Ginseng novel bacillus for preventing and treating soybean cyst nematode disease and application

Abstract

The invention provides a novel ginseng bacillus for preventing and treating soybean cyst nematode disease and application, and relates to the technical field of agricultural production, the Latin of the novel ginseng bacillus is Neobibacillus ginsenensis, the preservation name is Sneb2539, the preservation place is China General Microbiological Culture Collection Center, the preservation date is April 2, 2025, and the preservation number is CGMCC NO.9687. The preservation number is CGMCC NO.9687. The preservation number is CGMCC NO.9787. The preservation number is CGMCC NO.9787. The preservation number is CGMCC NO.9787. The preservation number is CGMCC NO.9787. The preservation number is CGMCC NO.9787. The preservation number is CGMCC (China General Microbiological Culture Collection Center) No.34086. According to the invention, the fermentation liquor of the bacillus panaxatrii Sneb2539 found by the invention has certain contact activity on second-stage larvae of soybean cyst nematode; when the fermentation liquor is used for carrying out root irrigation treatment on soybean seedlings or preparing coated soybean seeds, the soybean cyst nematode disease can be effectively prevented and treated, the development of the soybean cyst nematode in soybean roots can be delayed, and the vital index and the root length of the seeds can be remarkably increased.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural production, in particular to a novel Bacillus ginsengii strain for preventing and treating soybean cyst nematode disease and its application. Background Art

[0002] Soybean is an important crop, providing a sustainable source of protein and oil worldwide. The soybean cyst nematode, belonging to the order Heteroderae, family Heteroderae, genus Heteroderae, is an absolutely parasitic, advanced nematode. It can infect soybeans three to six times within a single growing season, making it one of the most destructive pathogens in soybean production. Currently, soybean cyst nematode disease can cause a 10% to 30% yield reduction, and in severe cases, a 70% to 90% reduction, or even total crop failure. Even without obvious symptoms, soybean cyst nematode disease can result in a 10% to 20% reduction in soybean yield. Global economic losses from this disease amount to $2.6 billion annually. Furthermore, soybean cyst nematodes can infect soybeans throughout their entire growing season. The nematode primarily attacks the roots, preventing them from effectively absorbing nutrients. This leads to nutrient deficiencies in the aboveground part of the plant, resulting in yellowing, shrunken, and stunted soybeans, as well as shrunken pods and seeds, and even pod failure. Therefore, soybean cyst nematode disease has become the main factor limiting the increase in soybean yield, and the prevention and control of soybean cyst nematode disease is quite challenging.

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

[0004] The present invention aims to provide a novel strain of Neobacillus ginsengisoli for controlling soybean cyst nematode disease and its application. By screening a novel strain of Neobacillus ginsengisoli from soil, targeting soybean cyst nematodes and showing high anti-nematode activity, this strain provides a new resource for the research and development of novel biocontrol agents effective against soybean cyst nematodes, thereby reducing or replacing the use of chemical agents for soybean cyst nematode disease and alleviating the environmental damage and human health threats posed by these agents.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a novel Bacillus ginsengisoli for preventing and treating soybean cyst nematode disease. The novel Bacillus ginsengisoli is named Neobacillus ginsengisoli in Latin, has a preservation name of Sneb2539, is stored at the General Microbiology Center of the China Culture Collection Administration of Microorganisms, has a preservation date of April 2, 2025, and has a preservation number of CGMCC No. 34086.

[0007] The present invention also provides the use of the novel Bacillus ginsengii in the prevention and treatment of soybean cyst nematode disease.

[0008] The present invention also provides the use of the novel Bacillus ginsengii in the preparation of a product for preventing and treating soybean cyst nematode disease.

[0009] The present invention also provides a fermentation liquid of the Sneb2539 strain for preventing and treating soybean cyst nematode disease. The fermentation liquid of the Sneb2539 strain contains the novel Bacillus ginsengii.

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

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

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

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

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

[0015] The beneficial effects of the present invention are:

[0016] (1) The present invention found that the fermentation liquid of the novel Bacillus sp. Sneb2539 from ginseng soil has certain contact killing activity against the second-instar larvae of soybean cyst nematode.

