Nephromyces NJAU-HN1 and application thereof

Abstract

The application provides a strain of protozoan nephrospora NJAU-HN1 and an application thereof, and belongs to the technical field of agricultural microorganisms. The nephrospora NJAU-HN1 has a preservation number of CCTCC NO: C2025172. The nephrospora NJAU-HN1 can directly and effectively inhibit the survival of root-knot nematodes. Application of the nephrospora NJAU-HN1 or a culture thereof can significantly promote the growth of melon plants, increase the fresh weight, dry weight and root length of the plants. The nephrospora NJAU-HN1 provided by the application provides valuable microbial resources for the development of a new microbial preparation for green prevention and control of melon root-knot nematode disease and crop health promotion, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, and relates to plant transgenic biotechnology breeding, specifically to a protozoan, the kidney-shaped insect NJAU-HN1, and its application. Background Technology

[0002] melon( Cucumis melo L. Southern root-knot nematode (Sinium spp.) is a high-value-added global economic crop, playing a crucial role in my country's facility agriculture system and increasing farmers' income. Its excellent edible quality and market value make it an important choice for optimizing planting structures and promoting agricultural industrial upgrading. Under large-scale and continuous cropping production models, the southern root-knot nematode... Meloidogyne incognita Root-knot nematode disease, caused by nematodes, has become a major biological obstacle restricting the sustainable development of the melon industry.

[0003] Root-knot nematode disease is progressive and systemic. The pathogenic nematode, in its second instar larvae, invades the young roots of melons, establishing permanent feeding sites and inducing abnormal root tissue proliferation to form root knots. This severely damages the root system's anatomical structure and its absorption and transport functions. Infected plants exhibit root dysfunction, inhibited above-ground growth, leaf chlorosis and wilting, and decreased photosynthetic efficiency, ultimately leading to reduced fruit yield, deteriorated quality, an imbalanced sugar-acid ratio, and a significant decrease in commercial value. In greenhouse cultivation and continuously cropped fields, root-knot nematode populations continuously accumulate in the soil, with the disease worsening year by year, often causing localized yield losses and seriously threatening the stability and economic benefits of melon production.

[0004] Currently, the control of root-knot nematode disease in melons still relies primarily on chemical nematicides. While these agents are fast-acting, they generally suffer from high toxicity, high residue levels, and non-selectivity, leading to soil microecological imbalance and increased nematode resistance. With increasingly stringent global requirements for agricultural product quality and safety and sustainable agricultural development, the application of highly toxic chemical agents is strictly limited. Developing efficient, safe, and environmentally friendly green control technologies has become an urgent need for the industry.

[0005] Soil protists are a core component of the soil micro-food web, playing a vital role in regulating microbial community structure, accelerating organic matter decomposition and nutrient mineralization, and improving soil physical structure. *Nephrota* genus (… Colpoda Protozoa are widely distributed in various soil environments and are characterized by rapid reproduction, strong adaptability, and high activity in the rhizosphere, making them considered to have potential application value in the regulation of plant rhizosphere health. Their biocontrol mechanisms mainly involve directly preying on the eggs and larvae of pathogenic nematodes, thereby reducing the initial population of infection sources in the soil. However, systematic research and development of protozoan resources that possess the dual functions of "controlling root-knot nematode disease" and "promoting crop growth" are still lacking. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a protozoan *Nematodeus* strain NJAU-HN1 and its applications, particularly a novel *Nematodeus* strain that can simultaneously and efficiently control root-knot nematode disease in melons and significantly promote melon growth; it also provides the organic culture of this *Nematodeus* strain and its preparation method, as well as various applications of this strain and its culture in agriculture.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A protozoan, *Nephroplasma reniformis* NJAU-HN1 ( Colpoda steinii NJAU-HN1), which is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: C2025172, and deposit date of October 27, 2025;

[0009] The *Nephroplasma reniformis* strain NJAU-HN1 was isolated and screened from the rhizosphere soil of healthy melons at the Sanya Research Base of Nanjing Agricultural University. It was identified morphologically (encapsulated and viable characteristics) and molecularly (18S rDNA sequence analysis) as... Colpoda steinii ;

[0010] The protozoan *Nematodeus* NJAU-HN1 is used to control root-knot nematode disease in melons and / or directly promote melon growth.

