Bacillus megaterium nbnc-104 for controlling root-knot nematode, its compound microbial agent and application

Abstract

The present application belongs to the field of microbial pesticides, and relates to a bacillus megaterium NBNC-104 for preventing and treating root-knot nematodes, a compound microbial agent and application thereof. The preservation number of the bacillus megaterium strain NBNC-104 is CCTCC M 20242324. Laboratory bioassay data show that the bacillus megaterium NBNC-104 has high toxic killing activity and repellent effect on southern root-knot nematodes, and has synergistic nematode killing activity after being compounded with bacillus thuringiensis strain NBIN-863 and kunming streptomyces sp. NBNH-006, and the strain can effectively colonize in the rhizosphere soil environment within 30 days under potting conditions. Therefore, the strain has broad application prospects in agricultural production.

Description

Technical Field

[0001] This invention belongs to the field of microbial pesticides and relates to a strain of Bacillus megaterium NBNC-104 for controlling root-knot nematodes, its compound bacterial agents, and their applications. Background Technology

[0002] Nematode diseases, due to their insidious transmission, rapid reproduction, and long period of damage, have become a significant disease in modern agriculture in my country. The Food and Agriculture Organization of the United Nations (FAO) estimates that nematode diseases can cause crop yield losses of 12% to 20%. The pathogens of nematode diseases are plant parasitic nematodes, with the root-knot nematode (Meloidogyne spp.) being the most damaging. Globally, the average annual yield loss due to root-knot nematodes, directly or indirectly, is estimated at US$173 billion.

[0003] Root-knot nematodes can parasitize almost all vascular plants. Second-instar larvae emerge from their eggs and search for plant roots in the soil. Using their stylets and a series of secreted enzymes, they destroy the host's root system and burrow into the plant, establishing feeding sites at suitable locations. This induces the formation of giant cells, leading to root knots. Therefore, the window for controlling root-knot nematodes is very short, generally only two weeks, which increases the difficulty of their control.

[0004] Currently, the main method for controlling nematodes is chemical control. However, the application of single chemical agents can easily induce nematode resistance and poses risks to food and environmental safety. Biological control utilizes natural enemies of nematodes or nematicidal biological products to control nematodes. Biological control is environmentally friendly and pests are less likely to develop resistance, making it a promising alternative and enhancement to existing chemical control methods. With the increasing demand for green agricultural development in my country and the implementation of pesticide and fertilizer reduction initiatives, biological control has gradually become an important research hotspot in the development of new green nematicides. Summary of the Invention

[0005] In view of the limited existing biological control resources for nematodes, the primary objective of this invention is to provide a biocontrol bacterium for controlling root-knot nematodes. The strain is identified as *Priestia megaterium*, named *Priestia megaterium* NBNC-104, and its accession number is CCTCC NO: M20242324.

[0006] Another objective of this invention is the application of the aforementioned Bacillus megaterium and its compound bacterial agents in the preparation of agricultural nematicides for controlling root-knot nematodes.

[0007] To achieve the above objectives, the present invention adopts the following technical measures:

[0008] The applicant collected soil samples from a tomato greenhouse in Wuhan, Hubei Province, which had experienced an outbreak of root-knot nematode disease. High-temperature screening was used to isolate Bacillus strains, and a strain with high toxicity against root-knot nematodes was selected from the isolated strains through root-knot nematode bioassay and named NBNC-104. Single colonies of NBNC-104 grown on Lauria-Bertani agar plates are nearly circular, milky white, with a central dotted protrusion. Figure 1 Microscopic examination revealed that the cells were rod-shaped, relatively thick, arranged in chains, and spores were centrally located. Genomic mean nucleotide identity (ANI) alignment analysis showed that this strain shared 97.81% ANI similarity with the type strain of *Priestia megaterium* (NBRC 15308), thus identifying NBNC-104 as *Priestia megaterium*. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on October 24, 2024, with the following classification and name: *Priestia megaterium* NBNC-104; accession number: CCTCC NO: M20242324; location: Wuhan University, Wuhan, China.

[0009] The scope of protection of this invention includes:

[0010] Fermentation broth of Bacillus megaterium NBNC-104, wherein the fermentation broth contains live Bacillus megaterium NBNC-104.

[0011] A microbial compound, wherein the active ingredient of the compound includes Bacillus megaterium with accession number CCTCC NO: M20242324.

[0012] Preferably, the active ingredients of the compound described above include Bacillus megaterium NBNC-104 and Bacillus thuringiensis NBIN-863.

[0013] Preferably, the active ingredients of the above-mentioned compound include Bacillus megaterium NBNC-104 and Stizemonas kunmingensis NBNH-006.

