Culture method for improving reproductive capacity of indigenous nematodes and application of culture method

Abstract

The invention discloses a culture method for improving the reproductive capacity of indigenous nematodes and application of the culture method, and belongs to the technical field of indigenous nematode culture. According to the present invention, the Rosselomyces aquimaris L-3 strain is the indigenous nematode intestinal tract strain, is the new strain, can enrich the nutrition source of the indigenous nematode, can significantly improve the egg laying amount of the nematode fed with the strain in the early stage of the breeding period, and can improve the egg laying amount by 36.3% compared with the currently common E. coli OP50 bacteria, and can not produce any harm to the indigenous nematode; the provided culture method is convenient and efficient, the breeding speed of indigenous nematodes can be remarkably increased under the laboratory environment condition by adopting the culture method provided by the invention, and compared with the currently common E. coli OP50 bacteria, the extremely remarkable difference can be achieved in the second breeding day, the necessary nematode number can be provided for short-period experiments, and the related research process can be accelerated.

Description

Technical Field

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

[0002] Nematodes are a common soil animal distributed in soils around the world. There are many species of nematodes, which can be divided into four categories: bacteria-feeding nematodes, fungi-feeding nematodes, predatory nematodes and herbivorous nematodes. They also have the functions of regulating rhizosphere microbial communities and improving rhizosphere health, and are of special significance to soil ecology and agricultural pathogen prevention and control.

[0003] The study of indigenous bacteria-feeding nematodes occupies an important position in ecology and soil biology. These nematodes play a key role in maintaining soil health and ecological balance by feeding on bacteria in the soil and participating in nutrient cycling and organic matter decomposition. In recent years, with the deepening of understanding of microbial communities and soil ecosystems, researchers have gradually discovered the importance of indigenous nematodes in regulating the structure and function of soil microbial communities.

[0004] Although indigenous nematodes are of great significance in ecological research, their slow reproduction rate and long life cycle limit their widespread application in laboratory research. Acrobeloides The reproduction period of nematodes 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 complicated and time-consuming. Therefore, it is urgent to explore new methods to accelerate the growth and reproduction of indigenous nematodes in order to conduct experimental research more effectively. Summary of the invention

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

[0006] To achieve the above object, the present invention provides Rossellomorea aquimaris Application of L-3 strain in improving the reproductive capacity of indigenous nematodes, Rossellomorea aquimaris The L-3 strain is a Gram-positive bacterium and is classified as Rossellomorea aquimaris L-3 is a rod-shaped spore-forming bacterium, 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, and the deposit number is CCTCC NO: M 20241623.

[0007] Preferably, the Rossellomorea aquimarisThe culture method of L-3 strain is as follows: in an ultra-clean workbench, spread or inoculate on the surface / in the sterile LB solid or liquid culture medium, TSB solid or liquid culture medium or NGM solid culture medium, and culture statically or by shaking at 30°C.

[0008] A method for improving the reproductive capacity of indigenous nematodes, using the above 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.

[0009] Preferably, in step S1, the preparation method of NGM solid culture medium is: 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, and 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 the filter membrane in an ultra-clean workbench, mix well, and pour into a sterile culture dish.

[0010] Preferably, 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.

[0011] Preferably, 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 .

[0012] A method for improving the reproductive capacity of indigenous nematodes as described above is used in the cultivation of indigenous nematodes.