[0011] A culture of the aforementioned protozoan NJAU-HN1, wherein the culture comprises NJAU-HN1 and its metabolites.

[0012] A method for preparing a culture of the above-mentioned protozoan NJAU-HN1, wherein the preparation method is to inoculate the protozoan NJAU-HN1 into PAS culture medium, add inactivated microorganisms as feed, and culture to obtain a culture of the protozoan NJAU-HN1;

[0013] The PAS culture medium is prepared as follows: disodium hydrogen phosphate and potassium dihydrogen phosphate are added to water to prepare solution I; sodium chloride, magnesium sulfate heptahydrate and calcium chloride hexahydrate are added to water to prepare solution II; solutions I and II are sterilized separately and then mixed to obtain the PAS culture medium.

[0014] Furthermore, the content of disodium hydrogen phosphate in solution I is 0.28~0.29 g / L, and the content of potassium dihydrogen phosphate is 0.26~0.27 g / L;

[0015] Solution II contains 0.235~0.245 g / L sodium chloride, 0.75~0.85 mg / L magnesium sulfate heptahydrate, and 0.115~0.125 mg / L calcium chloride hexahydrate.

[0016] The volume ratio of solution I to solution II in the PAS culture medium is 1:0.95~1.05;

[0017] The microorganism in question is Escherichia coli.

[0018] The application of the aforementioned protozoan, *Nematodeus nematode* NJAU-HN1, in the control of root-knot nematode disease in melon and / or in directly promoting melon growth, especially in the control of southern root-knot nematode disease.

[0019] Furthermore, the application involves inoculating the NJAU-HN1 renal nematode into the soil where melons are grown to control root-knot nematode disease in melons and / or directly promote melon growth.

[0020] The application of the aforementioned protozoan, *Nematodea repens* NJAU-HN1, in the preparation of biological agents for the control of root-knot nematode disease in melon and / or for the direct promotion of melon growth.

[0021] The application of a culture of the aforementioned protozoan *Nematodeus* NJAU-HN1 in the control of root-knot nematode disease in melon and / or in directly promoting melon growth, especially in the control of southern root-knot nematode disease.

[0022] Furthermore, the application involves inoculating the culture into the soil where melons are grown to control melon root-knot nematode disease and / or directly promote melon growth.

[0023] Application of a culture of the aforementioned protozoan *Nematodea repens* NJAU-HN1 in the preparation of biological agents for the control of root-knot nematode disease in melon and / or for the direct promotion of melon growth.

[0024] The beneficial effects of the protozoan *Nephroplasma reniformis* strain NJAU-HN1 and its application are as follows:

[0025] Therefore, this invention isolated and screened a specific *N. reniformis* strain, NJAU-HN1, from the rhizosphere soil of healthy melons. This strain not only has a significant inhibitory effect on southern root-knot nematodes but also effectively promotes melon plant growth, achieving a synergistic effect of "controlling nematodes with insects and promoting growth with insects." This invention aims to disclose the taxonomic information, cultivation method, and agricultural application of this strain, providing new microbial resources and technical approaches for the green control and healthy cultivation of melon root-knot nematodes, and has important practical significance for promoting the green transformation of agriculture and sustainable ecological development.

