAATCCCCGGGCTTA

[0042] ACCCGGGAACGGCATCCAAGACTGGTTGGCTAGAGTCTCGTAGAGGGGGGTAGAATTCCACGTGT

[0043] AGCGGTGAAATGCGTAGAGATGTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGAC

[0044] TGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAA

[0045] ACGATGTCGATTTGGAGGCTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCG

[0046] CCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCTGTGGA

[0047] GCATGTGATTTATTTCGATGCAACGCGAAGACCTTACCTACTCTTGACATCCACGGATCAGGCTGA

[0048] GATGCCCGAGTGCATCGGCATCGTGAGACAGTGCTGCATGCTGTCGTCAGCTCGTGTGTGAATGTT

[0049] GGCTTAAGTCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCACGTGATGGTGGGAACTCAAG

[0050] GGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAG

[0051] TAGGGCTACACACGTGCTACAATGGCAGATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGAA

[0052] CTCATAAAGTCTGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAG

[0053] TAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGGGGGCGCTACCCCGAGGGTA。

[0054] 3. Ascaroside decomposition experiment:

[0055] (1) Take the Xenorhabdus nematophila strain A11 preserved at -80℃ and activate it in LB plates. Select single colonies of microorganisms and culture them in LB liquid medium at 25℃.

[0056] (2) After 3 days, take 1 mL of bacterial culture and centrifuge at 6000 rpm for 10 min at 10℃, and discard the supernatant;

[0057] (3) Add 1 mL of sterile ultrapure water, mix well, centrifuge and discard the supernatant;

[0058] (4) Collect the bacterial cells, add sterile ultrapure water to 1 mL, and then dilute with sterile ultrapure water 100 times for later use;

[0059] (5) Take 2 mL of diluted bacterial cells and add them to a 6 cm sterile culture dish, then add 30 μL of 1 μMascr#18.

[0060] (6) Use 2 mL of diluted bacterial cells and 30 μL of sterile ultrapure water as controls. Each treatment was repeated 3 times;

[0061] (7) After sealing the petri dish with sealing film, incubate it statically in the dark at 25°C;

[0062] (8) After 3 days, the liquid from each treatment was collected, centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane;

[0063] (9) The above filtrate was frozen in a -80°C freezer. After the sample was concentrated and dried by a vacuum freeze dryer, it was sent to the Guangdong Academy of Sciences Institute of Analysis and Testing to detect the concentration of ascr#18.

[0064] 4. Results of ascaroside decomposition:

[0065] The results showed that the content of ascr#18 in Xenorhabdusnematophila A11 was significantly lower than that in the control (sterile ultrapure water), indicating that Xenorhabdusnematophila A11 has the function of decomposing ascr#18 (Table 1).

[0066] Table 1. Content of ASTR#18

[0067]

[0068] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Nematophyte pathogenic bacilli ( Xenorhabdus nematophila )A11, its accession number is: GDMCC No: 63492.

2. A microbial agent, characterized in that, Contains the nematode pathogenic bacillus as described in claim 1 ( Xenorhabdus nematophila )A11.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent also includes excipients acceptable to the bacterial agent.

4. The nematode-pathogenic bacillus described in claim 1 ( Xenorhabdus nematophila The application of the bacterial agent according to claim A11 or claim 2 in the decomposition of ascaroside ASCRIZ#18; the structural formula of ASCRIZ#18 is shown below: 。 5. The application according to claim 4, characterized in that, It is used in the preparation of products that decompose ascaroside ASCRI#18.

6. A method for decomposing ascaroside ASCRIZ#18, characterized in that, It is the nematode pathogenic bacillus described in claim 1 ( Xenorhabdus nematophila A11 is used to decompose ascaroside ascr#18.

7. The method according to claim 6, characterized in that, Includes the nematode pathogenic bacillus described in claim 1 ( Xenorhabdus nematophila The steps for mixing A11 bacterial solution with ascaroside ascr#18.

8. The method according to claim 7, characterized in that, The bacterial solution is a solution containing nematode-pathogenic bacilli (Bacillus nematodes). Xenorhabdus nematophila A11 was obtained by mixing and culturing with LB liquid medium.

9. The application of the method of claim 6 in the decomposition of ascaroside ascr#18.

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