[0017] (2) Soaking and coating soybean seeds with the fermentation liquid of the novel Bacillus sp. Sneb2539 can significantly increase the seed vitality index and root length.

[0018] (3) In an indoor potted experiment, root irrigation of soybean seedlings or coating of soybean seeds with the fermentation liquid of the novel Bacillus sp. Sneb2539 from ginseng soil can effectively prevent and control soybean cyst nematode disease and delay the development of soybean cyst nematodes in soybean roots, without inhibiting soybean growth.

[0019] Preservation Instructions

[0020] The novel Bacillus ginsengisoli, the preservation name is Sneb2539, the Latin name is Neobacillus ginsengisoli, the preservation location is the General Microbiology Center of the China Culture Collection Administration, the preservation location is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the preservation date is April 2, 2025, and the preservation number is CGMCC No. 34086. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A technical roadmap for screening, identification and efficacy research of biocontrol bacteria for soybean cyst nematode control;

[0022] Figure 2 To construct the phylogenetic tree of strain Sneb2539 based on 16S rDNA sequence. DETAILED DESCRIPTION

[0023] The present invention provides a novel Bacillus ginsengisoli for preventing and treating soybean cyst nematode disease. The novel Bacillus ginsengisoli is named Neobacillus ginsengisoli in Latin, has a preservation name of Sneb2539, is stored at the General Microbiology Center of the China Culture Collection Administration of Microorganisms, has a preservation date of April 2, 2025, and has a preservation number of CGMCC No. 34086.

[0024] The present invention also provides the use of the novel Bacillus ginsengii in the prevention and treatment of soybean cyst nematode disease.

[0025] The present invention also provides the use of the novel Bacillus ginsengii in the preparation of a product for preventing and treating soybean cyst nematode disease.

[0026] The present invention also provides a fermentation liquid of the Sneb2539 strain for preventing and treating soybean cyst nematode disease. The fermentation liquid of the Sneb2539 strain contains the novel Bacillus ginsengii.

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

[0028] The novel Bacillus ginsengii is inoculated into a liquid culture medium for fermentation to obtain a fermentation liquid of the Sneb2539 strain.

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

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

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

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

[0033] The technical route for the screening, identification and efficacy research of biocontrol bacteria for soybean cyst nematode disease in this application is shown in Figure 1 .

[0034] The technical solutions provided by the present invention are 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 comprises the following components: 10.0 g of peptone, 5.0 g of yeast powder, 5.0 g of sodium chloride, 1.0 g of glucose, and 15-20 g of agar; the preparation method is as follows: the above raw materials are dissolved in 1 L of distilled water, and sterilized by wet heat at pH 7.0 ± 0.2 and 121°C for 30 min.

[0036] The LB liquid culture medium comprises the following components: 10.0 g of peptone, 5.0 g of yeast powder, 5.0 g of sodium chloride, and 1.0 g of glucose. The preparation method is as follows: the above raw materials are dissolved in 1 L of distilled water, and sterilized by moist heat at pH 7.0 ± 0.2 and 121°C for 30 min.

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

[0038] Example 1 Obtaining Neobacillus ginsengisoli Sneb2539 strain

[0039] Soil samples were collected from sweet potato fields in Ordos City, Inner Mongolia Autonomous Region (109.781°E, 39.608°N). 10 g of soil sample was weighed and placed in a 250 mL Erlenmeyer flask containing 90 mL of sterile water in a clean bench. The sample was diluted to 10 -2 , 10 -3 , 10 -4 , obtaining a soil suspension of the corresponding concentration. Take 25 μL of each soil suspension and spread it onto LB solid medium. Incubate at 37°C for 12 h. Pick a single colony for purification. Prepare a fermentation broth of the strain and mix it with an equal volume of 30% glycerol. Store frozen in a -80°C freezer.

[0040] Example 2 Cultivation of Neobacillus ginsengisoli Sneb2539 strain

[0041] First, the ginseng soil novel Bacillus (Neobacillus ginsengisoli) was fermented and cultured: Sneb2539 stored in the cryopreserved tube was activated on LB solid medium. After culturing for 12 hours, a single colony with good growth was picked and inoculated into 150 mL LB liquid medium and shaken. The fermentation temperature was 37 ° C, the shaker speed was 200 r / min, and the fermentation time was 12 hours. The concentration of 1.0×10 8 CFU / mL of Sneb2539 strain fermentation broth.

