Bacillus megatherium NBNC-104 for preventing and treating root-knot nematode as well as compound fungicide and application of bacillus megatherium NBNC-104

Abstract

The invention belongs to the field of microbial pesticides and relates to bacillus megatherium NBNC-104 for preventing and treating root-knot nematode as well as a compound bacterial agent and application of the bacillus megatherium NBNC-104. The preservation number of the bacillus megaterium strain NBNC-104 provided by the invention is CCTCC (China Center for Type Culture Collection) M 20242324. Laboratory bioassay data shows that the bacillus megatherium NBNC-104 has high toxic activity and avoidance effect on meloidogyne incognita, and has synergistic nematicidal activity after being compounded with the bacillus thuringiensis strain NBIN-863 and the Kunming Setinia Zerzeri NBNH-006, and the strain can be effectively planted in a rhizosphere soil environment within 30 days under a potting condition. Therefore, the strain has a wide application prospect in agricultural production.

Description

Technical Field

[0001] The present invention belongs to the field of microbial pesticides, and relates to a Bacillus megaterium NBNC-104 for controlling root-knot nematodes, its compound microbial agent and application. Background Art

[0002] Due to the characteristics of the pathogen transmission being concealed, rapid reproduction, and long harm period, nematode diseases have become important diseases in modern agriculture in China. According to the estimation of the Food and Agriculture Organization of the United Nations (FAO), the loss of crop yield caused by nematode diseases can reach 12% - 20%. The pathogen of nematode diseases is plant parasitic nematodes, among which root-knot nematodes (Meloidogyne spp.) cause the most serious harm. The average annual loss of global yield directly or indirectly caused by root-knot nematodes is estimated to reach 173 billion US dollars.

[0003] Root-knot nematodes can parasitize almost all vascular plants. The second-stage larvae of root-knot nematodes search for plant roots in the soil after emerging from the eggs, use the stylets and a series of secreted enzymes to damage the host root system and then drill into the plant body, establish feeding sites at appropriate positions, and induce the plant to form giant cells, resulting in the formation of root knots in the plant. Thus, it can be seen that the prevention and control window period of root-knot nematodes is very short, generally only two weeks, which also increases the difficulty of its prevention and control.

[0004] Currently, the main prevention and control means for nematodes is chemical control, but the application of a single chemical agent is likely to induce nematode resistance, and there are also problems of food and environmental safety hazards. Biological control is to use nematode natural enemies or biological natural products with nematocidal activity to control nematodes. Biological control has the characteristics of being environmentally friendly and pests being less likely to develop resistance, and has great potential to become a synergistic and alternative means to the existing chemical control methods. With the proposal of the demand for the green development of agriculture in China and the implementation of the action of reducing pesticides and fertilizers, biological control has gradually become an important research hotspot for the development of new green nematicides. Summary of the Invention

[0005] In view of the problem of limited biological control resources for nematodes, the primary object of the present invention is to provide a biocontrol bacterium for controlling root-knot nematodes. The strain is identified as Bacillus megaterium (Priestia megaterium), named Bacillus megaterium NBNC-104, and the preservation number is: CCTCC NO: M20242324.

[0006] Another object of the present invention is the application of the above-mentioned Bacillus megaterium and its compound microbial agent in the preparation of an agricultural nematicide for controlling root-knot nematodes.

[0007] In order to achieve the above object, the present invention takes the following technical measures:

[0008] The applicant collected soil samples from a tomato greenhouse in Wuhan, Hubei Province where root-knot nematode disease had occurred, isolated Bacillus strains by the high-temperature screening method, and screened a strain with high toxic activity against root-knot nematodes from the isolated strains, which was named NBNC-104. The single colony of strain NBNC-104 growing on Lauria-Bertani agar plate was nearly circular, milky white, with a dot-like protrusion in the center ( Figure 1 ), and the cell morphology observed under microscope was rod-shaped, relatively thick, arranged in chains, and the spores were formed in the middle. Through the analysis of average nucleotide identity (ANI) comparison of the genome, it was found that the ANI similarity of this strain with the type strain (NBRC 15308) of Bacillus megaterium (Priestia megaterium) was 97.81%, so NBNC-104 was identified as Bacillus megaterium. This strain was sent to the China Center for Type Culture Collection for preservation on October 24, 2024. The taxonomic name: Bacillus megaterium (Priestia megaterium) NBNC-104; the preservation number: CCTCC NO: M20242324; location: Wuhan University, Wuhan, China.

