A culture method for enhancing the reproductive ability of indigenous nematodes and its application

Abstract

The present invention discloses a cultivation method for improving the reproductive ability of indigenous nematodes and its application, belonging to the technical field of indigenous nematode cultivation. The Rossellomorea aquimaris L-3 strain provided by the present invention is an indigenous nematode intestinal strain and is a new strain, which can enrich the nutrient source of indigenous nematodes. The egg-laying amount of nematodes fed with this strain in the early stage of the reproductive period is significantly increased, which is 36.3% higher than that of the commonly used E.coli OP50 bacteria, and it will not cause any harm to indigenous nematodes; the provided cultivation method is convenient and efficient. By using the cultivation method provided by the present invention, under laboratory environmental conditions, the reproductive speed of indigenous nematodes can be significantly accelerated. Compared with the commonly used E.coli OP50 bacteria, a highly significant difference can be achieved as early as the second day of reproduction, which can provide the necessary number of nematodes for short-term experiments and accelerate the relevant research process.

Description

Technical Field

[0001] The present invention relates to the technical field of indigenous nematode culture, and in particular to a culture method for improving the reproductive ability of indigenous nematodes and its application. Background Art

[0002] Nematodes are common soil animals distributed in soils worldwide. They are diverse in species and can be classified into four categories: bacterivorous nematodes, fungivorous nematodes, predatory nematodes, and phytophagous nematodes. They also have functions such as regulating the rhizosphere microbial community and enhancing rhizosphere health, which are of special significance for soil ecology and the prevention and control of agricultural pathogens.

[0003] The research on indigenous bacterivorous nematodes has occupied an important position in ecology and soil biology. These nematodes participate in nutrient cycling and organic matter decomposition by feeding on bacteria in the soil, playing a key role in maintaining soil health and ecological balance. In recent years, with the deepening understanding of the microbial community and soil ecosystem, researchers have gradually discovered the importance of indigenous nematodes in regulating the structure and function of the soil microbial community.

[0004] Although indigenous nematodes are of great significance in ecological research, the problems of their slow reproduction rate and long life cycle limit their wide application in laboratory research. For example, the reproduction period of indigenous nematodes of the genus Acrobeloides distributed widely in soils worldwide can reach more than 40 days, and the life cycle can reach more than 50 days, which makes the cultivation and experimental design of nematodes complex and time-consuming. Therefore, it is urgent to explore new methods to accelerate the growth and reproduction of indigenous nematodes for more effective experimental research. Summary of the Invention

[0005] The purpose of the present invention is to provide a culture method for improving the reproductive ability of indigenous nematodes and its application, so as to solve the problems that the growth rate of indigenous nematodes is slow and the reproduction period is long under laboratory conditions, which affect the progress and design of nematode-related experiments.

[0006] To achieve the above purpose, the present invention provides Rossellomorea aquimaris The application of strain L-3 in improving the reproductive ability of indigenous nematodes, wherein the Rossellomorea aquimaris strain L-3 is a Gram-positive bacterium, classified and named as Rossellomorea aquimaris L-3, a rod-shaped spore-forming bacterium, was deposited at the China Center for Type Culture Collection on July 22, 2024. The deposit address is within Wuhan University near Luonan 1st Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is CCTCC NO: M 20241623.

[0007] Preferably, the Rossellomorea aquimarisThe culturing method of strain L-3 is as follows: in a laminar flow hood, spread or inoculate it on the surface / in the sterile LB solid or liquid medium, TSB solid or liquid medium or NGM solid medium, and then statically culture or shake culture it at 30°C.

