Micrococcus endophyticus and application thereof in inhibiting plant root-knot nematode

Abstract

The application discloses an endomicrospira strain and application thereof in inhibiting plant root-knot nematodes, and relates to the field of microorganisms. The strain is preserved in the China General Microbiological Culture Collection Center on March 16, 2026, and the preservation number is CGMCC NO. 37922. The nucleotide sequence of 16S rRNA of the strain is shown in SEQ ID NO. 1. It is verified through experiments that TM2 has a significant toxic effect on root-knot nematodes, and has a good application potential in the field of biological control of root-knot nematodes. The strain has high nematicidal activity, and the biological bacterial agent is environment-friendly and has strong stability, and therefore has important application value in green prevention and control and quality improvement and yield increase of facility vegetable root-knot nematodes.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to an endophytic micrococcus and its application in inhibiting plant root-knot nematodes. Background Technology

[0002] Plant pathogenic nematodes are one of the most important pathogenic organisms that seriously harm crops, among which root-knot nematodes are the most prominent. Meloidogyne spp., root-knot nematode Root-knot nematodes are the most serious pests. As an obligate, live-feeding parasite, they can infect most cultivated vegetables, such as tomatoes and cucumbers, causing a 30% reduction in vegetable yield annually. They pose a serious threat to various economic crops, including vegetables and fruit trees, resulting in huge losses in agricultural production and severely jeopardizing my country's food security. However, many traditional chemical nematicides have been gradually banned or strictly restricted due to their high toxicity, residues, resistance, and environmental risks. Against this backdrop, biological control, with its advantages of being environmentally friendly and having high safety for non-target organisms, has gained widespread attention and importance globally.

[0003] Existing biocontrol bacteria are mostly concentrated in Bacillus, Pseudomonas, and Serratia, while endophytic micrococci are rarely reported in the field of root-knot nematode biocontrol. Therefore, developing a microorganism with toxic effects against root-knot nematodes is of great significance. Summary of the Invention

[0004] In view of this, the main objective of the present invention is to provide an endophytic micrococcus and its application in inhibiting plant root-knot nematodes, aiming to solve the problems of chemical residues, poisoning of non-target organisms, and environmental pollution in the process of using chemical methods to control plant nematode diseases in the prior art.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, an endophytic micrococcus, named TM2, was deposited on March 16, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.37922.

[0006] In a second aspect, the present invention provides a root-knot nematode biocontrol agent, the biocontrol agent comprising the endophytic micrococci or their cultures, ferments, metabolites or lysates described above.

[0007] Furthermore, the antibacterial agent also includes an auxiliary agent, which includes at least one of a carrier, wetting agent, dispersant, disintegrant, binder, stabilizer, and synergist.

[0008] Furthermore, the carrier is at least one of kaolin, diatomaceous earth, silica, and soluble starch; The wetting agent is silica; The dispersant is calcium lignosulfonate; The disintegrant is at least one of calcium chloride and sodium chloride; The binder is at least one of sucrose, polyethylene glycol, soluble starch, polyvinyl alcohol, and dextrin. The stabilizer is at least one of calcium carbonate, chitin, and polyvinyl alcohol; The synergist is at least one of chitin and chitosan.

[0009] Further, by weight percentage, the antibacterial agent comprises: 5%~10% wetting agent, 2%~5% dispersant, 2%~5% disintegrant, 2%~5% binder, 1%~5% synergist and 3%~8% stabilizer, and 62%~85% carrier loaded with endophytic micrococcal fermentation broth.

[0010] In a third aspect, the present invention provides a method for preparing the fermented product, comprising inoculating the aforementioned endophytic micrococcus into a fermentation medium for fermentation culture to obtain the product.

[0011] Furthermore, by weight percentage, the components of the fermentation medium are: 0.1%~0.5% beef extract, 0.1%~0.2% sucrose, 1%~3% peptone and 0.2%~0.8% magnesium sulfate.

[0012] Furthermore, the pH of the fermentation medium is 7-9; and / or the inoculum size is 5% to 10% by volume; and / or the culture temperature is 24-32℃; and / or the shaking speed is 200~250 r / min; And / or fermentation time is 60~100 hours.

[0013] The present invention also provides the application of the endophytic micrococcus or the root-knot nematode biocontrol agent described above in the inhibition of plant root-knot nematodes.

[0014] This invention also provides a method for controlling plant root-knot nematodes. Apply the endophytic micrococci or any of the above-described microbial agents to the soil or plants.

[0015] The beneficial effects of this invention include at least the following: (1) The endophytic micrococcus TM2 obtained from the rhizosphere soil of this invention can achieve a corrected mortality rate of 100% after the fermentation broth is treated with root-knot nematodes for 24 hours; after the optimized fermentation broth is treated with root-knot nematodes for 24 hours, the corrected mortality rate can reach more than 99%, and the nematode-killing activity is significantly better than that of existing conventional biocontrol strains, showing excellent potential for root-knot nematode control.

[0016] (2) Through systematic single-factor experiments and multi-factor orthogonal experiments, this invention determined the optimal fermentation medium formula for endophytic micrococcus TM2 to be 0.3% beef extract, 0.15% sucrose, 2% peptone, and 0.5% magnesium sulfate. The raw materials used in this medium are all conventional industrial raw materials, which are widely available and inexpensive. By eliminating expensive high-value-added components, the fermentation production cost is significantly reduced, which is conducive to large-scale industrial production.