[0009] The protection scope of the present invention includes:

[0010] The fermentation broth of Bacillus megaterium NBNC-104, and the fermentation broth contains viable Bacillus megaterium NBNC-104.

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

[0012] For the above-mentioned compound, preferably, the active ingredient of the compound includes Bacillus megaterium NBNC-104 and Bacillus thuringiensis NBIN-863;

[0013] For the above-mentioned compound, preferably, the active ingredient of the compound includes Bacillus megaterium NBNC-104 and Stutzerimonas kunmingensis NBNH-006;

[0014] The preservation number of the above-mentioned Bacillus thuringiensis NBIN-863 is CCTCC NO: M2013612, which has been disclosed in CN103898025B, and the preservation number of Stutzerimonas kunmingensis NBNH-006 is CCTCC NO: M20242323.

[0015] For the above-mentioned compound, preferably, the effective bacterial concentration of Bacillus megaterium NBNC-104 and Bacillus thuringiensis NBIN-863 is 1-10:1-10.

[0016] For the above-mentioned compound, preferably, the effective bacterial concentrations of Bacillus megaterium NBNC-104 and Stutzerimonas kunmingensis NBNH-006 are 1-10:1-10.

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

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

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

[0020] For the above-mentioned application, preferably, the nematode is Meloidogyne incognita. Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0021] 1. The present invention provides that the fermentation broth of strain NBNC-104 has high toxicity and repellent effects on Meloidogyne incognita, and compounding with the known Bacillus thuringiensis strain NBIN-863 with high nematocidal activity and the novel nematocidal strain Stutzerimonas kunmingensis NBNH-006 can produce a synergistic effect. In the tomato pot experiment, the pot control effect of the fermentation broth of strain NBNC-104 on Meloidogyne incognita is 79.00%, compounding with Bacillus thuringiensis NBIN-863 can further increase the pot control effect to 87.09%, compounding with Stutzerimonas kunmingensis NBNH-006 can further increase the pot control effect to 89.25%, which is equivalent to the control effect of the commercially available nematocide abamectin· fosthiazate. The invention can provide new resources for the creation of novel microbial nematicides.

[0022] 2. Due to environmental factors, many microbial inoculants cannot be stably colonized in the soil, resulting in unstable control effects in the field application of microbial pesticides. The present invention detected the colonization ability of strain NBNC-104 in the rhizosphere soil and found that within 30 days of root irrigation, the colonization density of strain NBNC-104 in the rhizosphere soil can be maintained at 10 6 -10 7 cfu / g level, indicating that the strain can be colonized in the rhizosphere soil. This shows that compared with other biocontrol bacteria, the field control effect of strain NBNC-104 may be more stable, can provide a green and efficient control means for nematode control, is especially suitable for the production of organic agricultural products, and has important economic value and application prospects. Description of the Drawings

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

[0024] Figure 2 It is a schematic diagram of the effect of strain NBNC-104 on poisoning Meloidogyne incognita;

[0025] Wherein: A is the phenotype that the nematodes showed body stiffness after being treated with the fermentation broth of strain NBNC-104 of the present invention for 10 h; B is the phenotype that the nematodes were still alive after being treated with the control fermentation medium for 10 h. Detailed Embodiments

[0026] In order to better explain the present invention, the main content of the present invention will be further clarified below in conjunction with specific embodiments. However, the content of the present invention is not limited to the following embodiments. The technical solutions described in the present invention, unless otherwise specified, are all conventional technologies in the art, and the reagents or materials, unless otherwise specified, are all from commercial channels.

[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 City, Hubei Province where root-knot nematode disease had occurred, and used the high-temperature screening method to isolate Bacillus strains. The specific process was as follows: Weigh 1 g of soil sample and put it into a triangular flask containing 100 mL of sodium acetate medium. After shaking and mixing evenly, treat it at 80 °C for 10 min, then take 1 mL of the suspension for gradient dilution, and take 0.2 mL of each dilution gradient and coat it on Lauria-Bertani agar (formula: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 2% agar powder) plate, and culture it overnight at 28 °C. Then pick single colonies respectively and transfer them to 5 mL of Lauria-Bertani liquid medium, culture at 28 °C and 220 rpm for 10 h, and then add an equal volume of 50% glycerol and store at -80 °C. A total of 5 kinds of Bacillus strains were isolated.