[0014] The Bacillus thuringiensis NBIN-863 has the accession number CCTCC NO: M2013612 and has been disclosed in CN103898025B. The accession number of Kunming Stizemonas NBNH-006 is CCTCC NO: M20242323.

[0015] Preferably, in the above-described compound, the effective bacterial concentrations of Bacillus megaterium NBNC-104 and Bacillus thuringiensis NBIN-863 are 1-10:1-10.

[0016] Preferably, in the above-described compound, the effective bacterial concentrations of Bacillus megaterium NBNC-104 and Bacillus stizezii NBNH-006 are 1-10:1-10.

[0017] Application of Bacillus megaterium NBNC-104, Bacillus megaterium NBNC-104 fermentation broth or its compound in the control of nematodes.

[0018] Application of Bacillus megaterium NBNC-104, Bacillus megaterium NBNC-104 fermentation broth or its compound in the preparation of nematode insecticides.

[0019] Application of Bacillus megaterium NBNC-104, Bacillus megaterium NBNC-104 fermentation broth or its compound in the preparation of nematode repellents.

[0020] In the above-described application, preferably, the nematode is the southern root-knot nematode (Meloidogyne incognita). Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0021] 1. This invention provides a fermentation broth containing strain NBNC-104 that exhibits high toxic and repellent activity against the southern root-knot nematode. Furthermore, it demonstrates synergistic effects when combined with the known highly nematicidal Bacillus thuringiensis strain NBIN-863 and the novel nematicidal strain *Stizemonas kunmingensis* NBNH-006. In tomato pot experiments, the fermentation broth of strain NBNC-104 showed a pot control efficacy of 79.00% against the southern root-knot nematode. Combining it with Bacillus thuringiensis NBIN-863 further increased the pot control efficacy to 87.09%, and combining it with *Stizemonas kunmingensis* NBNH-006 further increased the pot control efficacy to 89.25%, comparable to the efficacy of the commercially available nematicide abamectin·thiazophos. This invention provides a new resource for the creation of novel microbial nematicides.

[0022] 2. Due to environmental factors, many microbial inoculants cannot stably colonize in soil, leading to unstable efficacy in the field application of microbial pesticides. This invention tested the colonization ability of strain NBNC-104 in rhizosphere soil and found that within 30 days of root irrigation, the colonization density of strain NBNC-104 in the rhizosphere soil could be maintained at 10. 6 -10 7 The cfu / g level indicates that the strain can colonize the rhizosphere soil. This suggests that compared to other biocontrol bacteria, strain NBNC-104 may have more stable field control efficacy, providing a green and efficient control method for nematodes, particularly suitable for organic agricultural production, and possessing significant economic value and application prospects. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the colony morphology of strain NBNC-104 on Lauria-Bertani agar plates.

[0024] Figure 2 This is a schematic diagram illustrating the effect of strain NBNC-104 on killing root-knot nematodes.

[0025] Wherein: A represents the phenotype of root-knot nematodes exhibiting body stiffness after 10 hours of treatment with the fermentation broth of strain NBNC-104 of the present invention; B represents the phenotype of root-knot nematodes still surviving after 10 hours of treatment with the control fermentation medium. Detailed Implementation

[0026] To better explain the present invention, the main contents of the present invention are further illustrated below with reference to specific embodiments, but the contents of the present invention are not limited to the following embodiments. Unless otherwise specified, the technical solutions described in the present invention are conventional techniques in the art, and the reagents or materials described, unless otherwise specified, are all from commercial sources.

[0027] Example 1:

[0028] Isolation, screening and identification of Bacillus megaterium NBNC-104

[0029] 1) Isolation of Bacillus strains: The applicant collected soil samples from a tomato greenhouse in Wuhan, Hubei Province, which had experienced an outbreak of root-knot nematode disease, and used a high-temperature screening method to isolate Bacillus strains. The specific procedure was as follows: 1g of soil sample was weighed and placed in an Erlenmeyer flask containing 100mL of sodium acetate medium. After shaking and mixing, the sample was treated at 80℃ for 10min. Then, 1mL of the suspension was serially diluted, and 0.2mL of each dilution was plated onto Lauria-Bertani agar plates (formula: 10g / L peptone, 5g / L yeast extract, 10g / L NaCl, 2% agar powder) and incubated overnight at 28℃. Single colonies were then picked and transferred to 5mL Lauria-Bertani liquid medium, incubated at 28℃ and 220rpm for 10h, and then an equal volume of 50% glycerol was added before storage at -80℃. A total of 5 Bacillus strains were isolated.