[0017] (3) The endophytic micrococcus TM2 of the present invention can decompose cellulose, secrete urease and esterase, exhibit siderophore activity and IAA synthesis ability, and has a certain plant growth-promoting ability. While controlling root-knot nematodes, it can promote plant growth, achieve the dual effect of "disease prevention + growth promotion", reduce the use of chemical pesticides and chemical fertilizers, and is conducive to the green and sustainable development of agriculture.

[0018] (4) The endophytic micrococcus TM2 of the present invention is derived from rhizosphere soil and is an environmentally friendly indigenous microorganism. It poses low risks to non-target organisms and ecological environment, and is suitable for development into a commercial biological control product, which meets the development requirements of green agriculture and ecological security. Attached Figure Description

[0019] Figure 1 The results of the initial screening of 56 strains against Caenorhabditis elegans.

[0020] Figure 2 The results of the second screening for root-knot nematodes.

[0021] Figure 3 The results of TM2 identification are shown in the strain colony morphology (A) and Gram staining (B).

[0022] Figure 4 This is a cluster analysis diagram of strain TM2.

[0023] Figure 5 The results of the optimization of the culture medium for endophytic micrococcus TM2 are as follows: (A) Culture medium type, (B) Carbon source type, (C) N source type, (D) Inorganic salt type, (E) Orthogonal experiment.

[0024] Figure 6 For the optimized fermentation results of Endophytic Micrococcus TM2, (A) liquid volume, (B) inoculum size, (C) pH, (D) rotation speed, (E) temperature, and (F) time.

[0025] Figure 7These are the results of a biocompatibility test. The ingredients are: 1: Kaolin; 2: Talc powder; 3: Wheat bran; 4: Soluble starch; 5: Diatomite; 6: Sodium alginate; 7: Chitin; 8: Polyvinyl alcohol (PVA); 9: Sodium carboxymethyl cellulose (CMC-Na); 10: Ammonium dihydrogen phosphate; 11: Calcium carbonate; 12: Xanthan gum; 13: Polyethylene glycol (PEG); 14: Sucrose; 15: Fumed silica; 16: Nekal; 17: Sodium chloride; 18: Sodium dodecylbenzene sulfonate (SDBS); 19: Calcium lignosulfonate. 20: lignosulfonate; 21: Vitamin C; 22: Indole-3-acetic acid (IAA); 23: Dextrin; 24: Magnesium aluminum silicate; 25: Urea; 26: Ammonium sulfate; 27: Sodium tripolyphosphate; 28: Chitosan; 29: Sodium dodecyl sulfate (SDS); 30: Calcium chloride; 31: Medium control. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0028] The following specific embodiments illustrate the solution proposed in this invention: Example 1: Isolation and Identification of Strains 1.1 Screening of strains In the initial laboratory studies, 56 bacterial strains were isolated from rhizosphere soil, including 31 bacterial strains, 11 fungal strains, and 14 Streptomyces strains. These strains were inoculated from a -80℃ freezer onto suitable culture media (bacteria on LB medium, fungi on PSA medium, and Streptomyces on ISP medium) and cultured at 28℃ for 3-5 days. The strains were then inoculated into suitable liquid culture media and cultured in a constant temperature shaking incubator at 28℃ and 180 rpm for 48 hours to obtain the fermentation broth for each strain. This broth was used to treat *C. elegans*, and the number of dead and surviving strains was recorded. Data analysis was performed using SPAA software to preliminarily screen for strains with better nematode-killing effects. Figure 1 Then, root-knot nematodes were used for secondary screening, and finally, strain TM2 with the best nematicidal activity was selected. Figure 2 (Table 1).

[0029] Table 1. Mortality data of strains with good nematicidal effects

[0030] 1.2 Identification of strains Single colonies of purified TM2 strain were picked, streaked onto LB agar, and incubated at 28 °C for 2-3 days. Using Bergey's Manual of Bacteriological Identification and Microbiology as references, the size, color, shape, elevation, texture, and edge characteristics of each colony were observed, described, and recorded. The colony morphology of endophytic micrococcus TM2 was observed. Figure 3 Cells are often arranged in pairs, tetrads, octads, or three-dimensional encapsulated clusters; Gram-positive cocci, forming round, raised, smooth, opaque yellow colonies, approximately 0.5–3.5 mm in diameter. Physiological and biochemical characteristics of strain TM2 were determined according to the "Methods for Plant Disease Research," and the results are shown in Table 2. Strain TM2 can decompose cellulose, secrete urease and esterase, exhibiting siderophore activity and IAA synthesis ability, and possesses certain plant growth-promoting capabilities; it does not hydrolyze starch, liquefy gelatin, or have phosphorus, potassium, or nitrogen-fixing functions; drug sensitivity results showed it was sensitive to tetracycline and rifampin, but resistant to streptomycin.

[0031] Table 2 Physiological and biochemical characteristics of strain TM2

[0032] Note: +: positive reaction; -: negative reaction; S, L, and R in the table represent sensitive, moderately sensitive, and resistant, respectively. The grading criteria are: inhibition zone ≥18mm is sensitive, inhibition zone between 12mm and 18mm is moderately sensitive, and inhibition zone ≤12mm is resistant.

[0033] The 16S rRNA of strain TM2 was analyzed to obtain the nucleotide sequence of strain TM2, and a phylogenetic tree was constructed. Figure 4 Based on the morphology and physiological and biochemical characteristics of the strain, strain TM2 was ultimately identified as an endophytic micrococcus. This strain was deposited on March 16, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.37922.