[0042] The fermented liquid can be directly used to irrigate soybean seedlings or coat soybean seeds. Both root irrigation and coating treatments can reduce the damage caused by cyst nematode disease to soybeans.

[0043] Example 3 Identification of Neobacillus ginsengisoli Sneb2539 strain

[0044] The taxonomic status 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 plate streak method, placed in a 37°C constant temperature incubator and cultured upside down for 12 hours, and the colony morphology, color, odor, etc. were observed.

[0046] Physiological and biochemical characteristics: In a clean bench, the neck of a physiological and biochemical tube (purchased from Qingdao Haibo Biotechnology Co., Ltd.) was broken open, and a fresh single colony was picked with an inoculation loop and inoculated into the tube. The tube was sealed with sealing film and placed in a 37°C constant temperature incubator. The culture time was incubated according to the instructions in the physiological and biochemical kit (Qingdao Haibo Biotechnology Co., Ltd.), and the corresponding reagents were added dropwise as required to observe the final results.

[0047] 16S rDNA sequence analysis: DNA was extracted according to the bacterial DNA extraction method of Sutyak et al. (Sutyak KE, Wirawan RE, Aroutcheva A A and Chikindas M L. 2008. Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens [J]. Journal of Applied Microbiology, 104, (1067-1074)). The 16S rDNA gene was amplified using the universal bacterial primers 27F (SEQ ID NO. 1: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO. 2: 5'-ACGGCTACCTTGTTACGACTT-3') using genomic DNA as a template. The 25μL PCR amplification system is calculated based on 25μL: 1μL each of upstream primer and downstream primer, 2μL DNA template, 12.5μL 2×Taq MasterMix (Dye olus), and 8.5μL ddH2O. The PCR amplification conditions are: 94℃ pre-denaturation for 1.5min, 94℃ denaturation for 0.5min, 58℃ annealing for 0.5min, 72℃ extension for 1min, 30 cycles, 72℃ extension for 5min, and storage at 4℃. After detection by 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 with the BLAST database in NCBI, and the phylogenetic tree was constructed using the neighbor-joining method using MEGA 7.0 software, see. Figure 2The strain's sequence was then submitted to NCBI's GenBank, where it received accession number (PV423516). The results showed that the strain with the highest homology to Sneb2539 (accession number PV423516) was Neobacillus ginsengisoli, with accession numbers MK014249.1 and MZ067923.1. Therefore, this strain was confirmed to be Neobacillus ginsengisoli.

[0048] Example 4 Experiment on the contact killing of soybean cyst nematodes by the fermentation liquid of Neobacillus ginsengisoli Sneb2539

[0049] The fermentation liquid 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 the Sneb2539 strain, which was used in a test on killing second-instar larvae of soybean cyst nematodes.

[0050] Cyst soil containing soybean cyst nematodes (Heteroderaglycines) was obtained by propagating soybean roots. Nematode eggs were screened out and transferred to a funnel with a filter cloth for incubation. The eggs were collected once a day and prepared into a nematode suspension.

[0051] To 400 μL of the fermentation supernatant of the Sneb2539 strain, 100 μL of a nematode suspension (containing 50 second-instar larvae of the soybean cyst nematode) was added. Nematode mortality was observed for 24 and 48 hours, and the total number of nematodes and the number of deaths were recorded. Mortality and adjusted mortality were calculated. A control group was treated with equal amounts of ddHO and LB liquid medium, respectively, instead of the Sneb2539 fermentation supernatant. All other treatment steps were the same.

[0052] Mortality rate (%) = (number of dead nematodes / total number of nematodes) × 100%; Formula 1

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

[0054] Table 1 Contact toxicity of novel Bacillus sp. Sneb2539 to 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 as tested by Duncan's method.

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

[0058] As shown in Table 1, the fermentation supernatant of the novel Bacillus ginsengii strain Sneb2539 against second-instar larvae of soybean cyst nematodes achieved a corrected mortality rate of 68.44% after 24 hours and 73.34% after 48 hours. These results indicate that the novel Bacillus ginsengii strain Sneb2539 can effectively kill second-instar larvae of soybean cyst nematodes by contact.