[0030] 2) Screening of strains with high toxicity against root-knot nematodes: The isolated Bacillus strains were respectively inoculated into 5 mL of Lauria-Bertani liquid medium and activated overnight at 28°C and 220 rpm, and then transferred to the 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 by the dilution counting method, and the spore concentration in each fermentation broth was adjusted to 1×10 6 CFU / mL and then reserved for use. The Meloidogyne incognita used in the experiment was preserved and passaged by the unit using potted tomatoes. Nematode egg masses were picked from the tomato roots infected with nematodes, and the egg masses were placed in a petri dish containing 15 mL of sterile water and incubated in the dark at 20°C. The hatching of root-knot nematodes was observed, and the nematodes were collected by centrifugation and the nematode concentration was adjusted to 3 - 4 nematodes / μL. 100 μL of the fermentation broth of each Bacillus strain was taken into a 96-well plate, and then 10 μL of the nematode suspension was added to each well. It was ensured by inverted microscopy that 30 - 40 nematodes were added to each well. The fermentation medium was used as a negative control, and each treatment group had 3 replicates. The 96-well plate was placed in an incubator at 20°C and cultured in the dark for 10 h, and then the nematode mortality in each treatment group was counted by microscopy. If the mortality in the control group was <5%, no correction of mortality was required; if the control mortality was between 5% and 20%, the corrected mortality was calculated according to the formula: corrected mortality = (treatment group mortality - control group mortality) / (1 - control group mortality)×100 to correct the mortality of the treatment group; if the control mortality >20%, the experiment needed to be repeated. Through the bioassay in the 96-well plate, the strain with the highest activity among the isolated Bacillus strains was named NBNC-104. When the spore concentration in the fermentation broth was 1×10 6 CFU / mL, the mortality of root-knot nematodes treated was 98.00% (±0.78%).

[0031] 3) Identification of strain NBNC-104: The single colony morphology of strain NBIF-104 growing on the Lauria-Bertani agar plate was nearly circular, milky white, with a dot-like protrusion in the center ( Figure 1) Under microscopic examination, the cell morphology was rod-shaped, relatively thick, arranged in chains, with central spores, and Gram-positive staining. Based on these morphological characteristics, the strain was initially identified as Bacillus. Subsequently, the high-quality genomic DNA of strain NBNC-104 was extracted using the Qiagen genomic extraction kit. The genome of strain NBNC-104 was sequenced using the second-generation high-throughput sequencing technology combined with the third-generation Oxford Nanopore sequencing technology. On the bacterial identification platform EzBioCloud (https: / / www.ezbiocloud.net / ), the genome of strain NBNC-104 was analyzed and compared using the Average Nucleotide Identity (ANI) Calculator. The results showed that the ANI similarity between strain NBNC-104 and the type strain NBRC 15308 of Priestia megaterium was 97.81%. In taxonomy, when the ANI similarity between two strains is greater than 95%, the two strains are considered to be of the same species. Therefore, strain NBNC-104 was identified as Priestia megaterium.

[0032] The above strain was sent to the China Center for Type Culture Collection for preservation on October 24, 2024. Taxonomic name: Priestia megaterium NBNC-104; Preservation number: CCTCC NO: M20242324; Location: Wuhan University, Wuhan, China.

[0033] Example 2:

[0034] Determination of the nematocidal activity of Priestia megaterium NBNC-104

[0035] The fermentation broth of Priestia megaterium NBNC-104 was prepared according to the fermentation culture method in Example 1. The spore count in the fermentation broth was adjusted to 1×10 6 CFU / mL as the stock solution (i.e., 1000 μL / mL in Table 1). The nematicidal activity of different gradients of the NBNC-104 fermentation broth against Meloidogyne incognita was detected by the bioassay method using 96-well plates described in Example 1. The results are shown in Table 1. The killed nematodes showed a rigid phenotype, as Figure 2 shown. Using the SPSS v22.0 (IBM) data processing software, the probit model equation for the probability of NBNC-104 fermentation broth killing root-knot nematodes was obtained as PROBIT(P) = -2.937 + 1.726X (the variable X was transformed using the logarithm with base 10). By observing the confidence interval (shown in Table 2), the LC 50The value was 50.32 μL / mL, and the 95% confidence interval was (25.84 μL / mL - 75.59 μL / mL), showing good nematicidal activity.