[0034] The nucleotide sequence of the 16S rRNA of this strain is shown in SEQ ID NO. 1, as follows: Example 2: The toxic effect of endophytic micrococcus TM2 fermentation broth on root-knot nematodes The in vitro toxic activity of endophytic micrococcus TM2 fermentation broth against isolated root-knot nematodes was determined by the immersion method. The specific method is as follows: A single colony of endophytic micrococcus TM2 preserved in LB medium (10 g / L peptone, 5 g / L yeast extract and 10 g / L NaCl, sterilized at 121℃ for 30 min) was inoculated into 100 mL of LB liquid medium and cultured at 28℃ and 180 r / min for 48 h to obtain the fermentation stock broth. The fermentation stock broth was then diluted with sterile water by 1:1 to obtain a 50% fermentation broth. The fermentation stock broth was centrifuged to obtain a bacterial precipitate (centrifugation speed was 12000 rpm for 5 min). The precipitate was washed twice with sterile water to remove the bacterial solution remaining on the bacterial cells. An equal amount of sterile water was added and mixed to obtain a bacterial suspension.

[0035] 100 μL of fermentation stock broth, 50% fermentation broth, bacterial suspension, LB liquid medium, sterile water, and 20 μL of root-knot nematode suspension (approximately 60 nematodes) were respectively placed in 96-well plates, with each treatment replicated three times. After incubation at 28℃ for 24 h, the nematodes were observed using an optical microscope. Stimulation with 1 mol / L NaOH resulted in stiffness, which was considered a sign of death. Sterile water was used as a blank control. Corrected mortality rates were calculated, and the results are shown in Table 3.

[0036] Table 3. Toxicity of Endophytic Micrococcus TM2 under different treatments against nematodes

[0037] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences (p<0.05), and the same applies below.

[0038] Table 2 shows that strain TM2 has a good toxic effect on nematodes, and the corrected mortality rate of root-knot nematodes increases with increasing concentration. Treatment of nematodes with the fermentation stock broth (48 h incubation) and 50% fermentation broth for 24 h resulted in corrected mortality rates of 100% and 95%, respectively, significantly higher than the control. The corrected mortality rate of nematodes treated with the bacterial suspension for 24 h was 77.13%.

[0039] Example 3: Optimization of fermentation conditions for Endophytic Micrococcus TM2 This embodiment uses the corrected mortality rate of nematodes as a metric to screen out the fermentation conditions with the best nematode-killing effect.

[0040] (1) Screening of fermentation culture medium TM2 seed culture was inoculated at a volume fraction of 1% into 100 mL of different culture media: LB, NB, Y, NYBD, YSP, and CM. Each treatment was replicated three times. After incubation at 28℃ and 180 r / min for 48 h with shaking, the OD of the fermentation broth was measured. 600 nm The nematicidal activity of the fermentation broth in each culture medium was determined using the immersion method. The specific steps were as follows: 100 μL of fermentation broth from each culture medium and 20 μL of nematodes (approximately 40-60 nematodes) were added to each well of a 96-well plate. Corresponding culture medium treatments and sterile water treatments were set up as controls. Each treatment was repeated three times. After incubation at 28℃ for 24 h, the nematodes were observed using an optical microscope. Root-knot nematodes were stimulated with 1 mol / L NaOH; stiffness was considered a sign of death. The OD values ​​of the fermentation broth were then analyzed. 600 nm The optimal culture medium was determined by the nematode-corrected mortality rate. Corrected mortality rate (%) = (treatment mortality rate - control mortality rate) / (1 - control mortality rate) * 100%.

[0041] The results are as follows Figure 5 A. As shown in Table 4. The results show that the culture medium type is a key factor affecting the nematicidal activity of TM2. Strains TM2 exhibited strong root-knot nematode-killing activity when cultured in NA, LB, and Y media, with NA media showing the best effect; however, the toxicity was significantly reduced in NYBD and YSP media. Therefore, NA media was selected for subsequent experiments.

[0042] Table 4. Effects of different culture media on the nematicidal activity of TM2.

[0043] (2) Screening of carbon sources Based on NB medium, soluble starch, glucose, mannitol, maltose, beef extract, sucrose, lactose, and corn flour were used to replace the carbon sources in NB medium, while other components remained unchanged, resulting in NB medium containing different carbon sources. Seed cultures were inoculated into liquid medium at a 1% inoculum volume and incubated at 28℃ and 180 r·min⁻¹. -1 After culturing for 48 h under the specified conditions, the OD of the fermentation broth of the strain was measured. 600 nm The nematicidal activity of the strain's fermentation broth was determined by the immersion method, with the specific steps being the same as in step (1).

[0044] The results are as follows Figure 5 B. As shown in Table 5. The results show that different carbon sources have a significant impact on the nematicidal effect of *Micrococcus TM2*. Among them, the culture medium with beef extract as the carbon source showed the highest corrected mortality rate of root-knot nematodes after 24 h of fermentation broth treatment, at 94.53%. However, the culture medium with sucrose as the carbon source showed a lower OD... 600 nm Since the value is the largest, beef extract and sucrose were chosen as the carbon source for the culture medium for subsequent experiments.

[0045] Table 5. Effects of different carbon sources on the nematicidal activity of TM2.

