A Delft acid-eating bacterium and its application in the decomposition of ASICS#18

Abstract

This invention discloses a strain of Delftia acidovorans and its application in the decomposition of ascaroside #18. The strain is named Delftia acidovorans 31D, with accession number GDMCC No: 63491. This invention is the first to demonstrate that Delftia acidovorans 31D can decompose ascaroside #18. This research provides a reference for improving the production level of ascaroside #18 by plant-parasitic nematodes.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Delft acidophilus and its application in the decomposition of ASICS#18. Background Technology

[0002] Ascarosides are ubiquitous signaling molecules in nematodes, playing a crucial role in regulating their aggregation, avoidance, mating, dauer formation, and dispersal. Various ascarosides, such as ascr#10, ascr#16, ascr#18, ascr#20, ascr#22, and ascr#26, exist in root-knot nematodes. Ascr#18 is found to be the most abundant in both the southern root-knot nematode *Meloidogyne incognita* and the Javan root-knot nematode *M. javanica*. Studies have shown that in plants, ascr#18 can induce signature defense responses, including the expression of MAMP-triggered immune-related genes, mitogen-activated protein kinase, and acid-mediated defense signaling pathways mediated by salicylic acid and jasmonic acid activation, increasing the resistance of Arabidopsis thaliana, tomato, potato, and barley to viruses, bacteria, oomycetes, fungi, and nematodes. The nematode *Caenorhabditis elegans* can metabolize exogenous ascr#10 and ascr#18 into ascr#9. Ascr#9 significantly promotes the spread of the southern root-knot nematode *M. incognita*.

[0003] *Delftia* sp. is a group of Gram-negative bacteria that play an important role in environmental pollution control. Some species have been reported to degrade various pollutants, including p-chloronitrobenzene, terephthalates, nitrite, and chlorimuron-methyl. Current research shows that *D. acidovorans*, an acid-eating bacterium from Delftia, can degrade aniline and microcystins. Furthermore, *D. acidovorans* significantly inhibits *Sclerotium moniliforme*, the causal agent of corn leaf blight, and *Burkholderia bakanae*, exhibiting antifungal activity. Despite numerous studies, no reports have been found regarding its degradation of ascaridone glycosides.

[0004] This invention is the first to discover that *D. acidovorans*, a companion bacterium to the southern root-knot nematode, has the ability to decompose ASICS#18. Summary of the Invention

[0005] The purpose of this invention is to provide a Delftia acidovorans strain capable of decomposing ascaroside ASCRIZ#18. The Delftia acidovorans 31D strain 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 Academy of Sciences, Postcode: 510070, China, Accession No.: GDMCC No.: 63491.

[0006] A second objective of this invention is to provide a microbial agent comprising Delftia acidovorans 31D.

[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 Delftia acidovorans 31D or its inoculum in the decomposition of ascaroside ASICS#18; the structural formula of ASICS#18 is shown below:

[0009]

[0010] Furthermore, it provides the application of Delftia acidovorans 31D or its inoculum in the preparation of products that decompose ascaroside ASTR#18.

[0011] The fourth object of the present invention is to provide a method for decomposing ascaroside ascr#18, which involves using Delftia acidovorans 31D to decompose ascaroside ascr#18.

[0012] The method includes the step of mixing Delftia acidovorans 31D bacterial culture with ascaroside ascr#18.

[0013] Preferably, the bacterial culture is obtained by mixing and culturing Delftia acidovorans 31D 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 *Delftia acidovorans* 31D can decompose ascaroside ASCRIZ#18. This research provides a reference for improving the production level of ascaroside ASCRIZ#18 by plant-parasitic nematodes.

[0017] Delftia acidovorans 31D 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. 63491. Attached Figure Description

[0018] Figure 1 The result is the result of the 16S rDNA sequence of strain 31D being compared with that in 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. Collection of Southern Root-Knot Nematodes:

[0026] Collect root segments of *Calamus stenoptera* infected with southern root-knot nematodes. Soak the segments in 5% sodium hypochlorite solution for 3 minutes, then collect the eggs from a 500-mesh sieve and incubate them with sterile water. Collect the hatched second-instar larvae after 3-5 days. After rinsing with sterile water, adjust the final concentration of the root-knot nematode suspension to 5000 μJ / mL with sterile water for later use.

[0027] 3. Isolation and identification of microorganisms:

[0028] (1) Add 4 mL of root-knot nematode suspension to a 6 cm sterile culture dish, seal the culture dish with sealing film, and place it at 25°C for static culture.

[0029] (2) Collect the nematode fluid 3 days later, take 0.1 mL of the supernatant, and dilute it 10 times and 100 times with sterile water for later use;

[0030] (3) Spread the diluted nematode liquid onto LB, HIA and PDA plates respectively, with an inoculation volume of 50 μL per plate, and streak 3 plates for each culture medium.