[0036] Table 1 Determination data of the toxicicidal activity of the fermentation broth of Bacillus megaterium NBNC-104 against Meloidogyne

[0037]

[0038] Table 2 Concentrations of the fermentation broth of NBNC-104 corresponding to different mortality probabilities of Meloidogyne and the 95% confidence interval (partial)

[0039]

[0040] To facilitate the subsequent detection of whether the strain NBNC-104 can be compounded with other nematicidal microbial agents, the known highly active strain Bacillus thuringiensis NBIN-863 (CCTCC NO: M2013612) was inoculated and prepared into a fermentation broth according to the fermentation and culture method of Example 1, and the LC of the fermentation broth of the strain NBIN-863 treating Meloidogyne for 10 h was measured according to the above method 50 The value was 77.19 μL / mL, and the 95% confidence interval was (63.64 μL / mL - 91.37 μL / mL).

[0041] Example 3:

[0042] Determination of the chemotactic effect of Bacillus megaterium NBNC-104 on Meloidogyne

[0043] The Bacillus megaterium NBNC-104 fermentation broth was prepared according to the fermentation culture method in Example 1 for detecting the chemotactic effect of the fermentation broth on Meloidogyne spp. Pour 1% water agar into a petri dish with a diameter of 3.5 cm, and draw lines on the back of the petri dish. The petri dish was divided 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 Meloidogyne spp. suspension in the middle of region B, where the number of Meloidogyne spp. 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 Meloidogyne spp. in each region with an inverted microscope and calculate the chemotaxis index of Meloidogyne spp. The chemotaxis index (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 tests for each treatment. The experimental results are shown in Table 3. The average C.I. value of the NBNC-104 fermentation broth treating Meloidogyne spp. is -0.44, indicating that the NBNC-104 fermentation broth has a strong repellent effect on Meloidogyne spp.

[0044] Table 3 Chemotactic effect of the NBNC-104 fermentation broth on Meloidogyne spp.

[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 detect whether strain NBNC-104 can be compounded with other nematode-killing microbial agents, in this study, the fermentation broth of NBNC-104 was compounded with the fermentation broth of the known highly active strain Bacillus thuringiensis NBIN-863 (CCTCC NO: M2013612), and a bioassay in 96-well plates was carried out. The number of spores in the fermentation broths of strain NBNC-104 and strain NBIN-863 was counted respectively, and the number of spores in the two fermentation broths was adjusted to the same amount (1×10 6 CFU / mL) by adding fresh medium. The two fermentation broths were mixed at a volume ratio of 1:1, and a compound microbial agent with a spore number ratio of 1:1 was obtained (the compound microbial agent at this concentration is 1000 μL / mL in Table 4). On the basis of preliminary tests, the compound microbial agent was diluted into gradient concentrations by adding fresh medium, and the toxicity of the compound microbial agent against Meloidogyne incognita was detected by bioassay in 96-well plates. The bioassay results are shown in Table 4. Using the SPSS v22.0 (IBM) data processing software, the probit model equation for the compound microbial agent with a spore number ratio of 1:1 to kill Meloidogyne incognita was obtained as PROBIT(P) = -3.348 + 2.076X (the variable X was transformed using the logarithm with base 10). By observing the confidence interval (as shown in Table 5), the LC 50 value of the compound microbial agent with a spore number ratio of 1:1 against Meloidogyne incognita for 10 h was 41.00 μL / mL, and the 95% confidence interval was (22.88 μL / mL - 58.07 μL / mL).