[0026] The *Nephroplasma reniformis* strain NJAU-HN1 provided in this invention was isolated and screened from the rhizosphere soil of healthy melons at the Sanya Research Base of Nanjing Agricultural University. It was identified as *Nephroplasma reniformis* by morphological (cyst and viable characteristics) and molecular biology (18S rDNA sequence analysis). Colpoda steinii ;

[0027] In vitro co-culture experiments confirmed that this strain can directly act on the second-instar larvae of the southern root-knot nematode, significantly reducing their survival rate; pot experiments confirmed that the application of the culture of this strain can effectively promote the growth of melon plants, as evidenced by a significant increase in plant fresh weight, dry weight and root length.

[0028] In summary, this invention discloses for the first time a *Nephroplasma gondii* strain NJAU-HN1 with clear dual effects of "disease control" and "growth promotion"; this strain and its products provide a novel and efficient microbial resource and solution for developing green control technologies and healthy cultivation programs for soil-borne diseases of melons. Attached Figure Description

[0029] Figure 1 The results of morphological feature observations in Embodiment 2 of the present invention are shown; wherein, Figure 1 The left image shows the morphological characteristics of the vegetative body of the protozoan NJAU-HN1, the middle image shows the morphological characteristics of the dormant cyst of the protozoan NJAU-HN1, and the right image shows the morphological characteristics of the reproductive cyst of the protozoan NJAU-HN1.

[0030] Figure 2 This is a phylogenetic tree of the 18S rDNA gene sequence of *Nephroticosa* NJAU-HN1 in Example 2 of the present invention;

[0031] Figure 3 The root-knot nematode in Example 3 of this invention Meloidogyne incognita Characterizing root-knot nematodes by adding NJAU-HN1 suspension of *Nematoda* and M9 buffer to the culture medium, respectively. Meloidogyne incognita Line graph showing the change in the number of surviving organisms during 24 hours of co-culture; where Mi represents the control group with only M9 buffer added, and Mi+Cs represents the treatment group with NJAU-HN1 suspension added; * indicates significance, more * indicates greater significance;

[0032] Figure 4 This is a comparison chart of the growth of melon plants in the pot experiment of Example 4 of the present invention, showing the growth of the treatment group and the control group. The three pots on the left are the treatment group with the addition of NJAU-HN1 culture of kidney-shaped insect, and the three pots on the right are the control group with only sterile water.

[0033] Figure 5 This is the result of the pot experiment in Example 4 of the present invention showing the effect of the kidney-shaped insect NJAU-HN1 on plant growth; wherein, Figure 5The left figure in the chart is a box plot showing the fresh weight of plants in the treatment group with added NJAU-HN1 and the control group after sampling, root washing, and surface moisture removal. The right figure is a box plot comparing the dry weight of plants in the treatment group with added NJAU-HN1 and the control group after drying roots and stems at 50°C for 48 hours. NJAU-HN1 represents the treatment group inoculated with NJAU-HN1, and CK represents the control group that was irrigated only and had no additives.

[0034] Figure 6 This is a comparison of root growth between the potted plant treatment group and the control group in Example 4 of the present invention; wherein Figure 6 The left image in the figure is a picture of the roots, and the right image is a box plot of root length comparison. In the left image, the roots on the left are the roots of melons inoculated with the NJAU-HN1 culture of *Nephrodisiac*, and the roots on the right are the roots of the control group of melons. In the right image, NJAU-HN1 represents the treatment group inoculated with NJAU-HN1 of *Nephrodisiac*, and CK represents the control group that is only irrigated and has no additives. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it.

[0036] The PAS culture medium was prepared as follows: Solution I and Solution II were prepared by taking 0.142 g of disodium hydrogen phosphate and 0.136 g of potassium dihydrogen phosphate, and adding water to a final volume of 500 mL. Solution II was prepared by taking 0.12 g of sodium chloride, 0.4 mg of magnesium sulfate heptahydrate, and 0.6 mg of calcium chloride hexahydrate, and adding water to a final volume of 500 mL. Solutions I and II were sterilized at 121 °C for 20 min, cooled to room temperature, and then mixed at a 1:1 ratio to obtain the PAS culture medium.

