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

Abstract

The invention belongs to the field of microbial pesticides, and relates to bacillus cereus for preventing and treating root-knot nematode, and a compound bacterial agent and application thereof. The preservation number of the bacillus cereus NBNT-070 disclosed by the invention is CCTCC (China Center For Type Culture Collection) NO: M20242322. Laboratory test data shows that the bacillus cereus NBNT-070 has high poison killing and attracting activity on meloidogyne incognita, and has synergistic nematode killing activity after being respectively compounded with the bacillus thuringiensis strain NBIN-863 and the Kunming scherzersonia zerzersonia NBNH-006, and the strain can be effectively planted in a rhizosphere soil environment within 30 days under a pot culture 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 cereus for controlling root-knot nematodes, its compound bactericide and application. Background Art

[0002] Due to the characteristics of the pathogen transmission being concealed, rapid reproduction, and long damage 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 damage. 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. After the second-stage larvae of root-knot nematodes emerge from the eggs, they search for plant roots in the soil, 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 on the plant. Thus, it can be seen that the control window period of root-knot nematodes is very short, generally only two weeks, which also increases the difficulty of its control.

[0004] Currently, the main 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 existing chemical control methods. With the proposal of the demand for 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 in the development of new green nematocides. 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 cereus, named Bacillus cereus NBNT-070, and the preservation number is: CCTCC NO: M20242322.

[0006] Another object of the present invention is the application of the above-mentioned Bacillus cereus and its compound bactericide 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 Honghu City, Hubei Province where root-knot nematode disease had occurred, isolated Bacillus strains using the high-temperature screening method, and screened a strain with high toxic activity against root-knot nematodes from the isolated strains, naming it NBNT-070. The single colonies of the NBNT-070 strain growing on Lauria-Bertani agar plates were nearly circular, large, irregular, milky white, and had folds on the surface ( Figure 1 ). Microscopic examination showed that the cell morphology was rod-shaped, arranged in short or long chains, and the spores were central or nearly central. Through genomic average nucleotide identity (ANI) alignment analysis, it was found that the ANI similarity between this strain and the type strain (ATCC14579) of Bacillus cereus was 98.62%. Thus, NBNT-070 was identified as Bacillus cereus. This strain was sent to the China Center for Type Culture Collection for preservation on October 24, 2024. The taxonomic name: Bacillus cereus NBNT-070; the preservation number: CCTCC NO: M20242322; location: Wuhan University, Wuhan, China.

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

[0010] The fermentation broth of Bacillus cereus NBNT-070, and the fermentation broth contains viable Bacillus cereus NBNT-070 bacteria.

[0011] A microbial compound, and the active ingredient of the compound includes Bacillus cereus with the preservation number CCTCC NO: M20242322.

[0012] For the above-mentioned compound, preferably, the active ingredient of the compound includes Bacillus cereus NBNT-070 and Bacillus thuringiensis NBIN-863;

[0013] For the above-mentioned compound, preferably, the active ingredient of the compound includes Bacillus cereus NBNT-070 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 concentrations of Bacillus cereus NBNT-070 and Bacillus thuringiensis NBIN-863 are 1-10:1-10.

[0016] For the above-mentioned compound, preferably, the effective bacterial concentrations of Bacillus cereus NBNT-070 and Stutzerimonas kunmingensis NBNH-006 are 1-10:1-10.

[0017] Use of Bacillus cereus NBNT-070, the fermentation broth of Bacillus cereus NBNT-070 or their compound in controlling nematodes.

[0018] Use of Bacillus cereus NBNT-070, the fermentation broth of Bacillus cereus NBNT-070 or their compound in preparing nematode insecticides.

[0019] Use of Bacillus cereus NBNT-070, the fermentation broth of Bacillus cereus NBNT-070 or their compound in preparing nematode attractants.