[0008] A culturing method for enhancing the reproductive ability of indigenous nematodes, using the above Rossellomorea aquimaris strain L-3, comprising the following steps:

[0009] S1. Prepare the NGM solid medium;

[0010] S2. Prepare Rossellomorea aquimaris the L-3 bacterial suspension;

[0011] S3. Drop the L-3 bacterial suspension prepared in step S2 onto the surface of the NGM solid medium prepared in step S1, spread it, and then statically culture it at 30°C for more than 6 h, and then add indigenous nematodes and culture them at 20 - 25°C. Rossellomorea aquimaris

[0012] Preferably, in step S1, the preparation method of the NGM solid medium is as follows: accurately weigh 2.5 g / L of peptone, 3.0 g / L of sodium chloride, and 17.0 g / L of agar, dissolve them with distilled water and make up the volume, sterilize at 121°C for 20 min, and after the solution temperature drops to 65°C, in a laminar flow hood, add 25 mL / L of phosphate buffer solution, 1 mL / L of 1 mol / L magnesium sulfate solution, 1 mL / L of 1 mol / L calcium chloride solution, and 1 mL / L of 5 g / L cholesterol solution through a filter membrane, mix well and pour it into a sterile petri dish.

[0013] Rossellomorea aquimaris Preferably, in step S2 Rossellomorea aquimaris the L-3 bacterial suspension is composed of strain L-3 cultured to the logarithmic phase and sterile LB liquid medium.

[0014] Preferably, the indigenous nematodes in step S3 are indigenous nematode eggs or nematodes in other stages, and the inoculation amount is 3 - 5 nematodes / cm Rossellomorea aquimaris .

[0015] An application of the culturing method for enhancing the reproductive ability of indigenous nematodes as described above in the culturing of indigenous nematodes.

[0016] Therefore, a culturing method for enhancing the reproductive ability of indigenous nematodes and its application provided by the present invention have the following specific technical effects:

[0017] Rossellomorea aquimaris (1) The strain L-3 provided by the present invention, which is an indigenous nematode intestinal strain and a new strain, can enrich the nutrient source of indigenous nematodes, and the egg-laying amount of nematodes fed with this strain in the early stage of the reproductive period is significantly increased, compared with the currently commonly used E. coli ​E. coli The OP50 bacteria increased by 36.3% and did not cause any harm to the indigenous nematodes.

[0018] (2) The culture method provided by the present invention is convenient and efficient. By using the culture method provided by the present invention, under laboratory environmental conditions, the reproduction speed of indigenous nematodes can be significantly accelerated. Compared with the currently commonly used E. coli OP50 bacteria, a highly significant difference can be achieved as early as the second day of reproduction, which can provide the necessary number of nematodes for short-term experiments and accelerate the relevant research process. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0020] Figure 1 It is a photograph of nematodes of the genus Panagrolaimus Rossellomorea aquimaris fed with the L-3 strain in the second embodiment of the present invention; Panagrolaimus

[0021] Figure 2 It is a photograph of the L-3 strain under an optical microscope in the second embodiment of the present invention; Rossellomorea aquimaris

[0022] Figure 3 It is the total number of eggs laid by a single nematode in the first 16 days before the reproductive period in the third embodiment of the present invention;

[0023] Figure 4 It is the number of eggs laid by a single nematode per day in the first 16 days before the reproductive period in the third embodiment of the present invention;

[0024] Figure 5 It is a photograph of the indigenous nematodes of the genus Acrobeloides and the indigenous nematodes of the genus Panagrolaimus E. coli after 5 days of reproduction in the OP50 bacterial solution and Rossellomorea aquimaris the L-3 bacterial solution in the third embodiment of the present invention; among them, part A is a photograph of the nematodes of the genus Acrobeloides after 5 days of reproduction in OP50; part B is a photograph of the nematodes of the genus Acrobeloides after 5 days of reproduction in L-3; part C is a photograph of the nematodes of the genus Panagrolaimus after 5 days of reproduction in OP50; part D is a photograph of the nematodes of the genus Panagrolaimus after 5 days of reproduction in L-3;

[0025] Figure 6 It is the statistical result of the population quantity change of the indigenous nematodes of the genus Acrobeloides after 5 days of reproduction in the third embodiment of the present invention;

[0026] Figure 7It is the statistical result of the population quantity change of the indigenous nematode Bursaphelenchus after 5 days of reproduction in Example 3 of the present invention.