[0050] Table 4 Toxicity determination data of the compound microbial agent (spore number 1:1) against Meloidogyne incognita

[0051]

[0052] Table 5 Concentrations of the compound microbial agent (spore number 1:1) corresponding to different mortality probabilities of Meloidogyne incognita and the 95% confidence interval (partial)

[0053]

[0054]

[0055] To evaluate the combined effect of the fermentation broth of NBNC-104 and the fermentation broth of NBIN-863, according to the main reference indexes for pesticide formula screening specified by the Institute for the Control of Agrochemicals, Ministry of Agriculture and Rural Affairs of China, the Sun Yunpei co-toxicity coefficient method was used to quantitatively analyze the co-toxic effect of the fermentation broth of NBNC-104 and the fermentation broth of NBIN-863 on Meloidogyne incognita. According to the LC 50 of the fermentation broth of NBNC-104 and the fermentation broth of NBIN-863 against Meloidogyne incognita for 10 h measured in Example 2 50, calculate the relative toxicity index respectively and then calculate the co-toxicity coefficient. The calculation formula is as follows:

[0056] Relative toxicity index (TI) = Standard agent LC 50 / Test agent LC 50 × 100;

[0057] Measured toxicity index of the compound agent (ATI) = Standard agent LC 50 / Compound agent LC 50 × 100;

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

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

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

[0061] Taking the NBNC-104 fermentation broth as the standard agent, the relative toxicity index TI values of the NBNC-104 fermentation broth and the NBIN-863 fermentation broth were calculated to be 100 and 65.19 respectively. The measured toxicity index ATI of the compound agent (spore ratio 1:1) was 122.73, and the theoretical toxicity index TTI of the compound agent was calculated to be 82.60, thus calculating the co-toxicity coefficient CTC to be 148.58. Since the co-toxicity coefficient > 120, the combination of the NBNC-104 fermentation broth and the NBIN-863 fermentation broth at a spore ratio of 1:1 has a significant synergistic effect.

[0062] According to the above experimental procedures and calculation methods, the nematicidal activities of the compound agents with different ratios of the NBNC-104 fermentation broth and the NBIN-863 fermentation broth and the co-toxicity coefficients of the compound agents were determined. The results are shown in Table 6. The results show that the combination of the NBNC-104 fermentation broth and the NBIN-863 fermentation broth has a synergistic nematicidal effect within the range of spore ratios of 1 - 10:1 - 10. When the two fermentation broths are combined at spore ratios of 1:5 and 1:10, the compound agents have an additive synergistic nematicidal effect. When the spore ratios of the two fermentation broths are 1:1, 5:1 and 10:1, the compound agents have a synergistic nematicidal effect. Among them, the co-toxicity coefficient of the compound agent with a spore ratio of 1:1 is the highest, indicating the strongest synergistic nematicidal effect.

[0063] Table 6 Co-toxicity coefficients of the compound bactericides of NBNC-104 and NBIN-863 with different spore number ratios against Meloidogyne

[0064]

[0065] Example 5:

[0066] Determination of the nematicidal activity of the compound bactericide of Bacillus megaterium NBNC-104 and Stutzerimonas kunmingensis NBNH-006

[0067] This study also detected the activity of the compound of the fermentation broth of NBNC-104 and the fermentation broth of the newly screened highly active strain Stutzerimonas kunmingensis NBNH-006 (CCTCC NO: M20242323) against Meloidogyne.

[0068] Stutzerimonas kunmingensis NBNH-006 was isolated from the bottom silt of the Dahei River in Hohhot, Inner Mongolia. Through bioassay in 96-well plates, the mortality rate of Meloidogyne treated with the fermentation broth of NBNH-006 at a cell concentration of 1×10 6 CFU / mL was 99.07% (±0.80%). This strain was sent to the China Center for Type Culture Collection for preservation on October 24, 2024. Taxonomic name: Stutzerimonas kunmingensis NBNH-006; Preservation number: CCTCC NO: M20242323; Location: Wuhan University, Wuhan, China.