[0020] For the above-mentioned use, 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 NBNT-070 has high toxicity and attraction activities against Meloidogyne incognita, and has synergistic nematocidal activities after being compounded with Bacillus thuringiensis strain NBIN-863 and Stutzerimonas kunmingensis NBNH-006 respectively. The data of pot experiments show that the control effect of strain NBNT-070 on root-knot nematodes on tomato pots is 75.91%, and the pot control effect after being compounded with Bacillus thuringiensis NBIN-863 is increased to 87.41%, and the pot control effect after being compounded with Stutzerimonas kunmingensis NBNH-006 is increased to 85.24%, which is equivalent to that of the commercially available nematocide abamectin· fosthiazate. This invention can provide new resources for the creation of new 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 NBNT-070 in rhizosphere soil and found that 30 days after root irrigation, the colonization density of strain NBNT-070 in rhizosphere soil can be maintained at 10 6 CFU / g level, indicating that this strain can colonize in rhizosphere soil. This shows that compared with other biocontrol bacteria, the field control effect of strain NBNT-070 may be more stable, which can provide a green and efficient control means for nematode control, especially suitable for the production of organic agricultural products, and has important economic value and application prospects. Description of the Drawings

[0023] Figure 1Schematic diagram of the colony morphology of strain NBNT-070 on Lauria-Bertani agar plate.

[0024] Figure 2 Schematic diagram of the effect of strain NBNT-070 on poisoning Meloidogyne incognita. Among them Figure 2 A shows the phenotype that the nematodes showed body stiffness after being treated with the fermentation broth of strain NBNT-070 of the present invention for 10 h, Figure 2 B shows the phenotype that the nematodes were still alive after being treated with the control fermentation medium for 10 h. Detailed implementation manners

[0025] 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 are all conventional techniques in the art unless otherwise specified. The reagents or materials are all from commercial channels unless otherwise specified.

[0026] Example 1:

[0027] Isolation, screening and identification of Bacillus cereus

[0028] 1) Isolation of Bacillus strains: The applicant collected soil samples from a tomato greenhouse in Honghu City, Hubei Province where Meloidogyne incognita 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. Take 0.2 mL of each dilution gradient and coat it on Lauria-Bertani agar (formula: 10 g / L peptone, 5 g / L yeast extract, 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. After culturing at 28 °C and 220 rpm for 10 h, add an equal volume of 50% glycerol and store it at -80 °C. A total of 4 Bacillus strains were isolated.

[0029] 2) Screening of strains with high toxicity against Meloidogyne incognita: The isolated Bacillus strains were respectively inoculated into 5 mL of Lauria-Bertani liquid medium, 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) respectively. Culture at 28 °C and 220 rpm for 24 h, calculate the spore concentration in each fermentation broth by the dilution counting method, and adjust the spore concentration in each fermentation broth to 10 6After that, it is reserved for use. The Meloidogyne incognita used in the experiment was preserved and passaged by our unit using potted tomatoes. Nematode egg masses were picked from the roots of nematode-infected tomatoes. The egg masses were placed in a petri dish containing 15 mL of sterile water and incubated 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 the fermentation broth of each Bacillus strain was added to a 96-well plate, and 10 μL of the nematode suspension was added to each well. It was ensured that 30-40 nematodes were added to each well by observing under an inverted microscope. 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 microscopic examination. If the mortality in the control group was <5%, the mortality did not need to be corrected; if the control mortality was between 5% and 20%, the corrected mortality of the treatment group was calculated according to the formula corrected mortality = (treatment group mortality - control group mortality) / (1 - control group mortality) × 100; 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 NBNT-070, and the spore concentration in the fermentation broth was 10 6 The corrected mortality of Meloidogyne incognita was 100.00% (±0%) when the CFU / mL was