[0030] 2) Screening of strains with high toxicity against root-knot nematodes: The isolated Bacillus strains were inoculated into 5 mL Lauria-Bertani liquid medium and activated overnight at 28°C and 220 rpm. They were then transferred to fermentation medium (3% soybean meal, 1.5% corn steep liquor, 1.5% corn starch, pH 7.0-7.5) and cultured at 28°C and 220 rpm for 24 h. The spore concentration in each fermentation broth was calculated using the dilution counting method, and the spore concentration in each fermentation broth was adjusted to 1 × 10⁻⁶ using fresh fermentation medium. 6 The CFU / mL concentration was set aside for later use. The southern rhizosphere nematodes used in the experiment were preserved and passaged in potted tomatoes at our institution. Nematode egg masses were collected from the roots of infected tomatoes and placed in a petri dish containing 15 mL of sterile water for incubation at 20°C in the dark. The hatching of root-knot nematodes was observed. Nematodes were collected by centrifugation, and the nematode concentration was adjusted to 3-4 nematodes / μL. 100 μL of each Bacillus fermentation broth was transferred to a 96-well plate, and 10 μL of nematode suspension was added to each well. Observation under an inverted microscope ensured that 30-40 nematodes were added to each well. Fermentation medium was used as a negative control, and each treatment group was replicated three times. After incubating the 96-well plates in the dark at 20℃ for 10 hours, the mortality rate of nematodes in each treatment group was statistically analyzed under a microscope. If the mortality rate in the control group was <5%, no correction was needed. If the mortality rate in the control group was between 5% and 20%, the mortality rate of the treatment group was corrected using the formula: (mortality rate of treatment group - mortality rate of control group) / (1 - mortality rate of control group) × 100. If the mortality rate of the control group was >20%, the experiment needed to be repeated. Based on the 96-well plate bioassay, the strain with the highest activity among the isolated Bacillus strains was named NBNC-104, and the spore concentration in the fermentation broth was 1 × 10⁻⁶. 6 The mortality rate of root-knot nematodes treated with CFU / mL was 98.00% (±0.78%).

[0031] 3) Identification of strain NBNC-104: Single colonies of strain NBIF-104 grown on Lauria-Bertani agar plates were nearly round, milky white, with punctate protrusions in the center. Figure 1Microscopic examination revealed that the cells were rod-shaped, relatively thick, arranged in chains, with centrally located spores, and Gram-positive. Based on these morphological characteristics, the strain was initially identified as Bacillus. Subsequently, high-quality genomic DNA of strain NBNC-104 was extracted using the Qiagen genome extraction kit. The genome of strain NBNC-104 was sequenced using second-generation high-throughput sequencing combined with Oxford Nanopore third-generation sequencing. The genome of strain NBNC-104 was analyzed and compared using the ANI Calculator tool on the EzBioCloud bacterial identification platform (https: / / www.ezbiocloud.net / ). The results showed that strain NBNC-104 had an ANI similarity of 97.81% with the type strain of *Priestia megaterium*, NBRC 15308. In taxonomy, when the ANI similarity between two strains is greater than 95%, they are considered to be the same species. Therefore, strain NBNC-104 was identified as *Priestia megaterium*.

[0032] The above-mentioned strain was deposited at the China Center for Type Culture Collection on October 24, 2024, and its classification name is Priestia megaterium NBNC-104; the accession number is CCTCC NO: M20242324; the location is Wuhan University, Wuhan, China.

[0033] Example 2:

[0034] Determination of nematicidal activity of Bacillus megaterium NBNC-104

[0035] The fermentation broth of Bacillus megaterium NBNC-104 was prepared according to the fermentation culture method in Example 1, and the number of spores in the fermentation broth was adjusted to 1×10⁻⁶ using fresh culture medium. 6 The toxic activity of different gradients of NBNC-104 fermentation broth against *Strombus heterophylla* was tested using the stock solution (1000 μL / mL in Table 1) at CFU / mL as described in Example 1 via the 96-well plate bioassay method. The results are shown in Table 1. The killed nematodes exhibited a rigid phenotype, as shown in Table 1. Figure 2 As shown. Using SPSS v22.0 (IBM) data processing software, the probabilistic unit model equation for the killing of root-knot nematodes by NBNC-104 fermentation broth was calculated as PROBIT(P)=-2.937+1.726X (the variable X was transformed using the logarithm with a base of 10). By observing the confidence intervals (as shown in Table 2), the LC-100 of NBNC-104 fermentation broth treated with root-knot nematodes for 10 hours was obtained. 50The value was 50.32 μL / mL, with a 95% confidence interval of (25.84 μL / mL - 75.59 μL / mL), demonstrating good nematicidal activity.

[0036] Table 1. Data on the toxic activity of Bacillus megaterium NBNC-104 fermentation broth against root-knot nematodes.

[0037]

[0038] Table 2. NBNC-104 fermentation broth concentrations and 95% confidence intervals corresponding to different mortality probabilities of root-knot nematodes (partial).

