Panaxicola novogranulibacter for preventing and treating soybean cyst nematode disease and application thereof

Abstract

The application provides a novel ginseng soil bacillus for preventing and treating soybean cyst nematode disease and an application thereof, relates to the technical field of agricultural production, and the Latin name of the novel ginseng soil bacillus is Neobacillus ginsengisoli, the preservation name is Sneb2539, the preservation place is the China General Microbiological Culture Collection Center, the preservation date is April 2, 2025, and the preservation number is CGMCC No.34086. The novel ginseng soil bacillus Sneb2539 discovered in the application has certain contact-killing activity on the second instar larvae of soybean cyst nematodes; the fermentation liquor can be used for root irrigation treatment on soybean seedlings or prepared into coated soybean seeds, so that the soybean cyst nematode disease can be effectively prevented and treated, the development of soybean cyst nematodes in soybean roots is delayed, and the vitality index and root length of seeds can be significantly increased.

Description

Technical Field

[0001] This invention relates to the field of agricultural production technology, and in particular to a novel Bacillus subtilis strain for the prevention and control of soybean cyst nematode disease and its application. Background Technology

[0002] Soybeans are an important crop, providing sustainable protein and oils worldwide. The soybean cyst nematode, belonging to the order Heterodermales, family Heterodermaceae, and genus Heteroderm, is an absolute parasitic, highly evolved nematode. It can infect soybeans 3-6 times within a single growing season, making it one of the most destructive pathogens in soybean production. Currently, soybean cyst nematode disease can cause yield reductions of 10%-30%, and in severe cases, 70%-90%, or even total crop failure. Even without obvious symptoms, soybean cyst nematode disease can lead to a 10%-20% decrease in soybean yield. Globally, the annual economic losses due to this disease reach as high as US$2.6 billion. Furthermore, the soybean cyst nematode can cause damage throughout the entire growth cycle of soybeans. It primarily damages the roots, preventing the roots from effectively absorbing nutrients, leading to malnutrition in the above-ground parts, resulting in symptoms such as yellowing, wrinkling, stunting, small pods and seeds, and even failure to form pods. Therefore, soybean cyst nematode disease has become a major factor limiting soybean yield, and the prevention and control of soybean cyst nematode disease is quite challenging.

[0003] Although most chemical nematicides have proven effective in controlling soybean cyst nematode disease, they also pose problems such as environmental pollution, damage to soil ecosystems, impacts on human and animal safety, and the development of resistance. Therefore, developing an environmentally friendly, safe, and highly effective control technology for soybean cyst nematode disease is crucial to reducing the use of chemical pesticides. Summary of the Invention

[0004] The purpose of this invention is to provide a novel *Neobacillus ginsengisoli* strain for the control of soybean cyst nematode disease and its applications. Targeting the soybean cyst nematode, *Neobacillus ginsengisoli* with highly efficient nematode-resistant activity was screened from soil. This provides a new resource for the research and development of novel and effective biocontrol agents against soybean cyst nematodes, reducing or replacing the use of chemical agents in the control of soybean cyst nematode disease, thereby reducing the environmental damage and threats to human health posed by chemical agents.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a strain of *Neobacillus ginsengisoli* for the prevention and control of soybean cyst nematode disease. The Latin name of *Neobacillus ginsengisoli* is Sneb2539, the deposit location is China General Microbiological Culture Collection Center (CGMCC), the deposit date is April 2, 2025, and the deposit number is CGMCCNo.34086.

[0007] The present invention also provides the application of the aforementioned *Bacillus ginseng* in the prevention and control of soybean cyst nematode disease.

[0008] The present invention also provides the application of the aforementioned *Bacillus ginseng* in the preparation of products for the prevention and control of soybean cyst nematode disease.

[0009] The present invention also provides a fermentation broth of Sneb2539 strain for the prevention and control of soybean cyst nematode disease, wherein the Sneb2539 strain fermentation broth contains the aforementioned *Bacillus ginseng*.

