Yanagimorpha sp. and its use in decomposing ascr#18

Abstract

The application discloses a Sphingobium yanoikuyae and application thereof in decomposing ascr#18. The Sphingobium yanoikuyae 25D is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on May 10, 2023, and the address of the GDMCC is the 5th floor of the building 59, the courtyard 100, the Middle of the Martyrs, Guangzhou, the postal code of the Guangdong Academy of Microbiology is 510070, and the preservation number is GDMCC No: 63454. It is found for the first time that the Sphingobium yanoikuyae 25D is a southern root-knot nematode accompanying bacterium and has the function of decomposing ascr#18.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a type of Sphingosine monocytogenes and its application in the decomposition of ASTR#18. Background Technology

[0002] Ascarosides are a class of pheromone substances composed of side chains of fatty acids linked to dideoxycarboxylic acid groups. Different types of ascarosides can be formed by altering the length of the side chains and different derivatives. They are ubiquitous in nematodes and can regulate various important biological activities such as foraging, mating, and aggregation. Currently, several ascarosides have been detected in *Strombus haemolyticus*, such as ascr#10, ascr#16, ascr#18, ascr#20, ascr#22, and ascr#26. Studies have shown that ascr#18 can induce plant defense responses and enhance plant immunity against plant-parasitic nematodes. Furthermore, both monocotyledonous and dicotyledonous plants can rapidly convert ascr#18 into the short-chain ascr#9. Ascr#9 has a repulsive effect on plant nematodes. Therefore, when Ascr#9 is excreted into the plant rhizosphere, it can act as a chemical signal to regulate the interaction between plants and nematodes, thereby reducing the infection rate of root-knot nematodes on plants.

[0003] Sphingobium yanoikuyae is a Gram-negative bacterium capable of degrading various polycyclic aromatic heterocyclic pollutants, such as carbazole, dibenzofuran, and dibenzothiophene. However, its degradation of ascaroside has not been reported. Summary of the Invention:

[0004] The first objective of this invention is to provide a *Sphingobium yanoikuyae* 25D strain that has the ability to decompose ASTR#18.

[0005] Sphingobium yanoikuyae 25D was deposited on May 10, 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. The accession number is GDMCC No: 63454.

[0006] A second objective of this invention is to provide a microbial agent containing the aforementioned *Sphingosine monocytogenes* 25D as an active ingredient.

[0007] A third objective of this invention is to provide the role of the aforementioned *Sphingosine monocytogenes* 25D in the decomposition of ascaroside.

[0008] Preferably, the ascaroside is ascr#18, and its structural formula is as follows:

[0009]

[0010] The fourth object of the present invention is to provide a method for decomposing ascaroside, which utilizes the above-mentioned Sphingosine Yanoide 25D to decompose ascaroside.

[0011] This invention is the first to discover that Sphingobium yanoikuyae 25D is an accompanying bacterium of southern root-knot nematodes and has the function of decomposing ascr#18.

[0012] Sphingobium yanoikuyae 25D was deposited on May 10, 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. The accession number is GDMCC No: 63454. Attached Figure Description

[0013] Figure 1 The sequencing results were compared with those of the species with the highest similarity in NCBI, which was Delftia acidovoranstrain JCM similarity. Detailed Implementation

[0014] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0015] Example 1

[0016] 1. Collection of Southern Root-Knot Nematodes:

[0017] Roots of watermelons severely infested with southern root-knot nematodes were collected. After washing the surface of the roots with tap water to remove mud and sand, the roots were cut into 1-2 cm sections. The roots were then soaked in a 5% sodium hypochlorite solution for 3 minutes to dissolve the nematode egg sacs and release the eggs. The eggs were washed with sterile water and collected from a 500-mesh sieve, then incubated at 26°C. After 3-5 days, the hatched second-instar larvae were collected, and the concentration was adjusted to 5000 IJs / mL for later use.

[0018] 2. Microbial isolation:

[0019] Sample collection:

[0020] (1) Take 4 mL of Southern Root-knot Nematode solution with a concentration of 5000 IJs / mL into a 6 cm sterile culture dish, seal it with sealing film and place it in a static culture at 26℃.

[0021] (2) After 3 days, collect the nematode fluid into a 15mL centrifuge tube and let it stand for 3 hours;

[0022] (3) Take 0.1 mL of supernatant into a 2 mL centrifuge tube and dilute it 10 times and 100 times with sterile water in sequence;

[0023] (4) Spread the diluted supernatant onto LB, HIA and PDA plates, with 50 μL of supernatant spread on each plate. Three plates were made for each culture medium. Incubate at 26°C.

[0024] (5) After 3 days, single colonies were picked from LB, HIA, and PDA plates for numbering, transfer, identification, and preservation (preserved in 15% glycerol at -80°C); the primers required for identification are as follows:

[0025] 27F(5,-AGAGTTTGATCCTGGCTCAG-30);

[0026] 1492R(5,-TACGGYTACCTTGTTACGACTT-30);

[0027] This yielded strain 25D.

[0028] (6) The PCR products were sent to a biotechnology company for testing, and the obtained sequences were compared with the NCBI database. The 16S rDNA sequence is shown in SEQ ID NO.1. The sequencing results were compared with the species with the highest similarity in NCBI database: *Delftia acidovoran* strain JCM. Similarity: 99.85% ( Figure 1 ).

[0029] Therefore, strain 25D of this invention belongs to Sphingobium yanoikuyae and is named Sphingobium yanoikuyae 25D. It was deposited on May 10, 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, with accession number GDMCC No: 63454.

[0030] 3. Experiments on the effects of Ascr#18 on microorganisms:

[0031] Sphingosine monocytogenes 25D strain, stored at -80℃, was activated and expanded using LB liquid medium, cultured on a shaker at 26℃. After 3 days, 1 mL of Sphingosine monocytogenes 25D bacterial suspension was centrifuged at 10℃ for 10 min, washed once with a sterile container, and brought to a final volume of 1 mL. The washed bacterial suspension was then diluted 10-fold and 100-fold with sterile water. 2 mL of the 100-fold diluted bacterial suspension and 30 μL of 1 μM ASICS (i.e., ASICS concentration of 5 μg / L in the bacterial suspension) were added to a 6 cm sterile culture dish. A control was prepared by adding 2 mL of sterile water and 30 μL of 1 μM ASICS (i.e., ASICS concentration of 5 μg / L in the water). Each treatment was repeated three times. After 3 days, the bacterial suspension was collected, centrifuged at 10℃ for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. The filtrate was stored at -80℃ overnight. The filtrate was taken out at -80℃, concentrated and dried in a vacuum freeze dryer, and sent to the Guangdong Academy of Sciences Analysis and Testing Institute. The concentration of ascaroside was detected after dilution with 1 mL of methanol.

[0032] 4. Experimental Results:

[0033] Table 1. Results of Ascaridin Concentration Detection

[0034]

[0035]

[0036] 16S rDNA sequencing results, SEQ ID NO.1:

[0037]

Claims

1. Sphingosine monocytogenes (Yano) Sphingobium yanoikuyae )25D, the accession number is: GDMCC No: 63454.

2. An inoculant characterized in that, A sphingan produced by Sphingobium yanoikuyae 25D.

3. Use of Sphingobium yanoikuyae 25D according to claim 1 for the decomposition of an ascaridole, said ascaridole being ascr#18.

4. A method of decomposing ascaridole, characterized by, 4. Decomposition of an ascaridole, said ascaridole being ascr#18, using Sphingobium yanoikuyae 25D according to claim 1.

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