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

[0060] The liquid fermentation method in Example 2 was used to prepare a 1.0×10 8 CFU / mL of Sneb2539 strain fermentation broth for later use.

[0061] The soybean variety is Liaodou No. 15; the seeds are sterilized with chlorine and set aside.

[0062] Instruments and appliances: steam sterilizer, electric constant temperature blower drying oven, electric constant temperature incubator, clean workbench, disposable culture dish, shaking incubator.

[0063] Sterilized seeds were soaked in the fermentation broth of the Sneb2539 strain for 3 minutes and then placed in sterile Petri dishes (90 mm diameter) lined with moistened sterile filter paper. Controls were treated with equal amounts of ddH2O and LB liquid medium, respectively, instead of the Sneb2539 fermentation broth. All other treatments were identical. Each group was replicated three times, with 25 seeds per replicate. The Petri dishes were incubated in a constant temperature and humidity incubator at 25°C. After 24 hours, the number of germinated seeds was counted daily. After six days of incubation, the germination index and germination rate were calculated. Root length was measured, and the vigor index was calculated.

[0064] Germination rate (%) = number of germinated seeds / number of test seeds × 100%; Formula 3

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

[0066] Seed vitality index = seedling length × germination index formula 5

[0067] Table 2 Effects of soybean seeds coated with novel Bacillus ginseng on germination and growth

[0068] Treatment group <![CDATA[ddH2O]]> LB liquid medium Sneb2539 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 vitality 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 as tested by Duncan's method.

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

[0071] As shown in Table 2, soybean seed vigor index and root length after treatment with the fermentation broth of the Sneb2539 strain significantly increased compared to the LB liquid medium control, by 23.77% and 24.35%, respectively. There were no significant differences in germination rate and germination index compared to the LB liquid medium control. This indicates that soaking and coating soybean seeds with the fermentation broth of the Sneb2539 strain increased seed vigor index and promoted radicle growth without inhibiting seed germination.

[0072] Example 6 Experiment on the efficacy of irrigating the roots of ginseng plants with the fermentation liquid of Neobacillus ginsengisoli Sneb2539 under potted conditions

[0073] The liquid fermentation method in Example 2 was used to prepare a 1.0×10 8 CFU / mL of Sneb2539 strain fermentation broth for later use.

[0074] Prepare nematode suspension according to the aforementioned nematode culture method and set aside.

[0075] The soybean variety is Liaodou No. 15; the seeds are sterilized using chlorine gas, cultured to the true leaf stage, and set aside.

[0076] The control group consisted of a root irrigation with ddH₂O and inoculation with second-instar larvae (J2) of the soybean cyst nematode, or a root irrigation with LB liquid medium and inoculation with second-instar larvae of the soybean cyst nematode. The treatment group consisted of a root irrigation with a fermentation solution of the Sneb2539 strain and inoculation with second-instar larvae of the soybean cyst nematode. Each seedling was irrigated with 10 mL of the Sneb2539 fermentation solution. In the control group, each seedling was irrigated with 10 mL of ddH₂O or LB liquid medium, and 2 mL of a second-instar larvae suspension (approximately 1,000 second-instar larvae of the soybean cyst nematode) was added to each seedling 24 hours later. Each group had nine replicates, with one seedling per replicate, and the plants were randomly arranged. After 15 days of cultivation, the plant height, root length, root fresh weight and plant fresh weight of soybean plants, 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 the second instar larvae of soybean cyst nematode, the number of J3 is the number of the third instar larvae of soybean cyst nematode, and the number of J4 is the number of the fourth instar larvae of soybean cyst nematode) were investigated, and 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 + number of J3 + number of J4 + number of cysts; Formula 6

[0078] Number of nematodes per gram of root = total number of nematodes / fresh weight of root; Formula 7

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

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

[0081] J3 / total number (%) = J3 number / total number of nematodes × 100%; Formula 10

[0082] J4 / total number (%) = J4 number / total number of nematodes × 100% Formula 11

[0083] Table 3 Effects of root irrigation with Bacillus novelta in ginseng soil on soybean cyst nematode infection

[0084] Treatment group Root fresh weight (g) Total number (items) Nematodes per gram of root Nematode inhibition rate (%) <![CDATA[ddH2O]]> 3.12±0.28b 114.22±30.21a 36.23±6.78a - LB liquid 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 as tested by Duncan's method.