[0031] (4) After 3 days, single colonies on the plate were picked and transferred to another plate to obtain strain D31. Single colonies of strain D31 were then identified by PCR using the following primers:

[0032] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0033] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3';

[0034] (5) 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 D31 is Delftia acidovorans.

[0035] Therefore, the strain D31 of this invention belongs to Delftia acidovorans and is named Delftia acidovorans 31D. It 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, with accession number GDMCC No: 63491.

[0036] The 16S rDNA sequencing results are shown in SEQ ID NO.1, specifically:

[0037] TTGCCCTTACACATGCAGTCGAACGGTAACAGGTCTTCGGACGCTGACGAGTGGCGAACGGGTGA

[0038] GTAATACATCGGAACGTGCCCAGTCGTGGGGGATAACTACTCGAAAGAGTAGCTAATACCGCATA

[0039] CGATTCTGAGGATGAAAGCGGGGGACCTTCGGGCCTCGCGCGATTGGAGCGGCCGATGGCAGATTA

[0040] GGTAGTTGGTGGGATAAAAGCTTACCAAGCCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGC

[0041] CACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATG

[0042] GGCGAAAGCCTGATCCAGCAATGCCGCGTGCAGGATGAAGGCCTTCGGGTTGTAAACTGCTTTTG

[0043] TACGGAACGAAAAAGCTTCTCCTAATACGAGAGGCCCATGACGGTACCGTAAGAATAAGCACCGG

[0044] CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGT

[0045] AAAGCGTGCGCAGGCGGTTATGTAAGACAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCAT

[0046] TTGTGACTGCATGGCTAGAGTACGGTAGAGGGGGATGGAATTCCGCGTGTAGCAGTGAAATGCGT

[0047] AGATATGCGGAGGAACACCGATGGCGAAGGCAATCCCCTGGACCTGTACTGACGCTCATGCACGA

[0048] AAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTGGTT

[0049] GTTGGGAATTAGTTTTCTCAGTAACGAAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGCC

[0050] GCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGTTTAATTC

[0051] GATGCAACGCGAAAAACCTTACCCACCTTTGACATGGCAGGAAGTTTCCAGAGATGGATTCGTGC

[0052] TCGAAAGAGAACCTGCACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT

[0053] AAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCTACATTTAGTTGGGCACTCTAATGAGACT

[0054] GCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATAGGTGGGGCT

[0055] ACACACGTCATACAATGGCTGGTACAGAGGGTTCCAACCCGCGAGGGGGAGCTAATCCCATAAA

[0056] ACCAGTCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGC

[0057] GGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGCGGGTCTCGCCAGAAGTAGGTAGCCTAACCGCAAGGAGGGCGCTACCCACGTCCGG.

[0058] 4. Ascaridin decomposition experiment:

[0059] (1) The Delftia acidovorans 31D strain preserved at -80℃ was activated in LB plates, and single colonies of microorganisms were selected and cultured in LB liquid medium at 25℃.

[0060] (2) After 3 days, take 1 mL of bacterial solution and centrifuge at 6000 rpm for 10 min at 10℃. Remove the supernatant, add sterile ultrapure water to wash once, collect the bacterial cells, add sterile ultrapure water to 1 mL, and then dilute with sterile ultrapure water 100 times for later use.

[0061] (3) Add 2 mL of diluted bacterial cells to a 6 cm sterile culture dish, and add 30 μL of 1 μM Mascor#18. Set up a control with 2 mL of diluted bacterial cells and 30 μL of sterile ultrapure water. Repeat each treatment in 3 culture dishes;

[0062] (4) After sealing the petri dish with sealing film, place it in the dark at 25°C for static incubation;

[0063] (5) Collect the liquid from each treatment after 3 days, centrifuge, and filter the supernatant through a 0.22 μm filter membrane into a 15 mL centrifuge tube;

[0064] (6) The above filtrate was frozen overnight at -80℃. The sample was concentrated to dryness using a vacuum freeze dryer and sent to the Guangdong Academy of Sciences Analysis and Testing Institute to detect the concentration of ascr#18.

[0065] 5. Results of ascaroside decomposition:

[0066] The results showed that the content of ascr#18 in Delftia acidovorans 31D was significantly lower than that in the control (sterile ultrapure water), indicating that Delftia acidovorans 31D has the function of decomposing ascr#18 (Table 1).

[0067] Table 1. Content of ASTR#18

[0068]

[0069] 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. Delftia acidovorans 31D, with accession number GDMCC No: 63491.

2. A microbial agent, characterized in that, It contains Delftia acidovorans 31D as described in claim 1.

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

4. The application of Delftia acidovorans 31D as described in claim 1 or the bacterial agent as described in 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, The Delftia acidovorans 31D strain described in claim 1 is used to decompose ascaroside ascr#18.

7. The method according to claim 6, characterized in that, The step includes mixing the Delftia acidovorans 31D bacterial solution as described in claim 1 with ascaroside ascr#18.

8. The method according to claim 7, characterized in that, The bacterial culture was obtained by mixing Delftia acidovorans 31D with LB liquid medium.

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

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