[0046] (3) Screening of nitrogen sources Based on NB medium containing beef extract and sucrose, nitrogen sources in the medium were replaced by 1% urea, yeast extract, potassium nitrate, soybean flour, beef extract, tryptone, ammonium sulfate, and peptone, respectively, while other components remained unchanged, resulting in NB medium containing different nitrogen sources. Each treatment was repeated three times. After incubation at 28℃ and 180 r / min for 48 h with shaking, the OD of the fermentation broth was measured. 600 nm The nematicidal activity of the strain's fermentation broth was determined by the immersion method, with the specific steps being the same as in step (1).

[0047] The results are as follows Figure 5 C. As shown in Table 6. The results show that, when using different nitrogen sources as culture medium components, the corrected mortality rate of root-knot nematodes in the fermentation broth of *Micrococcus TM2*, from highest to lowest, was: tryptone, peptone, beef extract, soybean flour, yeast extract, ammonium sulfate, urea, and potassium nitrate. This indicates that strain TM2 utilizes organic nitrogen sources more efficiently than inorganic nitrogen sources. Among these, there was no significant difference in the corrected mortality rate of nematodes when tryptone and peptone were used as nitrogen sources, and peptone was chosen as the nitrogen source for subsequent experiments due to its low production cost.

[0048] Table 6. Effects of different nitrogen sources on the nematicidal activity of TM2.

[0049] (4) Screening of inorganic salts NB medium containing beef extract, sucrose, and peptone was supplemented with 0.5% sodium chloride, calcium chloride, copper sulfate, calcium carbonate, magnesium sulfate, dipotassium hydrogen phosphate, potassium chloride, ammonium sulfate, and disodium hydrogen phosphate to obtain NB medium containing different inorganic salts. Each treatment was repeated three times. After incubation at 28℃ and 180 r / min for 48 h with shaking, the OD of the fermentation broth was measured. 600 nm The nematicidal activity of the strain's fermentation broth was determined by the immersion method, with the specific steps being the same as in step (1).

[0050] The results are as follows Figure 5 Table 7 shows that the root-knot nematode-killing activity of strain TM2 was significantly affected by the type of inorganic salt in the culture medium. The corrected mortality rate of root-knot nematodes was highest when magnesium sulfate was used as a medium component, exceeding that of other inorganic salt treatments. Therefore, magnesium sulfate was selected as the inorganic salt for optimizing the component ratio in the culture medium.

[0051] Table 7. Effects of different inorganic salts on the nematicidal activity of TM2.

[0052] (5) Multi-factor orthogonal experiment Single-factor screening of the fermentation medium revealed that the optimal carbon source was beef extract and sucrose, the optimal nitrogen source was peptone, and the optimal inorganic salt was MgSO4. The four screened factors were then processed according to L9(3)... 4 The factors and levels of the orthogonal experiment were designed to compare the corrected mortality rate of root-knot nematodes under different component ratios and to determine the optimal component ratio of the fermentation medium. The factors and levels of the orthogonal optimization experiment of the endophytic micrococcus TM2 fermentation medium components are shown in Table 8.

[0053] Table 8 Factors and levels of orthogonal optimization experiment for the components of endophytic micrococcus TM2 fermentation medium

[0054] The results of the range analysis show (Table 9, Figure 5 (E) Beef extract had the greatest impact on the nematode activity of the fermentation broth of *Micrococcus endophyticus* TM2, followed by sucrose, while peptone had a smaller impact. The optimal ratio was A2B1C3D2. Since this ratio was not included in the orthogonal experimental design, to verify its nematode-killing effect, it was compared again with the group that showed the best nematode-killing effect in the orthogonal experiment (A2B2C3D1). The results are shown in Table 10. There was no significant difference in the nematode-killing effect between the two ratios, but the nematode-corrected mortality rate of the A2B1C3D2 ratio was higher (98.03%). Therefore, the optimal fermentation medium for *Micrococcus endophyticus* TM2 was determined to be 0.3% beef extract, 0.15% sucrose, 2% peptone, and 0.5% magnesium sulfate.

[0055] Table 9. Evaluation results of the range of optimized culture medium in orthogonal experiments

[0056] Table 10 Comparison of nematicide efficacy between the two combinations

[0057] (6) Screening of the optimal liquid volume Endophytic micrococcus TM2 was inoculated into the optimal fermentation medium obtained through screening above, and cultured at 28℃ and 180 r / min for 10 h to obtain seed culture. 30, 60, 90, 120, 150, and 180 mL of the optimal fermentation medium were then added to 250 mL Erlenmeyer flasks. Seed culture was inoculated into different volumes of medium at a 1% inoculation rate, and cultured at 28℃ and 180 r / min for 48 h. The OD of the fermentation broth obtained with different flask volumes was measured. 600 nmThe nematode mortality rate of different volumes of fermentation broth was determined using the immersion method. The specific steps were as follows: 100 μL of fermentation broth, NB medium, sterile water, and 20 μL of root-knot nematode suspension (approximately 40-60 nematodes) were placed in 96-well plates and incubated at 28℃ for 24 h. Observation was performed using an optical microscope. Root-knot nematodes were stimulated with 1 mol / L NaOH; stiffness was considered death. Each treatment was repeated three times. The OD value of the fermentation broth was then used as the nematode mortality rate. 600 nm The optimal liquid volume was determined using the value and the corrected mortality rate of nematodes as indicators.

[0058] The results are as follows Figure 6 A. As shown in Table 11. The results show that there is no significant difference in the mortality rate of root-knot nematodes when the liquid volume is 30-90 mL, and 90 mL is selected as the optimal liquid volume.