[0069] Prepare the fermentation broth of Stutzerimonas kunmingensis NBNH-006 (obtained by culturing in a conventional manner), and adjust the number of bacterial cells in the fermentation broth to 1×10 6 CFU / mL as the stock solution (i.e., 1000 μL / mL in Table 7). The nematicidal activity of different gradients of the fermentation broth of NBNH-006 against Meloidogyne incognita was detected by the 96-well plate bioassay method described in Example 2. The results are shown in Table 7. The killed nematodes showed a rigid phenotype. The probit model equation for the killing of Meloidogyne by the fermentation broth of NBNH-006 was calculated using SPSS v22.0 (IBM) data processing software as PROBIT(P) = -3.617 + 1.999X (the variable X was transformed using the logarithm with base 10). By observing the confidence interval (shown in Table 8), the LC 50 value of the fermentation broth of NBNH-006 for treating Meloidogyne for 10 h was 64.54 μL / mL, and the 95% confidence interval was (54.12 μL / mL - 75.25 μL / mL), showing good nematicidal activity.

[0070] Table 7 Toxicity assay data of the fermentation broth of Stenotrophomonas stutzeri NBNH-006 from Kunming against Meloidogyne spp.

[0071]

[0072]

[0073] Table 8 Concentrations of the fermentation broth of NBNH-006 and their 95% confidence intervals corresponding to different mortality probabilities of Meloidogyne spp. (partial)

[0074]

[0075] The number of bacterial cells in the fermentation broths of strains NBNC-104 and NBNH-006 were counted respectively, and the number of bacterial cells in the two fermentation broths was adjusted to the same amount (1×10 6 CFU / mL) by adding fresh medium. The two fermentation broths were mixed at a volume ratio of 1:1 to obtain a compound microbial agent with a cell number ratio of 1:1 (the compound microbial agent at this concentration is 1000 μL / mL in Table 9). Based on preliminary tests, the compound microbial agent was diluted into gradient concentrations by adding fresh medium, and the toxicity of the compound microbial agent against Meloidogyne spp. was detected using a 96-well plate bioassay. The bioassay results are shown in Table 9. Using the SPSS v22.0 (IBM) data processing software, the probit model equation for the compound microbial agent with a cell number ratio of 1:1 to kill Meloidogyne spp. was obtained as PROBIT(P) = -3.365 + 2.194X (the variable X was transformed using the logarithm with base 10). By observing the confidence interval (as shown in Table 10), the LC 50 value of the compound microbial agent with a cell number ratio of 1:1 against Meloidogyne spp. after 10 h was 34.18 μL / mL, and the 95% confidence interval was (26.78 μL / mL – 41.13 μL / mL).

[0076] Table 9 Toxicity assay data of the compound microbial agent (cell number 1:1) against Meloidogyne spp.

[0077]

[0078] Table 10 Concentrations of the compound microbial agent (cell number 1:1) and their 95% confidence intervals corresponding to different mortality probabilities of Meloidogyne spp. (partial)

[0079]

[0080] To evaluate the combined effect of the NBNC-104 fermentation broth and the NBNH-006 fermentation broth, according to 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 Sun Yunpei co-toxicity coefficient method was used to quantitatively analyze the co-toxic effect of the NBNC-104 fermentation broth and the NBNH-006 fermentation broth on root-knot nematodes. According to the LC 50 determined for the NBNC-104 fermentation broth and the NBNH-006 fermentation broth treating root-knot nematodes for 10 h in Example 2, and the LC 50 determined for the compound microbial agent (cell number ratio 1:1) treating root-knot nematodes for 10 h in this example, the relative toxicity index was calculated respectively and then the co-toxicity coefficient was calculated. The calculation formula is as follows:

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

[0082] Actual toxicity index of compound pesticide (ATI) = LC 50 of standard pesticide / LC 50 of compound pesticide × 100;

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

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

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

[0086] Taking the NBNC-104 fermentation broth as the standard pesticide, the relative toxicity index TI values of the NBNC-104 fermentation broth and the NBNH-006 fermentation broth were calculated to be 100 and 77.97 respectively. The actual toxicity index ATI of the compound microbial agent (spore number ratio 1:1) was 147.22, and the theoretical toxicity index TTI of the compound microbial agent was calculated to be 88.99. Thus, the co-toxicity coefficient CTC was calculated to be 165.43. Since the co-toxicity coefficient > 120, the compounding of the NBNC-104 fermentation broth and the NBIN-863 fermentation broth at a cell number ratio of 1:1 has a significant synergistic effect.