[0013] Therefore, the present invention provides a method for cultivating and applying the method to improve the reproductive capacity of indigenous nematodes, and the specific technical effects thereof are as follows: (1) The present invention provides Rossellomorea aquimaris The L-3 strain is a new strain of indigenous nematode intestinal strains that can enrich the nutritional sources of indigenous nematodes. The nematodes fed with this strain have significantly increased their egg production in the early reproductive period, which is better than the currently commonly used E. coli The OP50 bacteria increased by 36.3% without causing any harm to the indigenous nematodes; (2) The cultivation method provided by the present invention is convenient and efficient. The cultivation method provided by the present invention can significantly accelerate the reproduction speed of indigenous nematodes under laboratory conditions, which is different from the currently commonly used E. coli Compared with OP50 bacteria, extremely significant differences can be achieved on the second day of reproduction, which can provide the necessary number of nematodes for short-term experiments and accelerate the progress of related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0015] Figure 1 In the second embodiment of the present invention Rossellomorea aquimaris L-3 strain fed phagnum ( Panagrolaimus ) Photo of nematode; Figure 2 In the second embodiment of the present invention Rossellomorea aquimaris Optical microscopic photograph of strain L-3; Figure 3 is the total number of eggs laid by a single nematode in the first 16 days of the reproductive period in Example 3 of the present invention; Figure 4 is the number of eggs laid per day by a single nematode in the first 16 days of the reproductive period in Example 3 of the present invention; Figure 5 The indigenous nematodes of the genus Paratypharyngeus and the indigenous nematodes of the genus Pseudocyx in Example 3 of the present invention are E. coli OP50 bacterial solution and Rossellomorea aquimaris Photos of the nematodes propagated in L-3 bacterial solution for 5 days; Part A is a photo of the nematodes of the genus Pseudocloides propagated in OP50 for 5 days; Part B is a photo of the nematodes of the genus Pseudocloides propagated in L-3 for 5 days; Part C is a photo of the nematodes of the genus Pseudocloides propagated in OP50 for 5 days; Part D is a photo of the nematodes of the genus Pseudocloides propagated in L-3 for 5 days; Figure 6 is the statistical result of the population change of the indigenous nematode Pseudoclochaetes nematodes in Example 3 of the present invention after 5 days of reproduction; Figure 7 This is the statistical result of the population change of the indigenous nematode, the genus Pharynx, during 5 days of reproduction in Example 3 of the present invention.

[0016] Biomaterial Deposit Information Rossellomorea aquimaris The L-3 strain is a Gram-positive bacterium and is classified as Rossellomorea aquimarisL-3 is a rod-shaped spore-forming bacterium, 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, and the deposit number is CCTCC NO: M 20241623. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0018] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0019] The instruments, equipment, reagents and materials used in the examples were obtained from commercial sources; the indigenous nematode Pseudoclochaetes ( Acrobeloides ) The nematodes were isolated, identified and bred by the inventors themselves. The methods refer to “Mechanism of Response of Soil Nematode Acrobeloides to Drought Stress”, PhD dissertation of Lu Leilei, Nanjing Agricultural University, August 2023.

[0020] The culture medium and reagent information used in the examples are as follows: LB medium: 10 g tryptone, 5 g sodium chloride, 5 g yeast powder, 1 L distilled water, pH 7.0-7.2; TSB solid medium: trypsin powder 15g, NaCl 5g, soy peptone 5g, agar powder 15g, distilled water 1L; Phosphate buffer: 3.56 g of dipotassium hydrogen phosphate, 10.83 g of potassium dihydrogen phosphate, 100 mL of sterile distilled water; Magnesium sulfate solution: 12.03 g of anhydrous magnesium sulfate reagent, 100 mL of sterile distilled water; Calcium chloride solution: 11.38 g of anhydrous calcium chloride reagent, 100 mL of sterile distilled water; Cholesterol solution: 5 mg of cholesterol was dissolved in 1 mL of anhydrous ethanol; Nematode surface disinfectant: Mix sodium hypochlorite solution and 5 mol / L sodium hydroxide solution in a volume ratio of 2:1 and dilute 10 times; Nematode lysis solution: 1 ml sodium hypochlorite solution, 1 mL 5 mol / L sodium hydroxide solution, 7 ml sterile distilled water, prepared and used immediately; M9 buffer: 5 g sodium chloride, 3 g potassium dihydrogen phosphate, 6 g disodium hydrogen phosphate, add distilled water to 1 L, sterilize at 121 ° C for 20 min, and add 1 mL of 1 mol / L magnesium sulfate solution through a sterile filter membrane.

[0021] Embodiment 1 Prepare a nematode growth medium (NGM medium), the specific steps are as follows: Accurately weigh 2.5 g of peptone, 3.0 g of sodium chloride, and 17.0 g of agar, dissolve them in distilled water and make up to 1 L, sterilize at 121 ° C for 20 min, and after the solution temperature drops to 65 ° C, filter it through a sterile filter membrane in an ultra-clean workbench, 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 5 g / L cholesterol in anhydrous ethanol solution, mix well, and pour into a sterile culture dish.