[0030] 3) Identification of strain NBNT-070: The single colony of strain NBNT-070 growing on the Lauria-Bertani agar plate was nearly circular, large, irregular, milky white, and there would be folds on the surface ( Figure 1 ). By microscopic examination, the cell morphology was rod-shaped, arranged in short or long chains, with central or nearly central spores, and Gram staining was positive. Through these morphological characteristics, the strain was preliminarily identified as Bacillus. Subsequently, the high-quality genomic DNA of strain NBNT-070 was extracted using the Qiagen genomic extraction kit. The genome of strain NBNT-070 was sequenced using the second-generation high-throughput sequencing technology combined with the third-generation Oxford Nanopore sequencing technology. The genome of strain NBNT-070 was analyzed and compared on the bacterial identification platform EzBioCloud (https: / / www.ezbiocloud.net / ) using the average nucleotide identity alignment tool ANI Calculator. The results showed that the ANI similarity between strain NBNT-070 and the type strain ATCC 14579 of Bacillus cereus was 98.62%. In taxonomy, when the ANI similarity of two strains is greater than 95%, these two strains are considered to be the same species. Therefore, strain NBNT-070 was identified as Bacillus cereus.

[0031] The above strain was sent to the China Center for Type Culture Collection for preservation on October 24, 2024. The taxonomic name is Bacillus cereus NBNT-070; the preservation number is CCTCC NO: M20242322; the location is Wuhan University, Wuhan, China.

[0032] Example 2:

[0033] Determination of the chemotactic effect of Bacillus cereus NBNT-070 on Meloidogyne

[0034] Prepare the fermentation broth of Bacillus cereus NBNT-070 according to the fermentation culture method in Example 1 for detecting the chemotactic effect of the fermentation broth on Meloidogyne. 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 NBNT-070 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 suspension in the middle of region B, with about 100 Meloidogyne. 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 in each region under an inverted microscope and calculate the chemotactic index of Meloidogyne. Chemotactic index (C.I. value) = (number of nematodes in region A - number of nematodes in region C) / (number of nematodes in region A + 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 the sterilized medium without inoculation as the control treatment group, and conduct 5 parallel tests for each treatment. The test results (as shown in Table 1) show that the average C.I. value of the NBNT-070 fermentation broth treating Meloidogyne is 0.208, which is greater than 0.2, indicating that the NBNT-070 fermentation broth of the strain has a strong attracting effect on Meloidogyne.

[0035] Table 1 Chemotactic effect of the NBNT-070 fermentation broth on Meloidogyne

[0036]

[0037] Example 3:

[0038] Determination of the nematicidal activity of Bacillus cereus NBNT-070

[0039] Prepare the fermentation broth of Bacillus cereus NBNT-070 according to the fermentation culture method in Example 1, and adjust the number of spores in the fermentation broth to 1×10 6 CFU / mL as the stock solution (i.e., 1000 μL / mL in Table 2). Detect the nematicidal activity of different gradients of NBNT-070 fermentation broth against Meloidogyne incognita according to the 96-well plate bioassay method described in Example 1. The results are shown in Table 2. 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 killing probability of NBNT-070 fermentation broth against root-knot nematodes was obtained as PROBIT(P) = -3.937 + 3.615X (the variable X was transformed using the logarithm with base 10). By observing the confidence interval (shown in Table 3), the LC 50 value of NBNT-070 fermentation broth treating root-knot nematodes for 10 h was 12.28 μL / mL, and the 95% confidence interval was (9.12 μL / mL - 15.48 μL / mL), showing good nematicidal activity.

[0040] Table 2 Determination data of the nematicidal activity of Bacillus cereus NBNT-070 fermentation broth against root-knot nematodes

[0041]

[0042] Table 3 Concentrations of NBNT-070 fermentation broth corresponding to different mortality probabilities of root-knot nematodes and 95% confidence intervals (partial)

[0043]

[0044] To facilitate the subsequent detection of whether the strain NBNT-070 can be compounded with other nematicidal microbial agents, inoculate and prepare the fermentation broth of the known highly active strain Bacillus thuringiensis NBIN-863 (CCTCC NO: M2013612) according to the fermentation culture method in Example 1. Determine the LC 50 value of NBIN-863 fermentation broth treating root-knot nematodes for 10 h as 77.19 μL / mL, and the 95% confidence interval was (63.64 μL / mL - 91.37 μL / mL).