[0039]

[0040] To facilitate subsequent testing of whether strain NBNC-104 could be used in combination with other nematicidal microbial agents, a fermentation broth was prepared by inoculating the known highly active strain Bacillus thuringiensis NBIN-863 (CCTCC NO: M2013612) according to the fermentation culture method in Example 1. The LC50 of the fermentation broth containing strain NBIN-863 after 10 hours of treatment with root-knot nematodes was determined according to the above method. 50 The value was 77.19 μL / mL, with a 95% confidence interval of (63.64 μL / mL - 91.37 μL / mL).

[0041] Example 3:

[0042] Chemotaxis of Bacillus megaterium NBNC-104 against root-knot nematodes

[0043] The Bacillus megaterium NBNC-104 fermentation broth was prepared according to the fermentation culture method in Example 1 and used to detect the chemotactic effect of the fermentation broth on root-knot nematodes. Pour 1% water agar into a petri dish with a diameter of 3.5 cm, draw lines on the back of the petri dish, and divide the petri dish into three regions A, B, and C with widths of 1.5 cm, 0.5 cm, and 1.5 cm from left to right. Add 5 μL of the NBNC-104 fermentation broth at a position 0.5 cm away from the boundary in region A, add 5 μL of the sterilized medium without inoculation at a position 0.5 cm away from the boundary in region C, and add 5 μL of the root-knot nematode suspension in the middle of region B, where the number of root-knot nematodes is about 100. Place the petri dish on a horizontal table at 25 °C in the dark and let it stand still. After 2 h, count the number of root-knot nematodes in each region under an inverted microscope, and calculate the chemotaxis index of the root-knot nematodes. Chemotaxis Index Value (abbreviated as C.I. value) = (the number of nematodes in region A - the number of nematodes in region C) / (the number of nematodes in region A + the number of nematodes in region C). If the C.I. value ≥ 0.2, it indicates that the test sample has a strong attracting effect on nematodes; if 0.1 ≤ C.I. value < 0.2, it indicates that the test sample has a certain attracting effect on nematodes; if -0.1 ≤ C.I. value < 0.1, it is considered a random behavior of nematodes, indicating that the test sample has no chemotactic effect on nematodes; if -0.2 < C.I. value < -0.1, it indicates that the test sample has a certain repellent effect on nematodes; if the C.I. value ≤ -0.2, it indicates that the test sample has a strong repellent effect on nematodes. Use sterile water as the control treatment group, and conduct 5 parallel experiments for each treatment. The experimental results are shown in Table 3. The average C.I. value of the NBNC-104 fermentation broth treating root-knot nematodes is -0.44, indicating that the NBNC-104 fermentation broth has a strong repellent effect on root-knot nematodes.

[0044] Table 3 Chemotactic effect of the NBNC-104 fermentation broth on root-knot nematodes

[0045]

[0046]

[0047] Example 4:

[0048] Determination of the nematocidal activity of the compound bactericide of Bacillus megaterium NBNC-104 and Bacillus thuringiensis NBIN-863

[0049] To test whether strain NBNC-104 could be used in combination with other nematicide microbial agents, this study performed a 96-well plate assay after combining the fermentation broth of NBNC-104 with that of the known highly active strain Bacillus thuringiensis NBIN-863 (CCTCC NO: M2013612). The number of spores in the fermentation broths of strains NBNC-104 and NBIN-863 was counted separately, and the spore counts in both broths were adjusted to the same number (1×10⁻⁶) by adding fresh culture medium. 6 Two fermentation broths were mixed at a volume ratio of 1:1 (CFU / mL) to obtain a compound bacterial agent with a spore number ratio of 1:1 (this concentration of the compound bacterial agent is 1000 μL / mL as shown in Table 4). Based on the preliminary test, the compound bacterial agent was diluted to a gradient concentration by adding fresh culture medium, and the toxic activity of the compound bacterial agent against root-knot nematodes was detected by bioassay using 96-well plates. The bioassay results are shown in Table 4. The probability unit model equation for the killing of root-knot nematodes by the compound bacterial agent with a spore number ratio of 1:1 was calculated using SPSS v22.0 (IBM) data processing software as PROBIT(P) = -3.348 + 2.076X (the variable X was transformed using the logarithm with a base of 10). By observing the confidence interval (as shown in Table 5), the LC-value of the compound bacterial agent with a spore number ratio of 1:1 for 10 h of treatment with root-knot nematodes was obtained. 50 The value was 41.00 μL / mL, with a 95% confidence interval of (22.88 μL / mL–58.07 μL / mL).

[0050] Table 4. Data on the toxic activity of compound bacterial agents (spore count 1:1) against root-knot nematodes.

[0051]

[0052] Table 5. Concentrations of compound bacterial agents (1:1 spore count) and 95% confidence intervals corresponding to different mortality probabilities of root-knot nematodes (partial).