[0010] The present invention also provides a method for preparing the fermentation broth of the Sneb2539 strain, comprising the following steps: inoculating the novel Bacillus ginsenge in a liquid culture medium for fermentation to obtain the fermentation broth of the Sneb2539 strain.

[0011] Preferably, the liquid culture medium is LB liquid medium, the fermentation time is 10–14 h, the fermentation temperature is 35–39 °C, the fermentation speed is 100–300 r / min, and the concentration of the Sneb2539 strain fermentation broth is 8.0 × 10⁻⁶. 7 ~1.2×10 8 CFU / mL.

[0012] The present invention also provides the application of the fermentation broth of the Sneb2539 strain in alleviating soybean cyst nematode disease.

[0013] The present invention also provides a method for alleviating soybean cyst nematode disease using the fermentation broth of the Sneb2539 strain, comprising the following steps: drenching soybean seedlings with the fermentation broth of the Sneb2539 strain.

[0014] The present invention also provides a method for alleviating soybean cyst nematode disease using the fermentation broth of the Sneb2539 strain, comprising the following steps: coating soybean seeds with the fermentation broth of the Sneb2539 strain.

[0015] The beneficial effects of this invention are as follows:

[0016] (1) The present invention found that the fermentation broth of *Bacillus ginseng* Sneb2539 has a certain contact killing activity against second-instar larvae of soybean cyst nematodes.

[0017] (2) Soaking and coating soybean seeds with the fermentation broth of *Sneb. ginseng* can significantly increase the seed vigor index and root length.

[0018] (3) In indoor pot experiments, the fermentation liquid of Bacillus ginseng Sneb2539 can effectively prevent soybean cyst nematode disease and delay the development of soybean cyst nematodes in soybean roots when applied to soybean seedlings by root irrigation or coating soybean seeds, without inhibiting soybean growth.

[0019] Preservation Instructions

[0020] A novel *Neobacillus ginsengisoli* strain, with the accession name Sneb2539 and Latin name Neobacillus ginsengisoli, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on April 2, 2025. The accession number is CGMCC No. 34086. Attached Figure Description

[0021] Figure 1 A technical roadmap for screening, identifying, and studying the efficacy of biocontrol bacteria for the prevention and control of soybean cyst nematode disease;

[0022] Figure 2 A phylogenetic tree for strain Sneb2539 was constructed based on the 16S rDNA sequence. Detailed Implementation

[0023] This invention provides a strain of *Neobacillus ginsengisoli* for the prevention and control of soybean cyst nematode disease. The Latin name of *Neobacillus ginsengisoli* is Sneb2539, the deposit location is China General Microbiological Culture Collection Center (CGMCC), the deposit date is April 2, 2025, and the deposit number is CGMCCNo.34086.

[0024] The present invention also provides the application of the aforementioned *Bacillus ginseng* in the prevention and control of soybean cyst nematode disease.

[0025] The present invention also provides the application of the aforementioned *Bacillus ginseng* in the preparation of products for the prevention and control of soybean cyst nematode disease.

[0026] The present invention also provides a fermentation broth of Sneb2539 strain for the prevention and control of soybean cyst nematode disease, wherein the Sneb2539 strain fermentation broth contains the aforementioned *Bacillus ginseng*.

[0027] The present invention also provides a method for preparing the fermentation broth of the Sneb2539 strain, comprising the following steps:

[0028] The Sneb2539 strain fermentation broth can be obtained by inoculating the aforementioned novel Bacillus subtilis into a liquid culture medium for fermentation.

[0029] In this invention, the liquid culture medium is preferably LB liquid culture medium, the fermentation time is preferably 10-14 h, more preferably 12 h, the fermentation temperature is preferably 35-39 °C, more preferably 37 °C, the fermentation speed is preferably 100-300 r / min, more preferably 200 r / min, and the concentration of the Sneb2539 strain fermentation broth is preferably 8.0 × 10⁻⁶. 7 ~1.2×10 8 CFU / mL, further preferably 1.0 × 10⁻⁶ 8 CFU / mL.