[0045] Example 4:

[0046] Determination of the nematicidal activity of the compound microbial agent of Bacillus cereus NBNT-070 and Bacillus thuringiensis NBIN-863

[0047] To detect whether the strain NBNT-070 can be compounded with other nematode-killing microbial agents, in this study, the fermentation broth of NBNT-070 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 NBNT-070 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 a 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) = -5.679 + 4.590X (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 17.26 μL / mL, and the 95% confidence interval was (13.53 μL / mL - 21.55 μL / mL).

[0048] Table 4 Toxicity determination data of the compound microbial agent (spore number 1:1) against Meloidogyne incognita

[0049]

[0050] Table 5 Concentrations of the compound microbial agent (spore number 1:1) corresponding to different mortality probabilities of Meloidogyne incognita and 95% confidence intervals (partial)

[0051]

[0052] To evaluate the combined effect of the fermentation broth of NBNT-070 and the fermentation broth of NBIN-863, according to the main reference indicators for pesticide formulation screening specified by the Pesticide Inspection Institute of the 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 NBNT-070 and the fermentation broth of NBIN-863 on Meloidogyne incognita. According to the LC 50 of the fermentation broth of NBNT-070 and the fermentation broth of NBIN-863 against Meloidogyne incognita for 10 h measured in Example 3 50 and the LC

[0053] Relative toxicity index (TI) = standard agent LC 50 / test agent LC 50 × 100;

[0054] Measured toxicity index (ATI) of the compound agent = standard agent LC 50 / compound agent LC 50 × 100;

[0055] Theoretical toxicity index (TTI) of the compound agent = TI(A) × percentage content of single agent A in the compound agent + TI(B) × percentage content of single agent B in the compound agent;

[0056] Coefficient of co-toxicity (CTC) = measured toxicity index (ATI) of the compound agent / theoretical toxicity index (TTI) of the compound agent × 100.

[0057] When the coefficient of co-toxicity > 120, it is considered that the combination of two single agents has a synergistic effect; when 80 ≤ coefficient of co-toxicity ≤ 120, it is considered an additive effect; when the coefficient of co-toxicity < 80, it is considered an antagonistic effect.

[0058] Taking the NBNT-070 fermentation broth as the standard agent, the relative toxicity index TI values of the NBNT-070 fermentation broth and the NBIN-863 fermentation broth were calculated to be 100 and 15.91 respectively. The measured toxicity index ATI of the compound bactericide (spore number ratio 1:1) was 71.15, and the theoretical toxicity index TTI of the compound bactericide was calculated to be 57.96, thus calculating the coefficient of co-toxicity CTC to be 122.76. Since the coefficient of co-toxicity > 120, the combination of the NBNT-070 fermentation broth and the NBIN-863 fermentation broth at a spore number ratio of 1:1 has a significant synergistic effect.

[0059] According to the above test procedures and calculation methods, the nematicidal activities of the compound bactericides of different ratios of the NBNT-070 fermentation broth and the NBIN-863 fermentation broth against root-knot nematodes and the coefficients of co-toxicity of the compound bactericides were determined, and the results are shown in Table 6. The results show that when the NBNT-070 fermentation broth and the NBIN-863 fermentation broth are combined within the range of spore number ratios of 1-10:1-10, when the spore number ratios of the two are 1:10 and 1:5, they have an additive effect; when the spore numbers of the two fermentation broths are 1:1, 5:1 and 10:1, the compound bactericides have a synergistic effect, and the compound bactericide with a spore number ratio of 10:1 has the highest coefficient of co-toxicity, indicating the strongest synergistic nematicidal effect.