[0059] Table 11 Effect of different liquid volumes on the nematicidal efficacy of TM2

[0060] (7) Screening of the optimal inoculation amount Endophytic micrococcus TM2 was inoculated into the optimal fermentation medium obtained through screening above. A 250 mL fermentation flask was filled with 90 mL of the medium, and inoculation was carried out at inoculum rates of 1%, 3%, 5%, 7%, and 9%, respectively. The cultures were incubated at 28°C for 48 h, and the OD values ​​of the fermentation broths obtained at different inoculum rates were measured. 600 nm The nematode corrected mortality rate of the fermentation broth obtained from samples with different inoculation amounts was determined by the immersion method, and the specific steps were the same as in step (6).

[0061] The results are as follows Figure 6 B. As shown in Table 12. The results showed that different inoculation amounts had no significant effect on the efficacy of endophytic micrococcus TM2 in killing root-knot nematodes. However, the 7% inoculation amount resulted in the highest root-knot nematode mortality rate of 99.12%. Therefore, 7% was selected as the optimal inoculation amount.

[0062] Table 12 Effect of different inoculation amounts on the nematicidal efficacy of TM2

[0063] (8) Screening for optimal pH After adjusting the pH of the optimal fermentation medium obtained from the above screening to 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, the fermentation flasks were filled with 90 ml of the culture medium, and 7% of endophytic micrococci TM2 were inoculated. The cultures were then incubated at 28°C for 48 h. The OD values ​​of the fermentation broth at different pH values ​​were measured. 600 nm The values ​​were determined, and the nematode corrected mortality rate of fermentation broth at different pH values ​​was determined by immersion method, with the specific steps being the same as in step (6).

[0064] The results are as follows Figure 6 C. As shown in Table 13. The results show that the root-knot nematode activity of strain TM2 is highly sensitive to fermentation pH, and it is suitable for a slightly neutral to slightly alkaline environment. It has almost no activity at pH 5. When the initial pH is 8 and 8.5, the nematode correction mortality rate reaches more than 99%. Therefore, pH 8 or 8.5 is selected as the optimal initial pH value for the fermentation culture of endophytic micrococcus TM2.

[0065] Table 13 Effect of different pH values ​​on the nematicidal efficacy of TM2

[0066] (9) Screening of the optimal speed The optimal fermentation medium obtained from the above screening was adjusted to pH 8-8.5, the volume of the fermentation flask was 90 ml (250 mL), and the inoculum size was 7% of endophytic micrococci TM2. The shaking speeds were set to 140, 160, 180, 200, and 220 r / min, and the culture was carried out at 28℃ for 48 h. The OD values ​​of the fermentation broth obtained at different shaking speeds were measured. 600 nm The nematode corrected mortality rate of the fermentation broth obtained from samples taken at different time periods was determined by the immersion method, and the specific steps were the same as in step (6).

[0067] The results are as follows Figure 6 D. As shown in Table 14. The results show that different rotation speeds had no significant effect on the killing effect of endophytic micrococcus TM2 on root-knot nematodes. However, the root-knot nematode mortality rate was the highest at a rotation speed of 220 r / min, reaching 100%. Therefore, 220 r / min was selected as the optimal rotation speed.

[0068] Table 14 Effect of different rotation speeds on the nematicidal efficacy of TM2

[0069] (10) Screening of the optimal temperature The pH of the optimal fermentation medium obtained from the above screening was adjusted to 8-8.5. The volume of the fermentation flask was 90 ml (250 mL), and the inoculum size was 7% of endophytic micrococci TM2. The flasks were placed in shakers at temperatures of 24, 28, 32, 36, and 40 °C and cultured for 48 h with shaking. The shaker speed was set to 180 r / min. The OD values ​​of the fermentation broths obtained at different fermentation temperatures were measured. 600 nm The nematode corrected mortality rate of the fermentation broth obtained from samples taken at different time periods was determined by the immersion method, and the specific steps were the same as in step (6).

[0070] The results are as follows Figure 6E. As shown in Table 15. The results show that the nematode mortality rate was 100% when the temperature was between 24-32℃. When the temperature rose to 40℃, the nematode-killing activity of endophytic micrococcus TM2 decreased significantly, indicating that high temperature conditions were not suitable for its growth. Therefore, 24-32℃ can be selected as the optimal fermentation temperature for TM2.

[0071] Table 15 Effect of different fermentation temperatures on the nematicidal efficacy of TM2

[0072] (11) Screening of the optimal fermentation time The pH of the optimal fermentation medium obtained from the above screening was adjusted to 8-8.5. The volume of the fermentation flask was 90 ml, and the inoculum size was 7% of endophytic micrococci TM2. The culture was carried out at 28℃. During the fermentation period of 0-108 h, the OD of the fermentation broth was measured every 12 h. 600 nm The value was recorded until 108 hours later, and the nematode-corrected mortality rate of the fermentation liquid obtained from samples taken at different time periods was determined by the immersion method. The specific steps were the same as in step (6).

[0073] The results are as follows Figure 6 F. As shown in Table 16. The results showed that the nematicidal effect of the fermentation broth of Endophytic Micrococcus TM2 varied significantly with different fermentation times (P<0.05). The nematicidal activity of TM2 was significantly lower at 12 h and 24 h of fermentation; when the fermentation time was 72 h, 84 h, and 96 h, the corrected mortality rate of root-knot nematodes reached 100%. Therefore, 72 h was selected as the optimal fermentation time for Endophytic Micrococcus TM2.