[0030] The present invention also provides the application of the fermentation broth of the Sneb2539 strain in alleviating soybean cyst nematode disease.

[0031] The present invention also provides a method for alleviating soybean cyst nematode disease using the fermentation broth of the Sneb2539 strain, comprising the following steps: drenching soybean seedlings with the fermentation broth of the Sneb2539 strain.

[0032] The present invention also provides a method for alleviating soybean cyst nematode disease using the fermentation broth of the Sneb2539 strain, comprising the following steps: coating soybean seeds with the fermentation broth of the Sneb2539 strain.

[0033] The technical route for screening, identifying, and studying the efficacy of biocontrol bacteria for controlling soybean cyst nematode disease in this application is as follows: Figure 1 .

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] The LB solid culture medium used in this application consists of: 10.0g peptone, 5.0g yeast extract, 5.0g sodium chloride, 1.0g glucose, and 15-20g agar. The preparation method is as follows: dissolve the above raw materials in 1L of distilled water and sterilize by moist heat at 121℃ for 30min under pH 7.0±0.2.

[0036] The LB liquid culture medium consists of: 10.0g peptone, 5.0g yeast extract, 5.0g sodium chloride, and 1.0g glucose. The preparation method is as follows: dissolve the above raw materials in 1L of distilled water and sterilize by moist heat at 121℃ for 30min under pH 7.0±0.2.

[0037] The preparation method of 30% glycerol is as follows: mix 70 mL of glycerol with 30 mL of deionized water, and sterilize by moist heat at 121 °C for 30 min.

[0038] Example 1: Obtaining the novel ginsengisoli strain Sneb2539

[0039] Soil samples were taken from sweet potato fields in Ordos City, Inner Mongolia Autonomous Region (109.781°E, 39.608°N). 10g of soil sample was weighed and placed in a 250mL Erlenmeyer flask containing 90mL of sterile water in a clean bench, and then serially diluted to 10⁻⁶. -2 10 -3 10 -4 Soil suspensions of corresponding concentrations were obtained. 25 μL of each concentration of soil suspension was spread onto LB solid medium and cultured at 37 °C for 12 h. Single colonies were picked for purification, and the resulting fermentation broth was mixed with an equal volume of 30% glycerol and frozen at -80 °C.

[0040] Example 2: Culture of Neobacillus ginsengisoli Sneb2539 strain

[0041] First, *Neobacillus ginsengisoli* was fermented: Sneb2539, preserved in cryopreservation tubes, was activated on LB solid medium. After 12 hours of culture, a single colony with good growth was picked and inoculated into 150 mL of LB liquid medium. The fermentation temperature was 37℃, the shaker speed was 200 r / min, and the fermentation time was 12 hours, yielding a concentration of 1.0 × 10⁻⁶. 8 Fermentation broth of Sneb2539 strain CFU / mL.

[0042] This fermentation liquid can be directly applied to the roots of soybean seedlings or coated soybean seeds. Both root application and coating treatments can reduce the damage of cyst nematode disease to soybeans.

[0043] Example 3: Identification of Neobacillus ginsengisoli Sneb2539 strain

[0044] The taxonomic position of the strain was determined by morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis.

[0045] Morphological observation: The strain was activated on LB solid medium using the streak plate method, and then incubated upside down in a 37℃ constant temperature incubator for 12 hours. The colony morphology, color, odor, etc. were observed.

[0046] Physiological and biochemical characteristics: In a clean bench, break open the neck of the physiological and biochemical tube (purchased from Qingdao Haibo Biotechnology Co., Ltd.), pick up a fresh single colony with an inoculation loop and inoculate it into the tube, seal it with sealing film, place it in a 37°C constant temperature incubator, incubate it according to the instructions in the physiological and biochemical reagent kit (Qingdao Haibo Biotechnology Co., Ltd.), add the corresponding reagents as required, and observe the final results.