[0060] Table 6 Coefficients of co-toxicity of the compound bactericides of NBNT-070 and NBIN-863 with different spore number ratios against root-knot nematodes

[0061]

[0062] Example 5:

[0063] Determination of the nematicidal activity of the compound microbial agent of Bacillus cereus NBNT-070 and Stutzerimonas kunmingensis NBNH-006

[0064] To detect whether strain NBNT-070 can be used in combination with other nematicidal microbial agents, in this study, the fermentation broth of NBNT-070 was combined with the fermentation broth of the known highly active strain Stutzerimonas kunmingensis NBNH-006 (CCTCC NO: M20242323) screened during the same period, and a 96-well plate bioassay was carried out.

[0065] Stutzerimonas kunmingensis NBNH-006 was isolated from the bottom sludge of the Dahei River in Hohhot, Inner Mongolia. Through a 96-well plate bioassay, the mortality rate of Meloidogyne incognita treated with the cell concentration of 1×10 6 CFU / mL in the fermentation broth of NBNH-006 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.

[0066] 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 with fresh medium 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 3. The results are shown in Table 7. The killed nematodes showed a rigid phenotype. The probit model equation for the killing probability of the fermentation broth of NBNH-006 against Meloidogyne incognita was calculated using the 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 incognita 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.

[0067] Table 7 Data on the nematicidal activity of the fermentation broth of Stutzerimonas kunmingensis NBNH-006 against Meloidogyne incognita

[0068]

[0069] Table 8 Concentration of NBNH-006 fermentation broth and 95% confidence interval corresponding to different mortality probabilities of Meloidogyne spp. (partial)

[0070]

[0071] The number of bacterial cells in the fermentation broths of strains NBNT-070 and NBNH-006 was counted respectively. By adding fresh medium, the number of bacterial cells in the two fermentation broths was adjusted to the same amount (1×10 6 CFU / mL). The two fermentation broths were mixed at a volume ratio of 1:1, and a compound microbial agent with a cell number ratio of 1:1 was obtained (the compound microbial agent at this concentration is 1000 μL / mL in Table 9). 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 spp. was detected by bioassay using 96-well plates. 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) = -10.101 + 7.471X (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 22.49 μL / mL, and the 95% confidence interval was (18.55 μL / mL - 27.27 μL / mL).

[0072] Table 9 Determination data of the toxicity of the compound microbial agent (cell number ratio 1:1) against Meloidogyne spp.

[0073]

[0074] Table 10 Concentration of the compound microbial agent (cell number 1:1) and 95% confidence interval corresponding to different mortality probabilities of Meloidogyne spp. (partial)

[0075]

[0076] To evaluate the combined effect of the fermentation broth of NBNT-070 and the fermentation broth of NBNH-006, referring to Example 4, the relative toxicity index was calculated respectively and then the co-toxicity coefficient was calculated.

[0077] Taking the NBNT-070 fermentation broth as the standard agent, the relative toxicity index TI values of the NBNT-070 fermentation broth and the NBNH-006 fermentation broth were calculated to be 100 and 19.03 respectively. The measured toxicity index ATI of the compound microbial agent (the ratio of spore number to cell number is 1:1) was 54.60, and the theoretical toxicity index TTI of the compound microbial agent was calculated to be 59.52. Thus, the co-toxicity coefficient CTC was calculated to be 91.73. The compounding of the NBNT-070 fermentation broth and the NBNH-006 fermentation broth at a ratio of spore number to cell number of 1:1 has an additive synergistic effect.