[0053]

[0054]

[0055] To evaluate the combined effect of NBNC-104 and NBIN-863 fermentation broths, the co-toxicity of these two broths against root-knot nematodes was quantitatively analyzed using the Sun Yunpei co-toxicity coefficient method, based on the main reference indicators for pesticide formulation screening specified by the Institute for the Control of Agrochemicals, Ministry of Agriculture and Rural Affairs of China. The LC-values ​​of the NBNC-104 and NBIN-863 fermentation broths after 10 hours of treatment with root-knot nematodes were determined according to Example 2. 50 And the LC-index of the compound bacterial agent (spore count ratio 1:1) treated with root-knot nematodes for 10 h as determined in this embodiment. 50The relative toxicity index is calculated separately, and then the co-toxicity coefficient is calculated. The calculation formula is as follows:

[0056] Relative toxicity index (TI) = LC of standard reagent 50 / LC of the test reagent 50 ×100;

[0057] Actual Toxicity Index (ATI) of Compound Drugs = LC50 of Standard Drugs 50 LC-combined drugs 50 ×100;

[0058] Theoretical toxicity index (TTI) of compound drug = TI(A) × percentage content of single agent A in compound drug + TI(B) × percentage content of single agent B in compound drug;

[0059] Co-toxicity coefficient (CTC) = Actual toxicity index (ATI) of compound drug / Theoretical toxicity index (TTI) of compound drug × 100.

[0060] When the co-toxicity coefficient is >120, the combination of the two single agents is considered to have a synergistic effect; when 80≤co-toxicity coefficient≤120, it is considered to have an additive synergistic effect; and when the co-toxicity coefficient is <80, it is considered to have an antagonistic effect.

[0061] Using NBNC-104 fermentation broth as the standard reagent, the relative toxicity indices (TI) of NBNC-104 and NBIN-863 fermentation broths were calculated to be 100 and 65.19, respectively. The measured toxicity index (ATI) of the compound inoculum (spore number ratio 1:1) was 122.73, while the theoretical toxicity index (TTI) of the compound inoculum was calculated to be 82.60. Therefore, the co-toxicity coefficient (CTC) was calculated to be 148.58. Since the co-toxicity coefficient > 120, the 1:1 compounding of NBNC-104 and NBIN-863 fermentation broths exhibits a significant synergistic effect.

[0062] Following the experimental steps and calculation methods described above, the killing activity of compound microbial agents containing different ratios of NBNC-104 fermentation broth and NBIN-863 fermentation broth against root-knot nematodes and the co-toxicity coefficient of the compound microbial agents were determined. The results are shown in Table 6. These results indicate that the compound microbial agents containing NBNC-104 fermentation broth and NBIN-863 fermentation broth exhibit synergistic nematicidal effects when combined within a spore number ratio range of 1–10:1–10. When the two fermentation broths are combined at spore number ratios of 1:5 and 1:10, the compound microbial agents exhibit additive synergistic nematicidal effects. When the two fermentation broths are combined at spore number ratios of 1:1, 5:1, and 10:1, the compound microbial agents exhibit synergistic nematicidal effects. The compound microbial agent with a spore number ratio of 1:1 has the highest co-toxicity coefficient, indicating that its synergistic nematicidal effect is the strongest.

[0063] Table 6. Co-toxicity coefficients of NBNC-104 and NBIN-863 compound inoculants with different spore number ratios against root-knot nematodes.

[0064]

[0065] Example 5:

[0066] Nematicidal Activity Determination of a Compound Inoculum of Bacillus megaterium NBNC-104 and Bacillus stizezii NBNH-006 from Kunming

[0067] This study also tested the activity of NBNC-104 fermentation broth against root-knot nematodes after being combined with the fermentation broth of a novel, highly active strain of *Stizemonas kunmingensis* NBNH-006 (CCTCC NO: M20242323) screened at the same time.

[0068] Kunming Stizemonas NBNH-006 was isolated from the riverbed silt of the Dahei River in Hohhot, Inner Mongolia. A 96-well plate assay showed that the cell concentration of NBNH-006 in the fermentation broth was 1 × 10⁻⁶. 6 The mortality rate of root-knot nematodes treated with CFU / mL was 99.07% (±0.80%). This strain was deposited at the China Center for Type Culture Collection (CCTCC) on October 24, 2024, and is classified as: *Stutzerimonas kunmingensis* NBNH-006; accession number: CCTCC NO: M20242323; location: Wuhan University, Wuhan, China.