[0027] Biological material preservation information

[0028] Rossellomorea aquimaris Strain L-3 is a Gram-positive bacterium, classified and named Rossellomorea aquimaris L-3, a rod-shaped spore-forming bacterium, was preserved in the China Center for Type Culture Collection on July 22, 2024. The preservation address is within Wuhan University near Luonan 1st Road, Wuchang District, Wuhan City, Hubei Province. The preservation number is CCTCC NO: M 20241623. Detailed implementation manners

[0029] The technical solutions of the present invention are further described below through the accompanying drawings and examples.

[0030] In order to make the purpose, technical solutions and advantages of the present application clearer, more thorough and complete, the technical solutions of the present invention are clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of examples, aiming to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meanings as those generally understood by those of ordinary skill in the technical field to which this application belongs.

[0031] The instruments, equipment, reagents and materials used in the examples are all obtained through commercial channels; the indigenous nematode Acrobeloides ( Acrobeloides ) nematodes were isolated, identified and bred by the inventors themselves. For the method, see "Mechanism of the Response of the Soil Nematode Acrobeloides to Drought Stress", the doctoral dissertation of Lu Leilei, Nanjing Agricultural University, August 2023.

[0032] The information of the culture media and reagents used in the examples is as follows:

[0033] LB culture medium: 10 g of tryptone, 5 g of sodium chloride, 5 g of yeast extract, 1 L of distilled water, pH 7.0 - 7.2;

[0034] TSB solid culture medium: 15 g of tryptic soy protein, 5 g of NaCl, 5 g of soy peptone, 15 g of agar powder, 1 L of distilled water;

[0035] Phosphate buffer: 3.56 g of dipotassium hydrogen phosphate, 10.83 g of potassium dihydrogen phosphate, 100 mL of sterilized distilled water;

[0036] Magnesium sulfate solution: 12.03 g of anhydrous magnesium sulfate reagent, 100 mL of sterilized distilled water;

[0037] Calcium chloride solution: 11.38 g of anhydrous calcium chloride reagent, 100 mL of sterilized distilled water;

[0038] Cholesterol solution: 5 mg of cholesterol, dissolved in 1 mL of absolute ethanol;

[0039] Nematode surface disinfectant solution: Mix sodium hypochlorite solution and 5 mol / L sodium hydroxide solution in a volume ratio of 2:1 and dilute 10 times;

[0040] Nematode lysis solution: 1 mL of sodium hypochlorite solution, 1 mL of 5 mol / L sodium hydroxide solution, 7 mL of sterile distilled water, prepared and used immediately;

[0041] M9 buffer: 5 g of sodium chloride, 3 g of potassium dihydrogen phosphate, 6 g of disodium hydrogen phosphate, add distilled water to 1 L, sterilize at 121 °C for 20 min, and then add 1 mL of 1 mol / L magnesium sulfate solution after passing through a sterile filter membrane.

[0042] Example 1

[0043] Prepare a nematode growth medium (NGM medium), and the specific steps are as follows:

[0044] Accurately weigh 2.5 g of peptone, 3.0 g of sodium chloride, and 17.0 g of agar, dissolve with distilled water and make up to 1 L, sterilize at 121 °C for 20 min. After the solution temperature drops to 65 °C, in a laminar flow hood, add 25 mL of phosphate buffer, 1 mL of 1 mol / L magnesium sulfate solution, 1 mL of 1 mol / L calcium chloride solution, and 1 mL of absolute ethanol solution containing 5 g / L cholesterol through a sterile filter membrane. Mix well and pour into a sterile petri dish.

[0045] Example 2

[0046] Obtain Rossellomorea aquimaris Strain L-3, specifically as follows:

[0047] S21. Surface disinfect the indigenous nematode Aphelenchoides: Transfer the nematodes to a 1.5 mL centrifuge tube, add 1 mL of sterile distilled water to wash, centrifuge at 3000 rpm for 1 min, remove the supernatant until 0.1 mL of liquid remains in the centrifuge tube, then add 1 mL of sterile distilled water again to wash, and repeat the washing three times; then add 200 μL of M9 buffer and 400 μL of nematode surface disinfectant solution to the centrifuge tube with the remaining 0.1 mL of liquid, mix well, let stand for 2 min, centrifuge at 3000 rpm for 1 min, discard the supernatant, and wash the precipitate three times with M9 buffer.