[0087] According to the above experimental steps and calculation methods, the nematicidal activities of the compound microbial agents with different ratios of NBNC-104 fermentation broth and NBNH-006 fermentation broth against Meloidogyne incognita and the co-toxicity coefficients of the compound microbial agents were determined. The results are shown in Table 11. The results indicate that the compounding of NBNC-104 fermentation broth and NBNH-006 fermentation broth within the range of cell number ratios of 1-10:1-10 has a synergistic nematicidal effect. When the two fermentation broths are compounded at a cell number ratio of 1:5 and 10:1, the compound microbial agent has an additive synergistic nematicidal effect. When the cell number ratios of the two fermentation broths are 1:1, 5:1, and 1:10, the compound microbial agent has a synergistic nematicidal effect. Among them, the co-toxicity coefficient of the compound microbial agent with a cell number ratio of 1:1 is the highest, indicating the strongest synergistic nematicidal effect.

[0088] Table 11 Co-toxicity coefficients of the compound microbial agents of NBNC-104 and NBNH-006 with different cell number ratios against Meloidogyne incognita

[0089]

[0090] Example 6:

[0091] Pot experiment on the control effect of Bacillus megaterium NBNC-104 and its compound microbial agent against Meloidogyne incognita

[0092] To further evaluate the activity of Bacillus megaterium NBNC-104 against Meloidogyne incognita, a pot experiment was conducted to detect the control effect of NBNC-104 fermentation broth on Meloidogyne incognita. Tomato was used as the host plant for nematodes in the pot experiment. Seeds of susceptible tomato (Jinpeng No. 3) were planted in seedling plug trays filled with sterilized seedling substrate and cultured in a greenhouse at a temperature of 25 °C, a humidity of 60%, and a light / dark ratio of 16:8 for two weeks. Then the seedlings were transplanted into flower pots with a diameter of 14 cm filled with sterilized sand mixture (sand volume ratio of 2:1). When transplanting, 3000 second-instar larvae of Meloidogyne incognita were inoculated at the roots of each seedling.

[0093] The NBNC-104 fermentation broth prepared according to the fermentation and culture method in Example 1 was adjusted to a spore number of 10 in the fermentation broth 8At the level of CFU / mL, the seedlings were treated by root irrigation 1 day after inoculating nematodes. Each plant was irrigated with 50 mL of the fermentation broth, and the fresh fermentation medium was used as the blank control. The commercially available nematicide abamectin· fosthiazate (total active ingredient content 10%, abamectin content 2.5%, fosthiazate content 7.5%) was used as the positive control, and the dosage of the positive control was diluted according to the dosage indicated in the instruction manual. In addition, in Example 4, it was found that the compounding of the NBNC-104 fermentation broth and the NBIN-863 fermentation broth at a spore number ratio of 1:1 had the highest synergistic nematicidal activity. In Example 5, it was found that the compounding of the NBNC-104 fermentation broth and the NBNH-006 fermentation broth at a cell number ratio of 1:1 had the highest synergistic nematicidal activity. Therefore, in this example, the control effects of the NBNC-104 / NBIN-863 compound microbial agent and the NBNC-104 / NBNH-006 compound microbial agent at this ratio on root-knot nematodes were also tested under pot conditions, and the effective bacteria concentration of the compound microbial agent was also 10 8 CFU / mL.

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

[0095] The potted plants after root irrigation were placed in the greenhouse and watered every two days. 30 days after root irrigation, the tomatoes were carefully uprooted and washed with water to remove the substrate soil. The number of root knots on each plant was counted, and the weight of the tomato roots was weighed with a balance, and the number of root knots per gram of roots was calculated to represent the infection situation of root-knot nematodes.

[0096] The relative control effect of the treated sample on root-knot nematodes was calculated according to the formula: control effect = (number of root knots per gram of roots in the negative control - number of root knots per gram of roots in the treatment group) / (number of root knots per gram of roots in the negative control) * 100.