[0069] Fermentation broth of *Stizemonas kunmingensis* NBNH-006 was prepared (obtained by conventional culture method). The cell count in the fermentation broth was adjusted to 1×10⁶ using fresh culture medium. 6 CFU / mL was used as the stock solution (i.e., 1000 μL / mL in Table 7). The toxic activity of different gradients of NBNH-006 fermentation broth against *Strombus heterophylla* was detected using the 96-well plate bioassay method described in Example 2. The results are shown in Table 7; the killed nematodes exhibited a rigid phenotype. The probability unit model equation for the killing of *Strombus heterophylla* by NBNH-006 fermentation broth was calculated using SPSS v22.0 (IBM) software as PROBIT(P) = -3.617 + 1.999X (variable X was transformed using a logarithm with a base of 10). By observing the confidence intervals (as shown in Table 8), the LC-weighted average (LCI) of NBNH-006 fermentation broth treated with *Strombus heterophylla* for 10 h was obtained. 50 The value was 64.54 μL / mL, with a 95% confidence interval of (54.12 μL / mL - 75.25 μL / mL), demonstrating good nematicidal activity.

[0070] Table 7. Data on the toxic activity of Kunming Stizemonas NBNH-006 fermentation broth against root-knot nematodes.

[0071]

[0072]

[0073] Table 8. NBNH-006 fermentation broth concentrations and 95% confidence intervals corresponding to different mortality probabilities of root-knot nematodes (partial).

[0074]

[0075] The cell counts in the fermentation broths of strains NBNC-104 and NBNH-006 were counted separately, and the cell counts in both fermentation broths were adjusted to the same number (1×10⁻⁶) by adding fresh culture medium. 6 Two fermentation broths were mixed at a volume ratio of 1:1 (CFU / mL) to obtain a compound bacterial agent with a cell number ratio of 1:1 (this concentration of the compound bacterial agent is 1000 μL / mL as shown in Table 9). Based on the preliminary test, the compound bacterial agent was diluted to a gradient concentration by adding fresh culture medium, and its toxic activity against root-knot nematodes was detected using a 96-well plate bioassay. The bioassay results are shown in Table 9. The probability unit model equation for the 1:1 cell number ratio compound bacterial agent killing root-knot nematodes was calculated using SPSS v22.0 (IBM) data processing software as PROBIT(P) = -3.365 + 2.194X (variable X was transformed using a logarithm with a base of 10). By observing the confidence intervals (as shown in Table 10), the LC50 of the 1:1 cell number ratio compound bacterial agent treated with root-knot nematodes for 10 h was obtained. 50 The value was 34.18 μL / mL, with a 95% confidence interval of (26.78 μL / mL–41.13 μL / mL).

[0076] Table 9. Data on the toxic activity of compound bacterial agents (cell count 1:1) against root-knot nematodes.

[0077]

[0078] Table 10 shows the concentrations of compound bacterial agents (1:1 cell ratio) and 95% confidence intervals (partial) corresponding to different mortality probabilities of root-knot nematodes.

[0079]

[0080] To evaluate the combined effects of NBNC-104 and NBNH-006 fermentation broths, the co-toxicity of these broths against root-knot nematodes was quantitatively analyzed using the Sun Yunpei co-toxicity coefficient method, based on the main reference indicators for pesticide formulation screening specified by the Institute for the Control of Agrochemicals, Ministry of Agriculture and Rural Affairs of China. The LC-values ​​of the NBNC-104 and NBNH-006 fermentation broths after 10 hours of treatment with root-knot nematodes were determined according to Example 2. 50 And the LC-10 h calculus of the compound bacterial agent (cell ratio 1:1) used to treat root-knot nematodes as determined in this example. 50 The relative toxicity index is calculated separately, and then the co-toxicity coefficient is calculated. The calculation formula is as follows:

[0081] Relative toxicity index (TI) = LC of standard reagent 50 / LC of the test reagent 50 ×100;

[0082] Actual Toxicity Index (ATI) of Compound Drugs = LC50 of Standard Drugs 50 LC-combined drugs 50 ×100;

[0083] Theoretical toxicity index (TTI) of compound drug = TI(A) × percentage content of single agent A in compound drug + TI(B) × percentage content of single agent B in compound drug;

[0084] Co-toxicity coefficient (CTC) = Actual toxicity index (ATI) of compound drug / Theoretical toxicity index (TTI) of compound drug × 100.

[0085] When the co-toxicity coefficient is >120, the combination of the two single agents is considered to have a synergistic effect; when 80≤co-toxicity coefficient≤120, it is considered to have an additive synergistic effect; and when the co-toxicity coefficient is <80, it is considered to have an antagonistic effect.

[0086] Using NBNC-104 fermentation broth as the standard reagent, the relative toxicity indices (TI) of NBNC-104 and NBNH-006 fermentation broths were calculated to be 100 and 77.97, respectively. The measured toxicity index (ATI) of the compound inoculum (spore number ratio 1:1) was 147.22, while the theoretical toxicity index (TTI) of the compound inoculum was calculated to be 88.99. Therefore, the co-toxicity coefficient (CTC) was calculated to be 165.43. Since the co-toxicity coefficient > 120, the 1:1 cell number mixture of NBNC-104 and NBIN-863 fermentation broths exhibits a significant synergistic effect.