[0048] S22. After draining the water, freeze and grind the nematodes obtained in step S21 using liquid nitrogen. Immediately add 1 mL of sterile distilled water to 5 mg of the ground powder, vortex for 30 s to mix well, let stand for 10 min, take the supernatant and perform dilution coating on a TSB solid medium, and obtain various single colonies after culturing for 24 h.

[0049] S23. Use an inoculation loop to transfer the single colonies obtained in step S22 onto TSB solid medium respectively. If there are still different colonies on the new medium, streak each type of colony separately, and repeat this process until there is only one type of colony on the streaked medium, indicating that purification is complete.

[0050] S24. Measure the reproductive ability of indigenous nematodes for each of the purified strains. Use nematodes of the genus Panagrolaimus as the test nematodes. Inoculate each of the obtained strains into LB liquid medium and culture them in a shaker at 200 r / min and 30 °C for 24 h.

[0051] S25. Synchronize the indigenous nematodes: Rinse the nematodes from the medium with 900 μL of sterile water twice, collect the washing solution in a 1.5 mL centrifuge tube, centrifuge at 3000 rpm for 1 min, discard the supernatant, add 900 μL of nematode lysis solution, mix well and observe the lysis situation of the nematodes in the centrifuge tube at all times. Centrifuge at 5000 rpm in a timely manner for 1 min and discard the supernatant. Add 900 μL of M9 buffer to the precipitate and wash it 3 - 5 times at 4000 rpm, then discard the supernatant. Leave 100 μL of M9 buffer to cultivate and hatch the eggs.

[0052] S26. Feed the obtained eggs with the bacterial solutions of each strain and culture them until the L4 stage. Drop 25 μL of the corresponding bacterial solution in the center of the NGM medium, pick a single nematode into the bacterial solution drop in a sterile environment, and measure the number of eggs laid by a single nematode every day during the nematode reproductive period. If the nematode lays eggs in the medium on the same day, the nematode should be picked into a new 25 μL of the corresponding bacterial solution placed in the center of the NGM medium before counting to prevent the hatching and growth of eggs from interfering with the judgment of the original adult nematodes. Set 6 replicates for each treatment.

[0053] After 10 days, count the cumulative number of eggs laid, select the strain with the best effect for 16S rDNA identification. The result shows that the obtained strain is a bacterium of the genus Roseomonas, named Rossellomorea aquimaris L-3. Rossellomorea aquimaris A photo of the nematodes of the genus Panagrolaimus fed with strain L-3 is as Figure 1 shown. Rossellomorea aquimaris The 16S rDNA sequence of L-3 is shown in SEQ ID NO.1. It was deposited at the China Center for Type Culture Collection on July 22, 2024. The deposit address is: within Wuhan University, Wuhan, Hubei Province. The deposit number is: CCTCC NO: M 20241623. Rossellomorea aquimaris Strain L-3 is a Gram-positive bacterium, a rod-shaped spore-forming bacterium, and can grow normally in conventional bacterial media such as LB and TSB.

[0054] SEQ ID NO.1:

[0055] GGGCGGCGTGCTATAATGCAAGTCGAGCGGATTGATGGGAGCTTGCTCCCATCAGTCAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACTCATTTCCTCGCATGAGGAAATGTTGAAAGGTGGCTTTTAGCTATCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCAAGTGTAGCGGTGAAATGCGTAGATATTTGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGGAGTCCACGCCGTAAACGATGAGTGCTTAATGTTTAGGGGGTTTCCGCCCCTTAATGCTGCAGCTAACGCATTAAGCACTCCGCCCGGGGAGAACCGGCCCCAAAACTGAAACTCCCAGGGAATTACC

[0056] Under an optical microscope Rossellomorea aquimaris The photo of strain L-3 is asFigure 2 as shown

[0057] Example 3

[0058] Investigate the influence of the L-3 strain obtained in Example 2 Rossellomorea aquimaris on the reproductive ability of indigenous nematodes, specifically as follows:

[0059] S31. Inoculate the obtained Rossellomorea aquimaris L-3 strain and E. coli OP50 into LB liquid medium respectively, and culture them in a shaker at 200 r / min and 30 °C for 24 h.