[0074] Table 16 Effect of different fermentation times on the nematicidal efficacy of TM2

[0075] Example 4: Preparation of Endophytic Micrococcus TM2 Root-knot Nematode Biocontrol Agent (1) Compatibility determination of strain TM2 with various carriers and adjuvants To investigate the effects of different carriers and adjuvants on the growth of endophytic micrococcus TM2, 23 adjuvants were screened, including six carriers (kaolin, talc, diatomaceous earth, silica, wheat bran, and soluble starch) and 23 adjuvants (xanthan gum, CMC, soluble starch, dextrin, polyvinyl alcohol, polyethylene glycol, and sucrose), disintegrants (urea, ammonium sulfate, calcium chloride, ammonium dihydrogen phosphate, sodium chloride, and magnesium aluminum silicate), binders (xanthan gum, CMC, soluble starch, dextrin, polyvinyl alcohol, polyethylene glycol, and sucrose), stabilizers (sodium alginate, chitin, polyvinyl alcohol, CMC-Na, ammonium dihydrogen phosphate, and calcium carbonate), and synergists (chitin, vitamin C, chitosan, and indoleacetic acid). Solid culture media were prepared by mixing the tested carriers and auxiliaries, distilled water, and agar at a ratio of 5:100:1.5. After autoclaving for 30 min, the media were poured into 90 mm diameter petri dishes to prepare solid culture media containing different carriers and auxiliaries. After cooling and solidification, the fermentation broth was serially diluted and incubated at 28 °C for 48 h before counting the viable cells. NA medium was used as a control, and each treatment was repeated three times.

[0076] The results are as follows Figure 7 As shown in the figure. The results showed that kaolin, soluble starch, diatomaceous earth, chitin, polyvinyl alcohol, calcium carbonate, polyethylene glycol, sucrose, silica, sodium chloride, calcium lignosulfonate, dextrin, chitosan, and calcium chloride were all on the same order of magnitude as the control, indicating that they had no significant effect on the viable count of endophytic micrococci and could be further screened as effective components in the formulation.

[0077] (2) Screening of vectors The carrier in water-dispersible granules has a significant impact on the performance of the formulation. The optimal carrier is selected based on factors such as adsorption capacity, granulation difficulty, and price, ensuring it has little or no impact on bacterial growth. The carrier, selected through safety screening, is mixed thoroughly with the TM2 bacterial solution, stirred and kneaded until it "forms a clump when squeezed but crumbles easily when touched." The mixture is then passed through a 10-mesh sieve, granulated, and dried in a 52℃ oven for 1-2 hours to obtain the sample. Suitable fillers are then selected based on granulation difficulty and adsorption capacity.

[0078] The results are shown in Table 17. The results show that the adsorption capacity of different carriers for the fermentation broth of endophytic micrococcus TM2 varies significantly, with diatomaceous earth showing the highest adsorption capacity. Considering both granulation difficulty and particle fullness, diatomaceous earth exhibits the best overall performance due to its strong adsorption capacity, ease of granulation, and excellent particle fullness; therefore, diatomaceous earth was selected as the carrier for preparing TM2 water-dispersible granules.

[0079] Table 17. Vector Screening

[0080] (3) Screening of wetting agents and their dosage The aforementioned safety-screened wetting agent, silica, was mixed evenly with the carrier diatomaceous earth at proportions of 1%, 3%, 5%, 7%, and 9%, respectively. Endophytic micrococcus TM2 fermentation broth was added and the mixture was stirred and kneaded until it "forms a clump when kneaded but crumbles easily when touched." The mixture was then passed through a 10-mesh sieve, granulated, and dried in a 52℃ oven for 1-2 hours to obtain the sample. The wettability of the sample to water was determined (expressed as wetting time), and the amount of wetting agent was selected based on the wetting time. The wetting time was measured using a graduated cylinder test: 500 ml of 342 mg / ml hardness water was added to a 500 ml graduated cylinder. 1 g of sample was quickly poured into the graduated cylinder using a weighing dish without stirring, and a stopwatch was immediately started. The time it took for 99% of the sample to sink to the bottom of the cylinder was recorded. A time less than 1 minute was considered acceptable.

[0081] The results are shown in Table 18. The results indicate that within the experimental dosage range, as the amount of wetting agent added increases, the overall wetting time of the formulation decreases, while the wetting performance gradually improves. When the amount of wetting agent added reaches 7%, the wetting performance of the formulation is basically saturated. Therefore, 7% is selected as the optimal amount of wetting agent for this water-dispersible granule.

[0082] Table 18 Screening of wetting agent dosage

[0083] (4) Screening of dispersants and their dosage The safety-screened dispersant calcium lignosulfonate was mixed with 5% silica and diatomaceous earth carrier at proportions of 1%, 2%, 3%, 4%, and 5%, respectively. Endophytic micrococcus TM2 fermentation broth was added and stirred until the mixture was kneaded until it "forms a ball when kneaded but crumbles easily when touched." The mixture was then passed through a 10-mesh sieve, granulated, and dried in a 52℃ oven for 1-2 hours to obtain samples. The wettability and suspension rate of the samples were measured to screen for suitable dispersant dosage. The suspension rate was measured as follows: First, a sample was taken from a 200 mL beaker containing 50 mL of standard hard water. The sample was stirred at a constant speed by hand for 2 minutes and then placed in a 25℃ water bath for 4 minutes. The sample was then washed into a stoppered 250 mL graduated cylinder with standard hard water, water was added to the mark, the stopper was closed, and the cylinder was inverted 30 times within 1 minute, then allowed to stand for 30 minutes. Finally, 225 mL of water was washed out with a pipette, and the bottom 25 mL of suspension was transferred to a petri dish, dried, and the residue was weighed. The suspension rate is calculated using the following formula: [(m1-m2) / m1]*(10 / 9)*100.