[0078] According to the above test steps and calculation methods, the killing activities of the compound microbial agents of different ratios of the NBNT-070 fermentation broth and the NBNH-006 fermentation broth against root-knot nematodes and the co-toxicity coefficients of the compound microbial agents were determined. The results are shown in Table 11. The results indicate that when the NBNT-070 fermentation broth and the NBNH-006 fermentation broth are compounded within the range of the ratio of spore number to cell number of 1-10:1-10, when the ratio of spore number to cell number of the two fermentation broths is 10:1, 1:1, 1:5, and 1:10, the compound microbial agent has an additive synergistic effect, and the co-toxicity coefficient of the compound microbial agent with a ratio of spore number to cell number of 5:1 is the highest, showing a synergistic effect.

[0079] Table 11 Co-toxicity coefficients of the compound microbial agents of NBNT-070 and NBNH-006 with different cell number ratios against root-knot nematodes

[0080]

[0081] Example 6:

[0082] Pot experiment for detecting the control efficacy of Bacillus cereus NBNT-070 and its two compound microbial agents against root-knot nematode disease

[0083] In order to further evaluate the activity of Bacillus cereus NBNT-070 against root-knot nematodes, a pot experiment was conducted to detect the control efficacy of the NBNT-070 fermentation broth against root-knot nematodes. Tomato was used as the host plant for nematodes to carry out the pot experiment. Seeds of susceptible tomato (Jinpeng No. 3) were planted in a seedling plug tray 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, which were filled with sterilized sand and soil mixture (the volume ratio of sand to soil is 2:1). When transplanting, 3000 second-instar larvae of Meloidogyne incognita were inoculated at the roots of each seedling.

[0084] The NBNT-070 fermentation broth prepared according to the fermentation and culture method in Example 1 was adjusted to make the spore number in the fermentation broth reach 10 8CFU / mL. One day after inoculating the nematodes, the seedlings were treated by root irrigation with 50 mL of the fermentation broth for each plant. Fresh fermentation medium was used as the blank control, and the commercially available nematicide abamectin thiazolophos (total active ingredient content 10%, abamectin content 2.5%, thiazolophos content 7.5%) was used as the positive control. The dosage of the positive control was diluted according to the dosage indicated in the instruction manual. Additionally, in Example 4, it was found that the compounding of the NBNT-070 fermentation broth and the NBIN-863 fermentation broth at a spore number ratio of 10:1 had the highest synergistic nematicidal activity, and in Example 5, it was found that the compounding of the NBNT-070 fermentation broth and the NBNH-006 fermentation broth at a cell number ratio of 5:1 had the highest synergistic nematicidal activity. Therefore, in this example, the control effects of the NBNT-070 / NBIN-863 compound bactericide and the NBNT-070 / NBNH-006 compound bactericide at this ratio on root-knot nematodes were also tested under pot conditions. The effective bacteria concentration of the compound bactericide was also 10 8 CFU / mL.

[0085] There were a total of 7 treatment groups (the NBNT-070 fermentation broth root irrigation treatment group, the NBIN-863 fermentation broth root irrigation treatment group, the NBNH-006 fermentation broth root irrigation treatment group, the NBNT-070 and NBIN-863 compound bactericide (spore number ratio 10:1) root irrigation treatment group, the NBNT-070 and NBNH-006 compound bactericide (cell number ratio 5:1) root irrigation treatment group, the chemical agent root irrigation treatment group, and the fresh fermentation medium root irrigation treatment group), with 5 replicates for each treatment group.

[0086] The potted plants after root irrigation were placed in the greenhouse and watered every two days. 30 days after root irrigation, the tomato plants were carefully uprooted and rinsed 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 to calculate the number of root knots per gram of roots on average, which was used to represent the infection of root-knot nematodes.

[0087] 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.