[0047] 16S rDNA sequence analysis: DNA was extracted according to the bacterial DNA extraction method described by Sutyak et al. (Sutyak KE, Wirawan RE, Aroutcheva AA and Chikindas ML. 2008. Isolation of the Bacillus subtilis antimicrobialpeptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens[J]. Journal of Applied Microbiology, 104, (1067-1074).). Using genomic DNA as a template, 16S rDNA gene amplification was performed using universal bacterial primers 27F (SEQ ID NO.1: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO.2: 5'-ACGGCTACCTTGTTACGACTT-3'). The 25 μL PCR amplification system consisted of: 1 μL each of upstream and downstream primers, 2 μL DNA template, 12.5 μL 2×Taq MasterMix (Dye olus), and 8.5 μL ddH2O. The PCR amplification conditions were: 94℃ pre-denaturation for 1.5 min, 94℃ denaturation for 0.5 min, 58℃ annealing for 0.5 min, 72℃ extension for 1 min, 30 cycles, followed by a final extension at 72℃ for 5 min, and storage at 4℃. After 1.0% agarose gel electrophoresis, the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared and analyzed against the NCBI BLAST database. A phylogenetic tree was constructed using MEGA 7.0 software and the Neighbor-joining method. (See figure). Figure 2The strain sequence was then submitted to NCBI's GenBank, obtaining accession number (PV423516). The results showed that the strain with the highest homology to Sneb2539 (accession number PV423516) was *Neobacillus ginsengisoli*, accession numbers MK014249.1 and MZ067923.1. Therefore, this strain was identified as *Neobacillus ginsengisoli*.

[0048] Example 4: Contact killing test of fermentation broth of Neobacillus ginsengisoli Sneb2539 against soybean cyst nematodes.

[0049] The fermentation broth of the strain in Example 2 was centrifuged at 8000 r / min for 5 min, and the supernatant was collected to obtain the fermentation supernatant of strain Sneb2539, which was used for the experiment of poisoning second-instar larvae of soybean cyst nematodes.

[0050] Soybean root propagation was used to obtain cyst soil containing soybean cyst nematodes (Heteroderaglycines). After sieving out the nematode eggs, they were transferred to a funnel with a filter cloth for hatching. The eggs were collected once a day to prepare a nematode suspension.

[0051] Take 400 μL of Sneb2539 strain fermentation supernatant and add 100 μL of nematode suspension (containing 50 second-instar larvae of soybean cyst nematodes). Treat for 24 h and 48 h, observe nematode mortality, and record the total number of nematodes and the number of dead nematodes. Calculate the mortality rate and corrected mortality rate. At the same time, treat with equal volumes of ddH2O and LB liquid medium to replace the Sneb2539 strain fermentation supernatant as a control group, with other treatment steps being the same.

[0052] Mortality rate (%) = (Number of dead nematodes / Total number of nematodes) × 100%; Equation 1

[0053] Corrected mortality rate (%) = (nematode mortality rate in treatment group - nematode mortality rate in control group) / (1 - nematode mortality rate in control group) × 100% Equation 2

[0054] Table 1. Contact killing effect of *Bacillus ginseng* Sneb2539 on second-instar larvae of soybean cyst nematode.

[0055]

[0056] Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level according to Duncan's test.

[0057] Note:Date are mean±SD.Different letters in the same column indicate significant difference at P<0.05levelbyDuncan's test.

[0058] Table 1 shows that the corrected mortality rate of second-instar soybean cyst nematode larvae in the fermentation supernatant of *Bacillus ginseng* strain Sneb2539 reached 68.44% at 24 hours and 73.34% at 48 hours. The results indicate that *Bacillus ginseng* strain Sneb2539 can effectively kill second-instar soybean cyst nematode larvae through contact.

[0059] Example 5: Seed germination test of Neobacillus ginsengisoli Sneb2539 strain

[0060] The liquid fermentation culture method described in Example 2 above yielded a concentration of 1.0 × 10⁻⁶. 8 Fermentation broth of Sneb2539 strain CFU / mL, for later use.

[0061] The soybean variety is Liaodou 15; the seeds were sterilized using chlorine gas and are ready for use.