[0060] S32. Synchronize the indigenous nematodes: Wash the nematodes from the medium with 900 μL of sterile water twice, collect the washing solution in a 1.5 mL centrifuge tube, centrifuge at 3000 rpm for 1 min, discard the supernatant, add 900 μL of nematode lysis solution, mix well and observe the lysis of nematodes in the centrifuge tube at any time. Centrifuge at 5000 rpm in time for 1 min and discard the supernatant. Add 900 μL of M9 buffer to the precipitate, wash it 3 - 5 times at 4000 rpm, and discard the supernatant. Leave 100 μL of M9 buffer to cultivate and hatch the eggs.

[0061] S33. Feed the obtained eggs with Rossellomorea aquimaris the L-3 strain and E. coli OP50 bacterial solution respectively, and culture them until the L4 stage. Drop 25 μL of the corresponding bacterial solution in the center of the NGM medium, pick a single nematode into the bacterial solution drop in a sterile environment, and measure the number of eggs laid by a single nematode every day during the nematode reproduction period. If the nematode lays eggs in the medium on the same day, the nematode should be picked into a new 25 μL of the corresponding bacterial solution placed in the center of the NGM medium before counting to prevent the hatching and growth of eggs from interfering with the judgment of the original adult worms. Set 6 replicates for each treatment.

[0062] S34. The total number of eggs laid by a single nematode in the first 16 days before the reproduction period is as Figure 3 shown, and the number of eggs laid by a single nematode per day in the first 16 days before the reproduction period is as Figure 4 shown, indicating that feeding the indigenous nematode Acrobeloides with the L-3 strain provided by the present invention Rossellomorea aquimaris performs well in the laboratory reproduction experiment and can increase the number of eggs laid by nematodes by 36.3% in the early reproduction stage.

[0063] S35. Select indigenous nematodes Acrobeloides ( Acrobeloides ), Panagrolaimus ( Panagrolaimus ), after synchronizing the nematodes by the method of step S32, use Rossellomorea aquimaris L-3 bacterial solution, E. coliThe OP50 bacterial solution was fed to nematodes at the L4 stage. The nematodes on the culture dish were washed into a centrifuge tube with sterile water and washed three times. 100 μL of the L-3 bacterial solution or OP50 bacterial solution was added to the NGM solid medium and spread. It was statically cultured at 30 °C for more than 6 h. Approximately 50 nematodes were added to the NGM nematode culture dishes after spreading and culturing, with 6 replicates for each bacterial solution and each type of nematode. Optical microscope photos of the culture dishes under different treatments after 5 days are as shown in Figure 5 shown, and the statistical results of the population quantity change of the indigenous nematode *Acrobeloides* after 5 days of reproduction are as shown in Figure 6 shown; the statistical results of the population quantity change of the indigenous nematode *Panagrolaimus* after 5 days of reproduction are as shown in Figure 7 shown.

[0064] It can be seen that compared with the commonly used E. coli OP50 bacteria, feeding Rossellomorea aquimaris the L-3 strain can significantly accelerate the reproduction rate of indigenous nematodes. The indigenous nematode *Acrobeloides* can reach a significant difference on the 3rd day of reproduction ( P = 0.02), and reach a highly significant difference on the 5th day of reproduction ( P = 0.007). The indigenous nematode *Panagrolaimus* can reach a highly significant difference on the 2nd day of reproduction ( P < 0.02). Using the L-3 strain provided by the present invention, a large number of indigenous nematodes can be obtained in a short time, which facilitates the related research on indigenous nematodes. Rossellomorea aquimaris Therefore, the L-3 strain provided by the present invention is an intestinal strain of indigenous nematodes and is a new strain. It can enrich the nutrient source of indigenous nematodes. The egg-laying amount of nematodes fed with this strain in the early stage of the reproduction period is significantly increased, which is 36.3% higher than that of the commonly used