[0088] The results are shown in Table 12. Under pot culture conditions, the number of root knots per gram of roots of the plants treated with the fermentation broth of Bacillus cereus NBNT-070 by root irrigation was 66.69±12.97, and the relative control efficacy was 75.91%. For the plants treated with the compound microbial agent prepared by mixing the fermentation broth of Bacillus cereus NBNT-070 with that of Bacillus thuringiensis NBIN-863 at a spore number ratio of 10:1 by root irrigation, the number of root knots per gram of roots was 34.84±6.89, and the relative control efficacy was 87.41%. For the plants treated with the compound microbial agent prepared by mixing the fermentation broth of Bacillus cereus NBNT-070 with that of Stutzerimonas kunmingensis NBNH-006 at a spore number and cell number ratio of 5:1 by root irrigation, the number of root knots per gram of roots was 40.86±4.33, and the relative control efficacy was 85.24%. The control efficacies of the two compound agents were comparable to that of the commercially available chemical nematicide. The results indicate that Bacillus cereus NBNT-070 and its compound microbial agent have good control efficacy against root-knot nematodes under pot culture conditions and have significant development value.

[0089] Table 12 Pot culture control efficacy of the fermentation broth of Bacillus cereus NBNT-070 and its two compound microbial agents against root-knot nematodes

[0090]

[0091]

[0092] Different lowercase letters after the data in the same column indicate significant differences at the 5% level

[0093] Example 7:

[0094] Detection of the colonization ability of Bacillus cereus NBNT-070 in tomato rhizosphere soil

[0095] To facilitate the detection of the colonization ability of Bacillus cereus NBNT-070 in the soil environment, the strain NBNT-070 was screened for resistance using Lauria-Bertani medium containing different concentrations of rifampicin to obtain an artificially mutagenized strain with rifampicin resistance. Through 96-well plate bioassay 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. Prepare the fermentation broth of this resistant mutant strain according to the fermentation culture method in Example 1, and adjust the spore concentration in the fermentation broth to 10 8 CFU / mL, and irrigate the tomatoes inoculated with root-knot nematodes under pot culture conditions. Sample the tomato rhizosphere soil in batches within 30 days. After weighing the sampled soil, use the gradient dilution and plate coating method to screen and count the resistant mutant strains colonized in the soil using plates containing 50 μg / mL rifampicin. The results show that the abundance of the strain NBNT-070 in the tomato rhizosphere soil on the 1st day was 10 7CFU / g level. As time increased, the number of strains decreased. By the 30th day, the colonization amount of strain NBNT-070 in the rhizosphere soil was 1.78×10 6 CFU / g. This result shows that within 30 days after pot irrigation with the bacterial suspension, the colonization abundance of Bacillus cereus NBNT-070 in the soil remained at 10 6 -10 7 CFU / g, indicating that strain NBNT-070 can stably colonize in the rhizosphere soil of tomatoes.

Claims

1. An isolated strain of Bacillus cereus ( Bacillus cereus )NBNT-070, the deposit number of the Bacillus cereus is: CCTCC NO: M20242322.

2. The fermentation broth of Bacillus cereus NBNT-070 according to claim 1, wherein the fermentation broth contains live bacteria of Bacillus cereus NBNT-070.

3. A compound, wherein the active ingredient of the compound comprises Bacillus cereus with a deposit number of CCTCC NO: M20242322.

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 concentration of the Bacillus cereus NBNT-070 and the Bacillus thuringiensis NBIN-863 is 1-10:1-10.

6. The compound according to claim 4, characterized in that The effective bacterial concentration of the Bacillus cereus NBNT-070 and the Kunming Stitzerella NBNH-006 is 1-10:1-10.

7. Use of the Bacillus cereus NBNT-070 according to claim 1, the fermentation liquid of Bacillus cereus NBNT-070 according to claim 2, or the compound according to claim 3 in controlling nematodes.

8. Use of the Bacillus cereus NBNT-070 according to claim 1, the fermentation liquid of Bacillus cereus NBNT-070 according to claim 2, or the compound according to claim 3 in the preparation of a nematode insecticide.

9. Use of the Bacillus cereus NBNT-070 according to claim 1, the fermentation liquid of Bacillus cereus NBNT-070 according to claim 2, or the compound according to claim 3 in the preparation of a nematode attractant.

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

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