[0062] Instruments and equipment: steam sterilizer, electric thermostatic drying oven, electric thermostatic incubator, clean workbench, disposable petri dishes, shaking incubator.

[0063] After sterilization, seeds were soaked in the Sneb2539 strain fermentation broth for 3 minutes, then placed in sterile Petri dishes (d = 90 mm) lined with moistened sterile filter paper. Simultaneously, equal volumes of ddH2O and LB liquid medium were used as control groups to replace the Sneb2539 strain fermentation broth; all other treatment steps were the same. Each group was repeated three times, with 25 seeds per replicate. The Petri dishes were incubated in a constant temperature and humidity incubator at 25°C. Germination was counted after 24 hours, once daily. After 6 days of incubation, the germination index and germination rate were calculated, and root length was measured to calculate the vigor index.

[0064] Germination rate (%) = (Number of germinated seeds / Number of seeds tested) × 100%; Equation 3

[0065] Germination index = Σ(number of germinated germinations at different times of day / corresponding number of germination days); Equation 4

[0066] Seed vigor index = seedling length × germination index (Formula 5)

[0067] Table 2. Effects of Bacillus subtilis coating on soybean seeds on their germination and growth.

[0068] Germination rate / % 86.67±6.11a 85.33±6.11a 88.00±8.00a Germination Index 28.08±3.06a 24.84±1.22a 23.67±1.97a Seed Vigor Index 659.02±71.88b 737.05±36.28b 912.24±76.06a Root length / cm 23.47±6.07c 31.00±4.77b 38.55±6.55a

[0069] Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level according to Duncan's test.

[0070] Note:Date are mean±SD.Different letters in the same column indicate significant difference at P<0.05levelbyDuncan's test.

[0071] Table 2 shows that the seed vigor index and root length of soybeans treated with Sneb2539 fermentation broth were significantly increased compared with the LB liquid medium control group, increasing by 23.77% and 24.35%, respectively; the germination rate and germination index were not significantly different compared with the LB liquid medium control group. This indicates that soaking and coating soybean seeds with Sneb2539 fermentation broth improves seed vigor index, promotes radicle growth, and has no inhibitory effect on soybean seed germination.

[0072] Example 6: Experiment on the efficacy of root irrigation with fermentation broth of Neobacillus ginsengisoli Sneb2539 strain in potted plants under potted conditions.

[0073] The liquid fermentation culture method described in Example 2 above yielded a concentration of 1.0 × 10⁻⁶. 8 Fermentation broth of Sneb2539 strain CFU / mL, for later use.

[0074] Prepare a nematode suspension according to the aforementioned nematode culture method, and set it aside for later use.

[0075] The soybean variety is Liaodou 15; the seeds were sterilized with chlorine and cultured until the true leaf stage for later use.

[0076] The control groups were drenched in ddH2O and inoculated with second-instar soybean cyst nematode larvae (J2), and drenched in LB liquid medium and inoculated with second-instar soybean cyst nematode larvae, respectively. The treatment groups were drenched in Sneb2539 strain fermentation broth and inoculated with second-instar soybean cyst nematode larvae. Each seedling was drenched in 10 mL of Sneb2539 strain fermentation broth, while each seedling in the control group was drenched in 10 mL of ddH2O or LB liquid medium. After 24 h, each seedling was given 2 mL of a suspension of second-instar larvae (approximately 1000 second-instar soybean cyst nematode larvae). Each group had 9 replicates, with 1 seedling per replicate, and all seedlings were randomly placed. After 15 days of cultivation, the plant height, root length, root fresh weight, and plant fresh weight of soybean plants were investigated, as well as the number of cysts and the number of nematodes of each instar in the roots (including the number of J2, J3, and J4, where the number of J2 is the number of second-instar larvae of soybean cyst nematodes, the number of J3 is the number of third-instar larvae of soybean cyst nematodes, and the number of J4 is the number of fourth-instar larvae of soybean cyst nematodes). The total number of nematodes, total number / gram weight, nematode inhibition rate, J2 / total number, J3 / total number, and J4 / total number were calculated.