[0065] OP50 bacteria, and it will not cause any harm to indigenous nematodes; the provided culture method is convenient and efficient. Using the culture method provided by the present invention, under laboratory environmental conditions, the reproduction rate of indigenous nematodes can be significantly accelerated. Compared with the commonly used Rossellomorea aquimaris OP50 bacteria, a highly significant difference can be reached preferably on the 2nd day of reproduction, which can provide the necessary number of nematodes for short-cycle experiments and accelerate the related research process. E. coli Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions of the present invention or make equivalent replacements, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention. E. coli OP50 bacteria, and a highly significant difference can be reached preferably on the 2nd day of reproduction, which can provide the necessary number of nematodes for short-cycle experiments and accelerate the related research process.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions of the present invention or make equivalent replacements, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Rossellomorea aquimaris The application of L-3 strain in improving the reproductive capacity of indigenous nematodes is characterized by: Said Rossellomorea aquimaris The L-3 strain is a Gram-positive, rod-shaped spore-forming bacterium. It was deposited in the China Center for Type Culture Collection on July 22, 2024. The deposit address is: Wuhan University, near Luonan 1st Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is CCTCC NO: M 20241623.

2. according to claim 1 Rossellomorea aquimaris The application of L-3 strain in improving the reproductive capacity of indigenous nematodes is characterized by: Said Rossellomorea aquimaris The culture method of L-3 strain is as follows: in a clean bench, spread it on the surface of sterile LB solid medium, TSB solid medium or NGM solid medium, and culture it statically at 30°C.

3. According to claim 1 Rossellomorea aquimaris The application of L-3 strain in improving the reproductive capacity of indigenous nematodes is characterized by: Said Rossellomorea aquimaris The culture method of L-3 strain is as follows: inoculate into sterile LB liquid culture medium or TSB liquid culture medium in a clean bench and culture at 30°C with shaking.

4. A method for cultivating native nematodes to improve their reproductive capacity, characterized in that: Using the method described in any one of claims 1 to 3 Rossellomorea aquimaris L-3 strain, comprising the following steps: S1. Prepare NGM solid culture medium; S2. Preparation Rossellomorea aquimaris L-3 bacterial suspension; S3, the step S2 prepared Rossellomorea aquimaris The L-3 bacterial suspension is dripped onto the surface of the NGM solid culture medium prepared in step S1, and after coating, it is incubated at 30°C for more than 6 hours, and then indigenous nematodes are added and cultured at 20-25°C.

5. A method for cultivating native nematodes to improve their reproductive capacity according to claim 4, characterized in that: In the step S1, the preparation method of NGM solid culture medium is as follows: accurately weigh 2.5 g / L of peptone, 3.0 g / L of sodium chloride, and 17.0 g / L of agar, dissolve them in distilled water and make up the volume, sterilize them at 121°C for 20 min, place them in a clean bench after the solution temperature drops to 65°C, add 25 mL / L of phosphate buffer, 1 mL / L of 1 mol / L magnesium sulfate solution, 1 mL / L of 1 mol / L calcium chloride solution, and 1 mL / L of 5 g / L cholesterol solution through a filter membrane, mix well, and pour into a sterile culture dish.

6. A method for cultivating native nematodes to improve their reproductive capacity according to claim 4, characterized in that: In step S2 Rossellomorea aquimaris L-3 bacterial suspension was cultured to the logarithmic phase Rossellomorea aquimaris L-3 strain and sterile LB liquid medium.

7. A method for cultivating native nematodes to improve their reproductive capacity according to claim 4, characterized in that: The indigenous nematodes in step S3 are indigenous nematode eggs or nematodes at other stages, and the inoculation amount is 3-5 nematodes / cm 2 .

8. Use of the cultivation method for improving the reproductive capacity of indigenous nematodes according to any one of claims 4 to 7 in the cultivation of indigenous nematodes.

Images