[0037] M9 buffer is prepared by taking 1.5g of potassium dihydrogen phosphate, 3.0g of disodium hydrogen phosphate, 2.5g of sodium chloride and 0.06g of magnesium sulfate, adding 500mL of distilled water, and sterilizing at 121℃ for 20min.

[0038] Example 1: Isolation and purification of protist NJAU-HN1

[0039] This invention involves collecting rhizosphere soil samples from healthy melon experimental plants at the Sanya Research Institute of Nanjing Agricultural University. The samples were collected at a depth of 0–20 cm, sealed in sterile self-sealing bags, and brought back to the laboratory. Protists were isolated using a soil dilution method, as detailed below:

[0040] Mix the rhizosphere soil sample from the melon crop thoroughly. Weigh 1g of soil into a 50mL centrifuge tube and add 30mL of sterile deionized water. Then, place the centrifuge tube in a shaker at 250rpm and 20℃ and shake for 15min to ensure thorough mixing of the rhizosphere soil and release of soil protozoa. Remove the centrifuge tube from the shaker and let it stand for 10min. Then, collect the supernatant and add it to a 96-well plate, along with inactivated Escherichia coli (OD200). 600 =0.04) was used as food, and then cultured at 20℃ in the dark for 2 days. The growth of the protists was examined under an inverted microscope at 100×, 200× and 400×, and serial dilutions were performed and cultured at 20℃ for 2 days. Finally, single protist cells were picked up by capillary tubes and transferred to new 96-well plates to obtain pure protist cultures.

[0041] Example 2 Identification of the protist NJAU-HN1:

[0042] The pure cultures of protists obtained in Example 1 were identified by a combination of morphological observation and molecular biological analysis. The results of the morphological observations are shown below. Figure 1 This indicates that the NJAU-HN1 renal worm is highly active in PAS culture medium, has cilia all over its body, and moves rapidly. Figure 1 (Left image in the image); its reproductive cysts are round and accompanied by continuous rotational movement ( Figure 1 (See the middle image in the image); while the dormant cysts are also round, but are in a quiescent state ( Figure 1 (The right image in the image). Figure 1 The paper demonstrates the typical morphological characteristics of the kidney-shaped worm NJAU-HN1 at different physiological stages.

[0043] Based on morphological analysis, genomic DNA was further extracted from the *Nephroticosa* species NJAU-HN1. Using this DNA as a template, PCR amplification was performed using commonly used eukaryotic 18S rDNA primers EukA(fwd) / EukB(rev). (EukA(fwd): 5'--AACCTGGTTGATCCTGCCAGT--3') and (EukB(rev): 5'--TGATCCTTCTGCAGGTTCACCTAC--3'). The PCR amplification system was 50µL: 25µL of 2× Mix, 2µL each of forward and reverse primers, and ddH2O to a final volume of 50µL. The PCR program was set as follows: 95℃ pre-denaturation for 3 min; followed by 40 cycles, each cycle consisting of 95℃ denaturation for 15 s, 55℃ annealing for 15 s, and 72℃ extension for 1.5 min; a final extension at 72℃ for 5 min after the cycles, and storage at 16℃. The PCR amplification products were detected and recovered by 1.5% agarose gel electrophoresis and then sent to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing. The sequencing results are shown in SEQ ID NO: 1.

[0044]

[0045] The 18S rDNA gene sequence obtained from sequencing was BLAST aligned in the NCBI Nucleotide Sequence Database, and a phylogenetic tree was constructed using MEGA software for phylogenetic analysis. The results are shown in [Figure number missing]. Figure 2 The comparison results show that the sequence of the kidney-shaped protozoan NJAU-HN1 of this invention is consistent with that of the model protozoan kidney-shaped protozoan. Colpoda steinii The similarity was the highest, reaching 99.18%. Based on this molecular systematic analysis result, the protozoan *Nephroplasma reniformis* NJAU-HN1 isolated in this invention was identified as... Colpoda steinii . Figure 2 A phylogenetic tree based on the 18S rDNA gene sequence is presented.