[0077] Total number of nematodes = number of J2 worms + number of J3 worms + number of J4 worms + number of cysts; Equation 6

[0078] Number of nematodes per gram of root = Total number of nematodes / Fresh weight of root; Equation 7

[0079] Nematode inhibition rate (%) = (Number of root-knot nematodes per gram in control group - Number of root-knot nematodes per gram in treatment group) / Number of root-knot nematodes per gram in control group × 100%; Equation 8

[0080] J2 / Total (%) = J2 number / Total number of nematodes × 100%; Equation 9

[0081] J3 / Total (%) = Number of J3 nematodes / Total number of nematodes × 100%; Equation 10

[0082] J4 / Total (%) = Number of J4 worms / Total number of nematodes × 100% Equation 11

[0083] Table 3. Effects of root irrigation with *Bacillus subtilis* from ginseng soil on soybean cyst nematode infection.

[0084] <![CDATA[ddH2O]]> 3.12±0.28b 114.22±30.21a 36.23±6.78a - LB liquid culture medium 3.29±0.21b 118.44±14.83a 36.18±5.21a - Sneb2539 3.59±0.19a 74.22±6.00b 20.72±1.78b 42.73

[0085] Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level according to Duncan's test.

[0086] Note:Datearemean±SD.Differentletters in the same column indicate significantdifferenceat P<0.05levelbyDuncan's test.

[0087] Table 4. Effects of root irrigation with *Bacillus subtilis* from ginseng soil on the development of soybean cyst nematodes.

[0088] Number of cysts 2.33±1.73a 1.78±1.48a 1.00±0.87a J2 (Article) 72.56±18.67a 76.67±11.55a 53.89±5.09b J3 (article) 26.11±8.16a 27.33±5.61a 14.67±3.61b J4 (item) 13.22±5.33a 12.67±3.81a 4.67±2.24b J2 / Total (%) 63.83±4.73b 64.77±5.38b 72.57±2.43a J3 / Total (%) 22.86±3.82a 23.10±3.92a 19.73±4.42a J4 / Total (%) 11.38±3.04a 10.66±2.60a 6.34±3.10b

[0089] Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level according to Duncan's test.

[0090] Note:Date are mean±SD.Different letters in the same column indicate significant difference at P<0.05levelbyDuncan's test.

[0091] Table 5. Effects of root irrigation with *Bacillus subtilis* from ginseng soil on soybean plant growth.

[0092]

[0093]

[0094] Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level according to Duncan's test.

[0095] Note:Date aremean±SD.Differentletters in the same columnindicatesignificantdifference atP<0.05level byDuncan's test.

[0096] As shown in Table 3, in the pot experiment under greenhouse conditions, the number of nematodes was significantly lower than that of the LB liquid medium control group after root irrigation with the fermentation broth of Sneb2539 strain in soybean seedlings, with a nematode inhibition rate of 42.73%. Therefore, in the pot experiment, *Bacillus ginseng* Sneb2539 can effectively control soybean cyst nematode disease.

[0097] Table 4 shows that in the greenhouse pot experiment, after root irrigation with the fermentation broth of strain Sneb2539 on soybean seedlings, the proportion of J2 significantly increased compared with the control group on LB liquid medium, while the proportion of J4 significantly decreased. Therefore, in the greenhouse pot experiment, *Bacillus ginseng* Sneb2539 can delay the development of soybean cyst nematodes in soybean roots.

[0098] Table 5 shows that in the pot experiment under greenhouse conditions, the root fresh weight of soybean seedlings treated with the fermentation broth of strain Sneb2539 increased significantly by 9.12% compared with the LB control group. Plant height, root length, and plant fresh weight showed no significant differences compared with the LB liquid culture medium control group. Therefore, in the greenhouse experiment, *Bacillus ginseng* strain Sneb2539 had no inhibitory effect on the growth of soybean seedlings.