[0097] The results are shown in Table 12. Under potting conditions, the number of root knots per gram of roots of the plants treated by drenching with the fermentation broth of *Bacillus megaterium* NBNC-104 was 58.14±6.09, and the relative control efficacy was 79.00%. For the plants treated by drenching with the compound microbial agent prepared by mixing the fermentation broth of *Bacillus megaterium* NBNC-104 and that of *Bacillus thuringiensis* NBIN-863 at a spore number ratio of 1:1, the number of root knots per gram of roots was 35.74±4.95, and the relative control efficacy was 87.09%. For the plants treated by drenching with the compound microbial agent prepared by mixing the fermentation broth of *Bacillus megaterium* NBNC-104 and that of *Stutzerimonas kunmingensis* NBNH-006 at a cell number ratio of 1:1, the number of root knots per gram of roots was 29.76±14.97, and the relative control efficacy was 89.25%, which was comparable to that of the commercially available chemical nematicide. The results indicate that *Bacillus megaterium* NBNC-104 and its compound microbial agents have good control efficacy against root-knot nematodes under potting conditions and have significant development value.

[0098] Table 12 Potting control efficacy of the fermentation broth of *Bacillus megaterium* NBNC-104 and its compound microbial agents against root-knot nematodes

[0099]

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

[0101] Example 7:

[0102] Detection of the colonization ability of *Bacillus megaterium* NBNC-104 in the rhizosphere soil of tomatoes

[0103] To facilitate the detection of the colonization ability of *Bacillus megaterium* NBNC-104 in the soil environment, the strain NBNC-104 was screened for resistance using Lauria-Bertani medium containing different concentrations of rifampicin to obtain an artificially mutagenized strain with rifampicin resistance. Through bioassay in 96-well plates and observation of colony morphology, it was found that the nematicidal activity and colony morphology of the resistant strain did not change significantly, and the 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 and culture method in Example 1, and the spore concentration in the fermentation broth was adjusted to 10 8 CFU / mL. Under potting conditions, tomatoes inoculated with root-knot nematodes were drenched. The rhizosphere soil of tomatoes was sampled in batches within 30 days. After weighing the sampled soil, the gradient dilution and plate coating method was used to screen for the resistant mutant strain colonized in the soil with plates containing 50 μg / mL rifampicin and count it. The results showed that the abundance of the strain NBNC-104 in the rhizosphere soil of tomatoes on the 1st day was 10 7 level. As time increased, the number of strains decreased. By the 30th day, the colonization amount of the strain NBNC-104 in the rhizosphere soil was 2.33×10 6CFU / g. The results showed that within 30 days after pot irrigation, the colonization abundance of *Bacillus megaterium* NBNC-104 in the soil remained at 10 6 -10 7 CFU / g, indicating that strain NBNC-104 could stably colonize in the rhizosphere soil of tomatoes.

Claims

1. An isolated strain of Bacillus megaterium ( Priestia megaterium )NBNC-104, the deposit number of the strain is: CCTCC NO: M20242324.

2. The fermentation broth of Bacillus megaterium NBNC-104 according to claim 1, wherein the fermentation broth contains live bacteria of Bacillus megaterium NBNC-104.

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

4. The composition according to claim 3, wherein the active ingredients of the composition comprise Bacillus thuringiensis NBIN-863 and / or Kunming Stitzerella NBNH-006, the deposit number of the Bacillus thuringiensis NBIN-863 is CCTCC NO: M2013612, and the deposit number of the Kunming Stitzerella NBNH-006 is CCTCC NO: M20242323.

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

6. The compound according to claim 5, characterized in that The effective bacterial concentrations of the Bacillus megaterium NBNC-104 and the Kunming Stitzerella monocytogenes NBNH-006 are 1-10:1-10.

7. Use of the Bacillus megaterium NBNC-104 according to claim 1, the fermentation liquid of Bacillus megaterium NBNC-104 according to claim 2, or the compound according to claim 3 in controlling nematodes.

8. Use of the Bacillus megaterium NBNC-104 according to claim 1, the fermentation liquid of Bacillus megaterium NBNC-104 according to claim 2, or the compound according to claim 3 in the preparation of a nematode insecticide.

9. Use of the Bacillus megaterium NBNC-104 according to claim 1, the fermentation liquid of Bacillus megaterium NBNC-104 according to claim 2, or the compound according to claim 3 in the preparation of a nematode repellent.

10. The use according to claim 7, 8 or 9, wherein the nematode is Meloidogyne incognita ( Meloidogyne Incognita ).

Images