[0087] Following the experimental steps and calculation methods described above, the killing activity of compound microbial agents containing different ratios of NBNC-104 fermentation broth and NBNH-006 fermentation broth against root-knot nematodes and the co-toxicity coefficient of the compound microbial agents were determined. The results are shown in Table 11. These results indicate that the compound microbial agents containing NBNC-104 fermentation broth and NBNH-006 fermentation broth exhibit synergistic nematicidal effects when combined within a cell number ratio range of 1–10:1–10. When the two fermentation broths are combined at cell number ratios of 1:5 and 10:1, the compound microbial agents exhibit additive synergistic nematicidal effects. When the two fermentation broths are combined at cell number ratios of 1:1, 5:1, and 1:10, the compound microbial agents exhibit synergistic nematicidal effects. The compound microbial agent with a cell number ratio of 1:1 has the highest co-toxicity coefficient, indicating that its synergistic nematicidal effect is the strongest.

[0088] Table 11. Co-toxicity coefficients of NBNC-104 and NBNH-006 compound bacterial agents with different cell number ratios against root-knot nematodes.

[0089]

[0090] Example 6:

[0091] Efficacy of Bacillus megaterium NBNC-104 and its compound inoculants against root-knot nematode disease in potted plants.

[0092] To further evaluate the activity of Bacillus megaterium NBNC-104 against root-knot nematodes, a pot experiment was conducted to test the control efficacy of NBNC-104 fermentation broth against root-knot nematodes. Tomato was used as the host plant for the nematode experiment. Seeds of susceptible tomato (Jinpeng No. 3) were planted in seedling trays filled with sterilized seedling substrate and cultured for two weeks in a greenhouse at 25℃, 60% humidity, and a light / dark ratio of 16:8. Afterward, the seedlings were transplanted into 14cm diameter pots filled with a sterilized sand-soil mixture (sand-soil volume ratio of 2:1). At the time of transplanting, 3000 second-instar larvae of southern root-knot nematodes were inoculated at the base of each seedling.

[0093] The NBNC-104 fermentation broth prepared according to the fermentation culture method in Example 1 was adjusted to have a spore count of 10. 8The CFU / mL level was determined by root drenching the seedlings one day after nematode inoculation. Each plant was drenched with 50 mL of fermentation broth. Fresh fermentation medium was used as a blank control, and a commercially available nematicide abamectin·thiazophos (total active ingredient content 10%, of which abamectin content is 2.5% and thiazophos content is 7.5%) was used as a positive control. The positive control was diluted according to the dosage instructions. Furthermore, in Example 4, it was found that the synergistic nematicidal activity of NBNC-104 fermentation broth and NBIN-863 fermentation broth at a spore ratio of 1:1 was the highest. In Example 5, it was found that the synergistic nematicidal activity of NBNC-104 fermentation broth and NBNH-006 fermentation broth at a cell ratio of 1:1 was the highest. Therefore, in this example, the efficacy of the NBNC-104 / NBIN-863 and NBNC-104 / NBNH-006 compound microbial agents at this ratio against root-knot nematodes under potted conditions was also tested. The effective bacterial concentration of the compound microbial agents was also 10. 8 CFU / mL.

[0094] There were a total of 7 treatment groups (NBNC-104 fermentation broth root irrigation treatment group, NBIN-863 fermentation broth root irrigation treatment group, NBNC-104 and NBIN-863 compound bacterial agent (spore ratio 1:1) root irrigation treatment group, NBNH-006 fermentation broth root irrigation treatment group, NBNC-104 and NBNH-006 compound bacterial agent (cell ratio 1:1) root irrigation treatment group, chemical agent root irrigation treatment group, and fresh fermentation medium root irrigation treatment group), with 5 replicates for each treatment group.

[0095] After root irrigation, place the potted plants in a greenhouse and water them every two days. 30 days after root irrigation, carefully uproot the tomatoes and rinse them with water to remove the substrate soil. Count the number of root knots on each plant and weigh the tomato roots using a balance to calculate the average number of root knots per gram of root, which indicates the extent of root-knot nematode infestation.

[0096] The relative control efficacy against root-knot nematodes in the treated samples was calculated using the following formula: Control efficacy = (Number of root knots per gram of root in the negative control group - Number of root knots per gram of root in the treated group) / (Number of root knots per gram of root in the negative control group) * 100.