[0099] Example 7: Experiment on the efficacy of coating treatment with fermentation broth of Neobacillus ginsengisoli Sneb2539 strain under pot cultivation conditions.

[0100] The liquid fermentation culture method described in Example 2 above yielded a concentration of 1.0 × 10⁻⁶. 8 Fermentation broth of Sneb2539 strain CFU / mL, for later use.

[0101] Prepare a nematode suspension according to the aforementioned nematode culture method, and set it aside for later use.

[0102] The soybean variety is Liaodou 15; the seeds were sterilized with chlorine and cultured until the true leaf stage for later use.

[0103] Control groups consisted of seeds coated with ddH2O and inoculated with second-instar soybean cyst nematode larvae, and seeds coated with LB liquid medium and inoculated with second-instar soybean cyst nematode larvae. Treatment groups consisted of seeds coated with Sneb2539 strain fermentation broth and inoculated with second-instar soybean cyst nematode larvae. Sterilized seeds were soaked in Sneb2539 strain fermentation broth for 3 minutes, while the control groups were soaked in an equal volume of ddH2O or LB liquid medium for 3 minutes. Seeds were then sown in sterilized sand, and at the true leaf stage, each seedling was treated with 2 mL of a suspension of second-instar larvae (approximately 1000 second-instar soybean cyst nematode larvae). Each group had 9 replicates, with 1 seedling per replicate, all randomly placed. After 15 days of cultivation, the plant height, root length, root fresh weight, and plant fresh weight of soybean plants were investigated, as well as the number of cysts and the number of nematodes of each instar in the roots (including the number of J2, J3, and J4, where the number of J2 is the number of second-instar larvae of soybean cyst nematodes, the number of J3 is the number of third-instar larvae of soybean cyst nematodes, and the number of J4 is the number of fourth-instar larvae of soybean cyst nematodes). The total number of nematodes, total number / gram weight, nematode inhibition rate, J2 / total number, J3 / total number, and J4 / total number were calculated.

[0104] Total number of nematodes = number of J2 worms + number of J3 worms + number of J4 worms + number of cysts; Equation 6

[0105] Number of nematodes per gram of root = Total number of nematodes / Fresh weight of root; Equation 7

[0106] Nematode inhibition rate (%) = (Number of root-knot nematodes per gram in control group - Number of root-knot nematodes per gram in treatment group) / Number of root-knot nematodes per gram in control group × 100%; Equation 8

[0107] J2 / Total (%) = J2 number / Total number of nematodes × 100%; Equation 9

[0108] J3 / Total (%) = Number of J3 nematodes / Total number of nematodes × 100%; Equation 10

[0109] J4 / Total (%) = Number of J4 worms / Total number of nematodes × 100% Equation 11

[0110] Table 6. Effects of root irrigation with *Bacillus subtilis* from ginseng soil on soybean cyst nematode infection.

[0111]

[0112] Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level according to Duncan's test.

[0113] Note:Datearemean±SD.Differentletters in the same column indicate significantdifferenceat P<0.05levelbyDuncan's test.

[0114] Table 7. Effects of root irrigation with *Bacillus subtilis* from ginseng soil on the development of soybean cyst nematodes.

[0115]

[0116]

[0117] Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level according to Duncan's test.

[0118] Note:Date are mean±SD.Different letters in the same column indicate significant difference at P<0.05levelbyDuncan's test.

[0119] Table 8. Effects of root irrigation with *Bacillus subtilis* from ginseng soil on soybean plant growth.

[0120] <![CDATA[ddH2O]]> 22.48±0.98a 17.75±2.45a 2.38±0.18a 3.24±0.10b LB liquid culture medium 22.96±1.25a 17.33±1.18a 2.66±0.39a 3.21±0.22b Sneb2539 22.83±1.23a 18.01±0.94a 2.75±0.50a 3.62±0.24a

[0121] Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level according to Duncan's test.

[0122] Note:Date are mean±SD.Different letters in the same column indicate significant difference atP<0.05level byDuncan's test.