[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 novelta in ginseng soil on the development of soybean cyst nematodes

[0088] Treatment group <![CDATA[ddH2O]]> LB liquid medium Sneb2539 Number of cysts 2.33±1.73a 1.78±1.48a 1.00±0.87a J2 (piece) 72.56±18.67a 76.67±11.55a 53.89±5.09b J3 (piece) 26.11±8.16a 27.33±5.61a 14.67±3.61b J4 (piece) 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 as tested by Duncan's method.

[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 spp. 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 as tested by Duncan's method.

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

[0096] As shown in Table 3, in the greenhouse potted experiment, nematode counts were significantly lower in soybean seedlings treated with the fermented liquid of Sneb2539 than in the LB liquid medium control group, with a nematode inhibition rate of 42.73%. Therefore, in this potted experiment, the novel Bacillus ginsengii strain Sneb2539 was effective in controlling soybean cyst nematode disease.

[0097] As shown in Table 4, in the greenhouse pot experiment, after root irrigation with the fermented liquid of the Sneb2539 strain in soybean seedlings, the proportion of J2 cells in the total number of seedlings increased significantly compared to the LB liquid medium control group, while the proportion of J4 cells in the total number of seedlings decreased significantly compared to the LB liquid medium control group. Therefore, in the greenhouse pot experiment, the novel Bacillus ginsengii strain Sneb2539 was able to delay the development of soybean cyst nematodes in soybean roots.

[0098] As shown in Table 5, in the greenhouse potted experiment, root irrigating soybean seedlings with the fermented liquid of Sneb2539 significantly increased root fresh weight by 9.12% compared to the LB control. Plant height, root length, and plant fresh weight showed no significant differences compared to the LB liquid medium control. Therefore, in the greenhouse experiment, the novel Bacillus ginsenosides Sneb2539 strain had no inhibitory effect on soybean seedling growth.

[0099] Example 7 Experiment on the efficacy of coating treatment of fermentation liquid of Neobacillus ginsengisoli Sneb2539 strain under potted conditions

[0100] The liquid fermentation method in Example 2 was used to prepare a 1.0×10 8 CFU / mL of Sneb2539 strain fermentation broth for later use.

[0101] Prepare nematode suspension according to the aforementioned nematode culture method and set aside.

[0102] The soybean variety is Liaodou No. 15; the seeds are sterilized using chlorine gas, cultured to the true leaf stage, and set aside.

[0103] The control group consisted of seedlings coated with ddH₂O and inoculated with second-instar larvae of the soybean cyst nematode, or seedlings coated with LB liquid medium and inoculated with second-instar larvae of the soybean cyst nematode. The treated group consisted of seedlings coated with Sneb2539 fermentation broth and inoculated with second-instar larvae of the soybean cyst nematode. Sterilized seeds were soaked in Sneb2539 fermentation broth for 3 minutes. The control group was soaked in an equal amount of ddH₂O or LB liquid medium for 3 minutes before being sown in sterilized sand. At the true leaf stage, 2 mL of a second-instar larvae suspension (approximately 1,000 second-instar larvae of the soybean cyst nematode) was added to each seedling. Each group consisted of nine replicates, with one seedling per replicate, all randomly arranged. After 15 days of cultivation, the plant height, root length, root fresh weight and plant fresh weight of soybean plants, 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 the second instar larvae of soybean cyst nematode, the number of J3 is the number of the third instar larvae of soybean cyst nematode, and the number of J4 is the number of the fourth instar larvae of soybean cyst nematode) were investigated, and 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 + number of J3 + number of J4 + number of cysts; Formula 6

[0105] Number of nematodes per gram of root = total number of nematodes / fresh weight of root; Formula 7

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

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

[0108] J3 / total number (%) = J3 number / total number of nematodes × 100%; Formula 10

[0109] J4 / total number (%) = J4 number / total number of nematodes × 100% Formula 11

[0110] Table 6 Effects of root irrigation with Bacillus novo in 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 as tested by Duncan's method.