[0022] Embodiment 2 get Rossellomorea aquimaris L-3 strains, as follows: S21. Disinfect the surface of the indigenous nematode Pseudocloides nematode: transfer the nematode 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 to wash again, and repeat the washing three times; then add 200 μL of M9 buffer and 400 μL of nematode surface disinfectant to the centrifuge tube with 0.1 mL of liquid remaining, 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.

[0023] S22. After controlling the water, use liquid nitrogen to freeze-grind the nematodes obtained in step S21, take 5 mg of the ground powder and immediately add 1 mL of sterile distilled water, vortex for 30 seconds to fully mix, and then let it stand for 10 minutes. Take the supernatant and dilute and spread it on TSB solid culture medium. After culturing for 24 hours, multiple single colonies are obtained.

[0024] S23, the single colonies obtained in step S22 are inoculated onto TSB solid culture medium using an inoculation loop. If there are still different colonies on the new culture medium, each colony is streaked, and this process is repeated until there is only one colony on the streaked culture medium, i.e., purification is completed.

[0025] S24. The purified strains were used to test the reproductive capacity of indigenous nematodes, and the genus Pseudochorium ( Panagrolaimus ) nematodes were used as the test nematodes. The obtained strains were inoculated into LB liquid culture medium and cultured in a shaking incubator at 200 r / min and 30°C for 24 h.

[0026] S25. Synchronize the native nematodes: add 900 μL sterile water twice to wash the nematodes from the culture medium, collect the washing solution in a 1.5 mL centrifuge tube, centrifuge at 3000 rpm for 1 min, remove the supernatant, add 900 μL nematode lysis solution, mix well and observe the lysis of the nematodes in the centrifuge tube at all times, centrifuge at 5000 rpm for 1 min and remove the supernatant, add 900 μL M9 buffer to the precipitate, wash at 4000 rpm for 3 to 5 times, and remove the supernatant. Keep 100 μL M9 buffer to cultivate and hatch eggs.

[0027] S26. Feed the obtained eggs with the bacterial solution of each strain and culture them to the L4 stage. Add 25 μL of the corresponding bacterial solution to the center of the NGM culture 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 breeding period. If the nematode lays eggs in the culture 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 culture medium before counting to prevent the hatching and growth of the eggs from interfering with the judgment of the original adult. Set up 6 replicates for each treatment.

[0028] After 10 days, the cumulative number of eggs laid was counted, and the strain with the best effect was selected for 16SrDNA identification. The results showed that the obtained strain was a Rosobacter genus and was named Rossellomorea aquimaris L-3, Rossellomorea aquimaris L-3 strain fed phagnum ( Panagrolaimus ) Photos of nematodes such as Figure 1 As shown, Rossellomorea aquimaris The 16SrDNA sequence of L-3 is shown in SEQ ID NO.1. It was deposited in the China Center for Type Culture Collection on July 22, 2024. The deposit address is: Wuhan University, Wuhan City, Hubei Province, and the deposit number is: CCTCC NO: M 20241623. Rossellomorea aquimaris The L-3 strain is a Gram-positive, rod-shaped, spore-forming bacterium that can grow normally in conventional bacterial culture media such as LB and TSB.

[0029] SEQ ID NO.1: GGGCGGCGTGCTATAATGCAAGTCGAGCGGATTGATGGGAGCTTGCTCCCATCAGTCAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACTCATTTCCTCGCATGAGGAAATGTTGAAAGGTGGCTTTTAGCTATCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCAAGTGTAGCGGTGAAATGCGTAGATATTTGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGGAGTCCACGCCGTAAACGATGAGTGCTTAATGTTTAGGGGGTTTCCGCCCCTTAATGCTGCAGCTAACGCATTAAGCACTCCGCCCGGGGAGAACCGGCCCCAAAACTGAAACTCCCAGGGAATTACC Under an optical microscope Rossellomorea aquimaris The photograph of strain L-3 is as Figure 2 shown

[0030] Example 3 Investigate the results obtained in Example 2 Rossellomorea aquimaris The effects of the L-3 strain on the reproductive capacity of indigenous nematodes are as follows: S31. Obtained Rossellomorea aquimaris L-3 strain and E. coli OP50 was inoculated into LB liquid culture medium and cultured in a shaking incubator at 200 r / min and 30°C for 24 h.