[0123] Table 6 shows that in the pot experiment under greenhouse conditions, the number of nematodes in soybean seedlings treated with the fermentation broth of strain Sneb2539 was significantly lower than that in the control group on LB liquid medium, with a nematode inhibition rate of 39.29%. Therefore, in the pot experiment, *Bacillus ginseng* Sneb2539 can effectively control soybean cyst nematode disease.

[0124] Table 7 shows that in the greenhouse pot experiment, the proportion of J2 in soybean seedlings treated with the fermentation broth of Sneb2539 was significantly increased compared to the control group on LB liquid medium, while the proportion of J4 was significantly decreased. Therefore, in the greenhouse pot experiment, *Bacillus ginseng* Sneb2539 can delay the development of soybean cyst nematodes in soybean roots.

[0125] Table 8 shows that in the pot experiment under greenhouse conditions, after soybean seeds were coated with fermentation broth of *Bacillus ginseng* Sneb2539, the root fresh weight of soybean seedlings increased significantly by 12.77% compared with the LB control. Plant height, root length, and plant fresh weight showed no significant differences compared with the LB liquid culture medium control group. Therefore, in the greenhouse experiment, *Bacillus ginseng* Sneb2539 had no inhibitory effect on soybean seedling growth.

[0126] As shown in the above embodiments, this invention provides a strain of *Neobacillus ginsengisoli* for the prevention and control of soybean cyst nematode disease and its application. The Latin name of *Neobacillus ginsengisoli* is Sneb2539, its deposit location is the China General Microbiological Culture Collection Center (CGMCC), the deposit date is April 2, 2025, and the accession number is CGMCC No. 34086. The fermentation broth of *Neobacillus ginsengisoli* Sneb2539 discovered in this invention exhibits certain contact toxicity against second-instar larvae of soybean cyst nematodes. Using this fermentation broth for root irrigation of soybean seedlings or for preparing coated soybean seeds can effectively prevent and control soybean cyst nematode disease, delay the development of soybean cyst nematodes in soybean roots, and significantly increase seed vigor and root length.

[0127] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A novel Bacillus subtilis strain for controlling soybean cyst nematode disease, characterized in that, The Latin name of the novel ginseng spore bacillus is Neobacillus ginsengisoli, the accession name is Sneb2539, the accession location is China General Microbiological Culture Collection Center, the accession date is April 2, 2025, and the accession number is CGMCCNo.34086.

2. The application of the novel Bacillus subtilis according to claim 1 in the prevention and control of soybean cyst nematode disease.

3. The application of the novel Bacillus subtilis according to claim 1 in the preparation of a product for the prevention and control of soybean cyst nematode disease.

4. A fermentation broth of Sneb2539 strain for controlling soybean cyst nematode disease, characterized in that, The Sneb2539 strain fermentation broth contains the novel Bacillus subtilis described in claim 1.

5. The method for preparing the fermentation broth of Sneb2539 strain according to claim 4, characterized in that, Includes the following steps: By inoculating the novel Bacillus ginseng described in claim 1 into a liquid culture medium for fermentation, the fermentation broth of strain Sneb2539 can be obtained.

6. The preparation method according to claim 5, characterized in that, The liquid culture medium is LB liquid medium, the fermentation time is 10–14 h, the fermentation temperature is 35–39 °C, the fermentation speed is 100–300 r / min, and the concentration of the Sneb2539 strain fermentation broth is 8.0 × 10⁻⁶. 7 ~1.2×10 8 CFU / mL.

7. The application of the Sneb2539 strain fermentation broth according to claim 4 in alleviating soybean cyst nematode disease.

8. A method for alleviating soybean cyst nematode disease using the fermentation broth of the Sneb2539 strain as described in claim 4, characterized in that, The steps include: drenching soybean seedlings with fermentation broth from the Sneb2539 strain.

9. A method for alleviating soybean cyst nematode disease using the fermentation broth of the Sneb2539 strain as described in claim 4, characterized in that, The process includes the following steps: coating soybean seeds with fermentation broth from the Sneb2539 strain.

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