[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 spp. in 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 as tested by Duncan's method.

[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 spp. from ginseng soil on soybean plant growth

[0120] Treatment group Plant height (cm) Root length (cm) Fresh weight per plant (g) Root fresh weight (g) <![CDATA[ddH2O]]> 22.48±0.98a 17.75±2.45a 2.38±0.18a 3.24±0.10b LB liquid 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 as tested by Duncan's method.

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

[0123] As shown in Table 6, in the greenhouse pot experiment, soybean seedlings coated with the fermentation broth of the Sneb2539 strain had significantly lower nematode counts than the LB liquid medium control group, with a nematode inhibition rate of 39.29%. Therefore, in this pot experiment, the novel Bacillus ginsengii strain Sneb2539 was effective in controlling soybean cyst nematode disease.

[0124] As shown in Table 7, in the greenhouse pot experiment, the proportion of J2 seedlings grown from soybean seeds coated with the fermentation broth of the Sneb2539 strain was significantly increased compared to the LB liquid medium control group, while the proportion of J4 seedlings was significantly decreased compared to the LB liquid medium control group. Therefore, in the greenhouse pot experiment, the novel Bacillus ginsengii strain Sneb2539 was able to delay the development of soybean cyst nematodes in soybean roots.

[0125] As shown in Table 8, in the greenhouse potted experiment, soybean seedlings coated with the fermentation broth of Bacillus ginsengii Sneb2539 significantly increased root fresh weight by 12.77% compared to the LB control. Plant height, root length, and plant fresh weight showed no significant differences compared to the LB liquid medium control. Therefore, in the greenhouse experiment, Bacillus ginsengii Sneb2539 had no inhibitory effect on soybean seedling growth.

[0126] As can be seen from the above examples, the present invention provides a novel Bacillus ginsengisoli strain for controlling soybean cyst nematode disease and its application. The novel Bacillus ginsengisoli strain is named Neobacillus ginsengisoli in Latin, deposited as Sneb2539, and deposited at the General Microbiology Center of the China Culture Collection Administration of Microorganisms on April 2, 2025, with a deposit number of CGMCC No. 34086. The fermentation liquid of the novel Bacillus ginsengisoli strain Sneb2539 discovered in the present invention has certain contact killing activity against second-instar larvae of soybean cyst nematodes. Using this fermentation liquid for root irrigation of soybean seedlings or preparing it into coated soybean seeds can effectively control soybean cyst nematode disease, delay the development of soybean cyst nematodes in soybean roots, and significantly increase the seed vitality index and root length.

[0127] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A novel strain of Bacillus ginsengii for preventing and treating soybean cyst nematode disease, characterized in that: The Latin name of the novel Bacillus ginsengisoli is Neobacillus ginsengisoli, the preservation name is Sneb2539, the preservation location is the General Microbiology Center of the China Culture Collection Administration, the preservation date is April 2, 2025, and the preservation number is CGMCC No. 34086.

2. Use of the novel Bacillus ginsengii strain according to claim 1 in the prevention and treatment of soybean cyst nematode disease.

3. Use of the novel Bacillus ginsengii strain according to claim 1 in the preparation of a product for preventing and controlling soybean cyst nematode disease.

4. A fermentation broth of the Sneb2539 strain for preventing and treating soybean cyst nematode disease, characterized in that: The fermentation broth of the Sneb2539 strain contains the novel Bacillus ginsengii according to claim 1.

5. The method for preparing the fermentation broth of the Sneb2539 strain according to claim 4, characterized in that: The steps include: The novel Bacillus ginsengii described in claim 1 is inoculated into a liquid culture medium for fermentation to obtain a fermentation liquid of the Sneb2539 strain.

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

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

8. A method for alleviating soybean cyst nematode disease using the fermentation liquid of the Sneb2539 strain according to claim 4, characterized in that: The method comprises the following steps: using the fermentation liquid of the Sneb2539 strain to irrigate the roots of soybean seedlings.

9. A method for alleviating soybean cyst nematode disease using the fermentation liquid of the Sneb2539 strain according to claim 4, characterized in that: The method comprises the following steps: coating soybean seeds with the fermentation liquid of the Sneb2539 strain.

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