[0097] The results are shown in Table 12. Under potted conditions, the average number of root knots per gram of root-knot nematodes was 58.14±6.09 in plants treated with the fermentation broth of Bacillus megaterium NBNC-104 at a 1:1 spore ratio, with a relative control efficacy of 79.00%. The average number of root knots per gram of root-knot nematodes was 35.74±4.95 in plants treated with the compound inoculant made from the fermentation broth of Bacillus thuringiensis NBIN-863 at a 1:1 spore ratio, with a relative control efficacy of 87.09%. The average number of root knots per gram of root-knot nematodes was 29.76±14.97 in plants treated with the compound inoculant made from the fermentation broth of Bacillus stizezii NBNH-006 at a 1:1 cell ratio, with a relative control efficacy of 89.25%, comparable to commercially available chemical nematicides. These results indicate that Bacillus megaterium NBNC-104 and its compound inoculants have good control efficacy against root-knot nematodes under potted conditions and possess significant development value.

[0098] Table 12. Potted plant control efficacy of Bacillus megaterium NBNC-104 fermentation broth and its compound inoculants against root-knot nematodes.

[0099]

[0100] Different lowercase letters in the same column indicate significant differences at the 5% level.

[0101] Example 7:

[0102] Colonization ability of Bacillus megaterium NBNC-104 in tomato rhizosphere soil was tested.

[0103] To facilitate the detection of the colonization ability of Bacillus megaterium NBNC-104 in the soil environment, resistance screening of strain NBNC-104 was performed using Lauria-Bertani medium containing different concentrations of rifampicin, obtaining artificially induced mutant strains with rifampicin resistance. After 96-well plate bioassay and colony morphology observation, it was found that the nematicidal activity and colony morphology of the resistant strain did not change significantly. This strain could grow normally on plates containing 50 μg / mL rifampicin. The fermentation broth of this resistant mutant strain was prepared according to the fermentation culture method in Example 1, and the spore concentration in the fermentation broth was adjusted to 10. 8 The CFU / mL level was measured by drenching the roots of tomatoes inoculated with root-knot nematodes under pot conditions. Rhizosphere soil samples were collected in batches over 30 days. After weighing, the soil samples were screened using a serial dilution plating method with plates containing 50 μg / mL rifampicin, and the resistant mutants colonized in the soil were counted. Results showed that the abundance of strain NBNC-104 in the tomato rhizosphere soil was 10 on day 1. 7 The number of strains decreased over time, and by day 30, the colonization rate of NBNC-104 strain in the rhizosphere soil was 2.33 × 10⁻⁶. 6CFU / g. This result showed that the abundance of Bacillus megaterium NBNC-104 in the soil remained at 10 for 30 days after potted root irrigation. 6 -10 7 The CFU / g ratio indicates that strain NBNC-104 can be stably established in the tomato rhizosphere soil.

Claims

1. An isolated strain of Bacillus megaterium ( Priestia megaterium The strain is NBNC-104, and its preservation number is CCTCC NO: M20242324.

2. The fermentation broth of Bacillus megaterium NBNC-104 as described in claim 1, wherein the fermentation broth contains live Bacillus megaterium NBNC-104.

3. A compound, wherein the active ingredient of the compound comprises Bacillus megaterium with accession number CCTCC NO: M20242324.

4. The compound according to claim 3, wherein the active ingredients of the compound are: Bacillus megaterium NBNC-104 and Bacillus thuringiensis NBIN-863, or Bacillus megaterium NBNC-104 and Stezia kunmingii NBNH-006, wherein the preservation number of Bacillus thuringiensis NBIN-863 is CCTCC NO: M2013612, and the preservation number of Stezia kunmingii NBNH-006 is CCTCC NO: M20242323.

5. The compound according to claim 4, characterized in that, The effective bacterial concentrations of Bacillus megaterium NBNC-104 and Bacillus thuringiensis NBIN-863 are 1~10:1~10.

6. The compound according to claim 5, characterized in that, The effective bacterial concentrations of Bacillus megaterium NBNC-104 and Bacillus stizezii NBNH-006 were 1~10:1~10.

7. The application of Bacillus megaterium NBNC-104 as described in claim 1, the fermentation broth of Bacillus megaterium NBNC-104 as described in claim 2, or the compound as described in claim 3 in the control of nematodes, wherein the nematode is Southern root-knot nematode (…). Meloidogyne incognita ).

8. The application of Bacillus megaterium NBNC-104 as described in claim 1, the fermentation broth of Bacillus megaterium NBNC-104 as described in claim 2, or the compound as described in claim 3 in the preparation of nematode insecticides, wherein the nematode is Southern root-knot nematode.

9. The application of Bacillus megaterium NBNC-104 as described in claim 1, the fermentation broth of Bacillus megaterium NBNC-104 as described in claim 2, or the compound as described in claim 3 in the preparation of nematode repellents, wherein the nematode is Southern root-knot nematode.

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