[0031] S32. Synchronize the native nematodes: add 900 μL sterile water twice to wash the nematodes from the culture medium, collect the washing solution in a 1.5 mL centrifuge tube, centrifuge at 3000 rpm for 1 min, remove the supernatant, add 900 μL nematode lysis solution, mix and observe the lysis of the nematodes in the centrifuge tube at all times, centrifuge at 5000 rpm for 1 min and remove the supernatant, add 900 μL M9 buffer to the precipitate, wash at 4000 rpm for 3 to 5 times, and remove the supernatant. Keep 100 μL M9 buffer to cultivate and hatch the eggs.

[0032] S33, the obtained eggs are used Rossellomorea aquimaris L-3 strain and E. coli OP50 bacterial solution was fed separately and cultured to the L4 stage. 25 μL of the corresponding bacterial solution was added to the center of the NGM medium, and a single nematode was picked into the bacterial solution drop in a sterile environment. The number of eggs laid by a single nematode was measured every day during the nematode breeding period. If the nematode lays eggs in the culture 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 culture medium before counting to prevent the hatching and growth of the eggs from interfering with the judgment of the original adult. Six replicates were set for each treatment.

[0033] S34. The total number of eggs laid by a single nematode in the first 16 days of the reproductive period is Figure 3 As shown in Figure 2, the number of eggs laid by a single nematode during the first 16 days of its reproductive period is Figure 4 As shown, it is shown that the use of the present invention provides Rossellomorea aquimaris The L-3 strain was fed to the indigenous nematode Pseudocephaga and performed well in laboratory breeding experiments, increasing the number of eggs laid by the nematodes by 36.3% in the early breeding stage.

[0034] S35, select the indigenous nematode Pseudocloides ( Acrobeloides )、Pelvichala( Panagrolaimus ) nematodes, after synchronizing the nematodes using the method of step S32, Rossellomorea aquimaris L-3 bacterial liquid, E. coliFeed the nematodes with OP50 bacterial solution until the nematodes are in the L4 stage. Wash the nematodes on the culture dish into a centrifuge tube with sterile water for three times. Spread 100 μL of L-3 bacterial solution or OP50 bacterial solution on the NGM solid medium, incubate at 30°C for more than 6 hours, and add about 50 nematodes to the NGM nematode culture dish after the coating culture. Repeat 6 times for each bacterial solution and each nematode. Optical microscope photos of the culture dishes under different treatments after 5 days are shown in the figure. Figure 5 The statistical results of the population changes of the indigenous nematode Pseudoclochaetes nematodes after 5 days of reproduction are shown in Figure 6 As shown; the statistical results of the population changes of the indigenous nematode genus Pharynx nematodes after 5 days of reproduction are shown as follows Figure 7 shown.

[0035] It can be seen that compared with the currently commonly used E. coli Compared with OP50 bacteria, Rossellomorea aquimaris The L-3 strain can significantly accelerate the reproduction rate of indigenous nematodes. The indigenous nematodes of the genus Pseudocloides can reach a significant difference on the third day of reproduction ( P =0.02), and reached a very significant difference on the 5th day of breeding ( P =0.007), the indigenous nematodes of the genus Pharynx reached a very significant difference on the second day of reproduction ( P <0.02). Rossellomorea aquimaris The L-3 strain can obtain a large number of indigenous nematodes in a short period of time, providing convenience for related research on indigenous nematodes.

[0036] Therefore, the present invention provides Rossellomorea aquimaris The L-3 strain is a new strain of indigenous nematode intestinal strains that can enrich the nutritional sources of indigenous nematodes. The nematodes fed with this strain have significantly increased their egg production in the early reproductive period, which is better than the currently commonly used E. coli The OP50 bacteria were increased by 36.3% without causing any harm to the indigenous nematodes; the culture method provided was convenient and efficient. The culture method provided by the present invention could significantly accelerate the reproduction speed of the indigenous nematodes under laboratory conditions, which was different from the currently commonly used E. coli Compared with OP50 bacteria, extremely significant differences can be achieved on the second day of reproduction, which can provide the necessary number of nematodes for short-term experiments and accelerate the progress of related research.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution 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, and 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 the filter membrane in an ultra-clean workbench, 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.

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