[0084] The results are shown in Table 19. The results show that as the amount of dispersant increases, the suspension rate of the formulation initially increases and then decreases. At concentrations of 1% and 2%, the suspension rate is low, and the dispersion and suspension effects are poor; the suspension rate reaches its highest value and the dispersion effect is optimal at a concentration of 3%; further increases in concentration lead to a gradual decrease in suspension rate, resulting in particle agglomeration and sedimentation. Therefore, 3% was selected as the optimal dosage of dispersant.

[0085] Table 19 Screening of Dispersant Dosage

[0086] (5) Screening of disintegrants and their dosage The safety-screened disintegrants (calcium chloride and sodium chloride) were mixed evenly with 5% silica, 4% calcium lignosulfonate, and diatomaceous earth as a carrier. Endophytic micrococcus TM2 fermentation broth was added and kneaded until the mixture "forms a clump when kneaded but crumbles easily when touched." The mixture was then passed through a 10-mesh sieve, granulated, and dried in a 52℃ oven for 1-2 hours to obtain the sample. After drying, the disintegration performance of the sample was measured to screen for suitable disintegrant types. The disintegrant was then regranulated at proportions of 1%, 2%, 3%, 4%, and 5%, and the disintegration performance of the samples was measured to screen for appropriate disintegrant dosages. The measurement method was as follows: 0.5g of sample was added to a 100mL stoppered graduated cylinder containing 90mL of distilled water at 25℃. The cylinder was then clamped in the middle, the opening was sealed, and the cylinder was rotated at 8 r / min along the center until the sample completely disintegrated in the water. The time recorded was the disintegration time.

[0087] The results are shown in Tables 20-21. The results show that, at the same dosage, sodium chloride has a significantly shorter disintegration time than calcium chloride, exhibiting superior disintegration performance. Therefore, sodium chloride was determined to be the suitable disintegrant. Based on this, further screening of sodium chloride dosage was conducted, setting the addition amount to 1%~5%. The results showed that as the dosage of sodium chloride increased, the disintegration time of the formulation continuously shortened, and the disintegration performance continuously improved. The decrease in disintegration time was significant at dosages of 1%-2%. When the dosage reached 3%, the increase in disintegration time tended to level off, and the difference in disintegration time at dosages of 3% and 4% was small, indicating that the disintegration effect had essentially reached saturation. Considering both the formulation production cost and the disintegration effect, the disintegrant dosage for this water-dispersible granule was determined to be 3% sodium chloride.

[0088] Table 20 Screening of Disintegrant Types

[0089] Table 21 Screening of disintegrant dosage

[0090] (6) Screening of adhesives and their dosage Binders impart strength and toughness to water-dispersible granules, preventing them from easily disintegrating into powder. The aforementioned safety-screened binders (sucrose, polyethylene glycol, soluble starch, polyvinyl alcohol, dextrin) were mixed evenly with 5% silica, 4% calcium lignosulfonate, 3% sodium chloride, and diatomaceous earth as a carrier. Endophytic micrococcus TM2 fermentation broth was added and kneaded until the mixture "forms a ball when kneaded but crumbles easily when touched." The mixture was then passed through a 10-mesh sieve and granulated. After granulation, the samples were dried in a 52℃ oven for 1-2 hours to obtain the final sample. The disintegration properties and particle strength of the samples were measured after drying to screen for the optimal binder type. Subsequently, the binder was granulated again at proportions of 1%, 2%, 3%, 4%, and 5%, and the disintegration properties and particle strength of the samples were measured to screen for suitable binder dosages.

[0091] The results are shown in Tables 22-23. The results indicate that, at the same dosage, the disintegration performance and particle strength of water-dispersible granules prepared with different binders varied. Sucrose and soluble starch exhibited good disintegration performance but poor particle strength; polyvinyl alcohol had good particle strength but poor disintegration performance; dextrin and polyethylene glycol combined good disintegration performance and particle strength, resulting in superior overall quality. However, polyethylene glycol is expensive, and considering the actual application requirements of the formulation, dextrin was selected as the suitable binder. Further screening of dextrin dosage revealed that as the dextrin dosage increased, the disintegration time of the formulation gradually lengthened, and the particle strength gradually increased. At a dosage of 1%–2%, the particle strength was insufficient, and the finished product was easily loose; when the dosage increased to 3%, the disintegration performance remained good, and the particle strength reached a moderate level; further increases to 4% and 5% significantly increased the disintegration time, decreased the disintegration performance, and resulted in harder particles, failing to meet the requirements for water-dispersible granules. Considering disintegration performance, particle strength, and production cost, the binder dosage for this water-dispersible granule was determined to be 3% dextrin.