[0046] The strain was classified as Nephroticosa NJAU-HN1. Colpoda steinii NJAU-HN1, Latin name Colpoda steinii It was deposited in October 2025 at the China Center for Type Culture Collection (address: Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province), with accession number CCTCC NO: C2025172 and deposit date of October 27, 2025.

[0047] Example 3: Co-culture experiment of protozoan NJAU-HN1 inhibiting the southern root-knot nematode, the pathogen of melon root-knot nematodes.

[0048] The tested melon root-knot nematode pathogen was the southern root-knot nematode ( Meloidogyne incognita The tested protozoan was *Nephroplasma reniformis* NJAU-HN1 from Example 2. Colpoda steinii NJAU-HN1), accession number: CCTCCNO: C2025172.

[0049] Preparation of *Nephrodisiac* NJAU-HN1 suspension: *Nephrodisiac* NJAU-HN1 was inoculated into PAS culture medium, and inactivated *Escherichia coli* (OD100) was added. 600 =0.04) was used as feed, and the cells were statically cultured at a constant temperature of 20℃ for 48 hours. After the culture was completed, 100µL of culture medium was taken and cell counting was performed under an inverted microscope. The concentration of NJAU-HN1 in *Nephrodisiac* was adjusted to 1×10⁻⁴ using M9 buffer. 4 cells / mL.

[0050] Preparation of a suspension of second-instar larvae of *Symplocos spp.* root-knot nematode*: Nematode eggs were isolated from the roots of melons infected with root-knot nematodes and incubated in sterile water at 28°C for 2 days to obtain second-instar larvae. The larval suspension was collected, centrifuged at 4500 rpm, and the supernatant was removed. This process was repeated to enrich the nematodes. 100 µL of the nematode suspension was used for microscopic counting, and finally diluted to 1 × 10⁻⁶ using M9 buffer. 3 nematodes / mL.

[0051] Set up a control group (Mi): take 50 μL of a concentration of 1×10 3 Second-instar Southern Root-knot Nematodes culture medium at nematodes / mL was mixed with 50 μL of sterile M9 buffer, and the resulting 100 μL mixture was added dropwise to a 96-well plate for a total of 3 replicates.

[0052] Set up a treatment group (Mi+Cs): take 50 μL of a concentration of 1×10 3 The concentration of second-instar southern root-knot nematode culture medium (nematodes / mL) was adjusted to 1×10⁻⁶ μL with 50 μL of M9 buffer. 4 Mix the NJAU-HN1 suspension of *Nephrodisiac* cells / mL, and add 100 μL of the resulting mixture to a 96-well plate for three replicates.

[0053] The growth of nematodes in the two experimental groups was observed under an inverted microscope. The number of surviving nematodes was recorded at 3h, 6h, 9h, 12h, 18h, and 24h. The results are as follows: Figure 3 As shown. Figure 3 For root-knot nematodes Meloidogyne incognita A line graph characterizing the change in the number of surviving root-knot nematodes (Meloidogyne incognita) after 24 hours of co-culture with NJAU-HN1 kidney-shaped nematodes and without kidney-shaped nematodes. Figure 3 The results showed that in the early stages of co-culture, *N. reniforme* NJAU-HN1 accelerated the growth of *Southern root-knot nematodes*. Meloidogyne incognita The death of the southern root-knot nematode, and ultimately the death of the southern root-knot nematode. Meloidogyne incognita The 24-hour survival rate of nematodes decreased by about 50%.

[0054] The above experiments demonstrate that the kidney-shaped parasite NJAU-HN1 of the present invention ( Colpoda steinii NJAU-HN1 can inhibit the growth of southern root-knot nematodes, and it has an inhibitory effect on the southern root-knot nematode Meloidogyneincognita, the pathogen of melon root-knot nematodes.

[0055] Example 4: Pot experiment on the promotion of melon growth by the kidney-shaped insect Colpoda steinii NJAU-HN1

[0056] The experiment set up two treatments: (1) a control group CK inoculated with an equal volume of sterile PAS culture medium; (2) inoculated with NJAU-HN1 renalis ( Colpoda steinii NJAU-HN1) and its culture were used in treatment groups Cs. Each treatment was repeated in triplicate.

[0057] The tested melon variety was Xinyugu, and the tested protozoan was Colpodasteinii NJAU-HN1 (exhibited in Example 2), preservation number: CCTCC NO: C2025172.

[0058] Preparation of NJAU-HN1 culture of *Nephrodisiac*: NJAU-HN1 ( Colpoda steinii NJAU-HN1 was inoculated into liquid PAS medium, and inactivated Escherichia coli (OD100) was added. 600 =0.04), placed in a constant temperature incubator at 20℃, and incubated statically. After 48 hours, 100 μL of culture medium was aspirated and counted under an inverted microscope to obtain the NJAU-HN1 culture of *Nephrodisiac*.

[0059] Cultivation of melon plants: To verify the direct growth-promoting effect of the NJAU-HN1 strain of *Nematodeus* on melon growth and to rule out the possibility of its indirect effect through the inhibition of soil pathogens (such as root-knot nematodes), this invention designed a rigorous pot-based control experiment, as follows:

[0060] The melon seedlings used in the pot experiment were collected from a local nursery in Sanya. Healthy, virus-free seedlings approximately 5 cm tall and free from pathogens were selected for transplanting. The potting soil was taken from the same topsoil layer (0-20 cm) at the Sanya Research Institute of Nanjing Agricultural University in Sanya. The collected soil underwent the following standardized sterilization and physicochemical property adjustment treatments: The soil was evenly spread and placed in a high-pressure steam sterilizer for moist heat sterilization (conditions: 121°C moist heat sterilization for 1 hour, for 2 consecutive days). After sterilization, it was aseptically sieved (2 mm aperture) to remove any plant debris, ensuring substrate homogeneity. Nutrient substrate and gravel (the weight ratio of soil, substrate, and gravel was 2:2:1; both substrate and gravel were tested and free of pathogens) were then added and mixed to restore the basic soil fertility level. The cultivation containers were sterilized 0.75-gallon pots. After transplanting, 10... 3 Each gram of soil was inoculated with NJAU-HN1 protozoan culture; the control group received an equal volume of sterile PAS culture medium free of protozoa. All potted plants were cultured in a sterile greenhouse for two weeks, after which plant growth was systematically observed, and the results are shown in the table below. Figures 4 to 6 .

[0061] Figure 4The three pots on the left show the growth status of melon plants in the treatment group inoculated with NJAU-HN1, indicating that NJAU-HN1 can have a beneficial effect on the growth of melon plants. Figure 4 The three pots on the right show the growth of melon plants in the control group that were only irrigated and had no additives. The results indicate that applying a culture of NJAU-HN1 (a type of nephrotic insect) promotes the growth of melon plants.

[0062] Figure 5 The left figure shows the fresh weight of the plants in the treatment group with added NJAU-HN1 and the control group after sampling, washing the roots and absorbing the surface moisture. It can be seen that the fresh weight of the melons in the culture with added NJAU-HN1 was significantly higher than that of the untreated control group. Figure 5 The right figure in the image is a box plot comparing the dry weight of the plants in the NJAU-HN1-inoculated group and the control group after drying the roots and stems at 50℃ for 48 hours. It can be seen that the dry weight of the melons cultured with NJAU-HN1 was significantly higher than that of the untreated control group. This indicates that the biomass of the plants inoculated with NJAU-HN1 was significantly increased compared to the control group.