[0092] Table 22 Screening of Adhesive Types

[0093] Table 23 Screening of Adhesive Dosage

[0094] (7) Screening of stabilizers and their dosage The stabilizers (calcium carbonate, chitin, and polyvinyl alcohol) selected through safety screening were mixed evenly with 5% silica, 4% calcium lignosulfonate, 3% sodium chloride, 3% dextrin, and diatomaceous earth as a carrier. Endophytic micrococcus TM2 fermentation broth was added and kneaded until the mixture "forms a ball when kneaded but crumbles easily when touched." The mixture was then passed through a 10-mesh sieve and granulated. After granulation, the mixture was dried in a 52℃ oven for 1-2 hours to obtain the sample. After drying, the most suitable stabilizer type was selected based on the stabilizing effect of the sample. Subsequently, the stabilizer was granulated again at proportions of 1%, 3%, 5%, 7%, and 9%, and the optimal stabilizer dosage was selected based on the stabilizing effect of the sample.

[0095] The results are shown in Tables 24-25. The results show that when the stabilizer dosage is 3%, polyvinyl alcohol has the longest wetting time but poor wetting effect; chitin has a shorter wetting time but poor stability; calcium carbonate has a moderate wetting time and good stability, exhibiting the best overall performance, therefore calcium carbonate was selected as the stabilizer. Further screening of calcium carbonate dosage showed that the stability of the formulation gradually improved with increasing calcium carbonate dosage; the stabilizer effect was poor at 1% dosage, moderate at 3% dosage, and good at 5% and above. At higher dosages, the stabilizer's effect tended to plateau, leading to raw material waste. Considering production costs, wetting performance, and stability, while also taking into account raw material utilization, the optimal stabilizer dosage for this water-dispersible granule was determined to be 5% calcium carbonate.

[0096] Table 24 Screening of Stabilizer Types

[0097] Table 25 Screening of stabilizer dosage

[0098] (8) Screening of synergists The aforementioned synergists (chitin and chitosan), which have undergone safety screening, were mixed at 3% with 5% silica, 4% calcium lignosulfonate, 3% sodium chloride, 3% dextrin, 5% calcium carbonate, and diatomaceous earth as a carrier. Endophytic micrococcus TM2 fermentation broth was added and kneaded until the mixture "forms a clump when kneaded but crumbles easily when touched." The mixture was then passed through a 10-mesh sieve, granulated, and dried in a 52℃ oven for 1-2 hours to obtain the sample. After drying, suitable synergists were screened based on the sample's toxicity against root-knot nematodes.

[0099] The results are shown in Table 26. The results indicate that at a treatment concentration of 0.2 g / ml, the corrected mortality rate of the nematicide formulation after adding the synergist was higher than that of the control group without the synergist. The corrected mortality rate in the chitosan group was 69.46%, showing a limited increase; the corrected mortality rate in the chitin group reached 74.19%, demonstrating a more prominent synergistic effect in insecticidal activity. Based on comprehensive screening, chitin was determined to be the synergist for this water-dispersible granule formulation.

[0100] Table 26 Screening of Synergists

[0101] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0102] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0103] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An endophytic micrococcus ( Micrococcus endophyticus ), characterized in that, The endophytic micrococcus was deposited on March 16, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO. 37922.

2. A root-knot nematode biocontrol agent, characterized in that, The antibacterial agent includes the endophytic micrococcus or its culture, fermentation product, metabolite or lysate as described in claim 1.

3. The root-knot nematode biocontrol agent according to claim 2, characterized in that, The antibacterial agent also includes adjuvants, which include at least one of the following: carrier, wetting agent, dispersant, disintegrant, binder, stabilizer, and synergist.

4. The root-knot nematode biocontrol agent according to claim 3, characterized in that, The carrier is at least one of kaolin, diatomaceous earth, silica, and soluble starch. The wetting agent is silica; The dispersant is calcium lignosulfonate; The disintegrant is at least one of calcium chloride and sodium chloride; The binder is at least one of sucrose, polyethylene glycol, soluble starch, polyvinyl alcohol, and dextrin. The stabilizer is at least one of calcium carbonate, chitin, and polyvinyl alcohol; The synergist is at least one of chitin and chitosan.

5. The root-knot nematode biocontrol agent according to claim 4, characterized in that, By weight percentage, the antibacterial agent comprises: 5%~10% wetting agent, 2%~5% dispersant, 2%~5% disintegrant, 2%~5% binder, 1%~5% synergist and 3%~8% stabilizer, and 62%~85% carrier loaded with endophytic micrococcal fermentation broth.

6. The method for preparing the fermented product according to claim 2, characterized in that, This includes inoculating the endophytic micrococcus described in claim 1 into a fermentation medium for fermentation culture to obtain the product.

7. The preparation method according to claim 6, characterized in that, The components of the fermentation medium, by weight percentage, are: 0.1%~0.5% beef extract, 0.1%~0.2% sucrose, 1%~3% peptone and 0.2%~0.8% magnesium sulfate.

8. The preparation method according to claim 6, characterized in that, The pH of the fermentation medium is 7-9; and / or the inoculum size is 5% to 10% by volume; and / or the culture temperature is 24-32℃; and / or the shaking speed is 200~250 r / min; And / or fermentation time is 60~100 hours.

9. The application of the endophytic micrococcus according to claim 1, or the root-knot nematode biocontrol agent according to any one of claims 2-5, in inhibiting plant root-knot nematodes.

10. A method for controlling plant root-knot nematodes, characterized in that, Apply the endophytic micrococcus as described in claim 1 or the microbial agent as described in any one of claims 2-5 to the soil or plants.

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