[0063] Figure 6 The left image shows a comparison of root length between the melon plants in the treatment group inoculated with NJAU-HN1 and the control group. The left side shows the root of the melon plant inoculated with NJAU-HN1 culture, and the right side shows the root of the control group. It can be seen that the root growth of the melon plant inoculated with NJAU-HN1 culture is significantly better than that of the control group. Figure 6 The right figure in the figure is a box plot comparing the root length of the treatment group with added NJAU-HN1 and the control group. The results show that the root growth of the culture inoculated with NJAU-HN1 is significantly better than that of the control group.

[0064] The above experiments demonstrate that the protozoan *Nephroplasma reniformis* NJAU-HN1 can directly promote the growth of melon plants.

[0065] It should be noted that the kidney-shaped insect NJAU-HN1 of the present invention can be applied to all melon varieties, including but not limited to "Xinyugu" in the embodiments of the present invention.

[0066] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. Those skilled in the art can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A protozoan, *Nephroplasma reniformis* NJAU-HN1 ( Colpoda steinii NJAU-HN1), which is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: C2025172, and deposit date of October 27, 2025; The protozoan *Nematodea nematode* NJAU-HN1 is used to control root-knot nematode disease in melons and / or directly promote melon growth. The promotion of melon refers to promoting the biomass of the melon plant and / or the root growth of the melon.

2. A culture of the protozoan *Nephroplasia elegans* NJAU-HN1 as described in claim 1, characterized in that, The culture was Nephroticosa NJAU-HN1 and its metabolites; The culture was prepared by inoculating the protozoan *Neopterygium glomerulosa* NJAU-HN1 into PAS culture medium and adding inactivated *Escherichia coli* as feed, and culturing to obtain a culture of the protozoan *Neopterygium glomerulosa* NJAU-HN1.

3. A method for preparing a culture of the protozoan *Nephrodisiac* NJAU-HN1 as described in claim 2, characterized in that, The PAS culture medium is prepared as follows: disodium hydrogen phosphate and potassium dihydrogen phosphate are added to water to prepare solution I; sodium chloride, magnesium sulfate heptahydrate and calcium chloride hexahydrate are added to water to prepare solution II; solutions I and II are sterilized separately and then mixed to obtain the PAS culture medium.

4. The preparation method according to claim 3, characterized in that, Solution I contains 0.28~0.29 g / L of disodium hydrogen phosphate and 0.26~0.27 g / L of potassium dihydrogen phosphate. Solution II contains 0.235~0.245 g / L sodium chloride, 0.75~0.85 mg / L magnesium sulfate heptahydrate, and 0.115~0.125 mg / L calcium chloride hexahydrate. The volume ratio of solution I to solution II in the PAS culture medium is 1:0.95~1.

05.

5. The application of the protozoan NJAU-HN1 as described in claim 1 in the control of root-knot nematode disease in melon and / or in directly promoting melon growth; The promotion of melon refers to promoting the biomass of the melon plant and / or the root growth of the melon.

6. The application according to claim 5, characterized in that, The application involves inoculating the NJAU-HN1 renal nematode into the soil where melons are grown to control root-knot nematode disease in melons and / or directly promote melon growth.

7. The use of the protozoan NJAU-HN1 as described in claim 1 in the preparation of a biological agent for controlling root-knot nematode disease in melon and / or directly promoting melon growth; The promotion of melon refers to promoting the biomass of the melon plant and / or the root growth of the melon.

8. The application of a culture of the protozoan NJAU-HN1 as described in claim 2 in the control of root-knot nematode disease in melon and / or in directly promoting melon growth; The promotion of melon refers to promoting the biomass of the melon plant and / or the root growth of the melon.

9. The application according to claim 8, characterized in that, The application involves inoculating the culture into the soil where melons are grown to control root-knot nematode disease in melons and / or directly promote melon growth.

10. The use of a culture of the protozoan *Nematodeus* NJAU-HN1 as described in claim 2 in the preparation of a biological agent for controlling root-knot nematode disease in melon and / or directly promoting melon growth; The promotion of melon refers to promoting the biomass of the melon plant and / or the root growth of the melon.

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