Methylotrophic bacillus n7, preparation and application thereof in prevention and control of soybean cyst nematode

Abstract

The application provides a methylotrophic bacillus N7, a preparation and application thereof in prevention and control of soybean cyst nematode, and belongs to the technical field of microorganisms. The methylotrophic bacillus N7 has a preservation number of CGMCC No.31391, and is obtained by mutagenizing an original strain N1 with 0.4% ethyl methanesulfonate. The methylotrophic bacillus N7 is suitable for a low-temperature soybean production area with a temperature of 5 to 20 DEG C, has high resistance to soybean cyst nematode, a cyst inhibition rate of a high-concentration group in a seedling stage is higher than 70%, a soybean yield in a mature stage is increased by 24.6% compared with a blank control, is non-toxic and non-residual, and is friendly to soil ecology.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a methyltrophic Bacillus N7, its preparation, and its application in the control of soybean cyst nematodes. Background Technology

[0002] Soybean cyst nematode (SCN) belongs to the genus *Cytocercaria* of the family Heterodermaceae, class Pleurocera, order Pachycercaria, superfamily Pachycercariae, phylum Nematoda. It is the most destructive soybean pathogen, characterized by high reproductive capacity, wide adaptability, and long cyst survival time. In my country, it has developed 11 physiological races and 12 highly virulent races, severely impacting soybean yield. The insidious nature of SCN outbreaks greatly complicates control efforts. Currently, traditional strategies for SCN control in production mainly include crop rotation with non-host crops, planting resistant soybean varieties, and chemical control. However, due to factors such as adjustments in soybean planting structure and economic benefits, traditional crop rotation models have limitations. Utilizing resistant varieties is the most economical and suitable method for SCN control; however, nematode-resistant soybean resources are scarce, and long-term planting of a single resistant variety leads to the continuous evolution of nematode race virulence in the field, gradually reducing or even eliminating the resistance of resistant varieties. Traditional chemical control methods such as nematicides are effective in controlling SCN, but due to their high toxicity and high residue, long-term use not only harms the ecological environment and disrupts the soil ecological balance, but also may lead to problems such as drug resistance in pathogenic nematodes.

[0003] In recent years, biological control technology has received increasing attention. Natural enemies of nematodes, including functional microorganisms such as *Verticillium chlamydosporum*, *Pasteurella lilacinus*, *Bacillus*, *Pasteurella multocida*, and *Streptomyces*, have been widely used in SCN control, achieving good results while ensuring ecological safety. However, compared with traditional chemical pesticides, functional microorganisms still face challenges in SCN control, such as lower inhibition rates and poorer stability.

[0004] Therefore, developing functional microorganisms with high insecticidal activity and stable efficacy is of great significance for the biological control of SCN. Summary of the Invention

[0005] In order to overcome the above-mentioned defects and deficiencies in the prior art, this application aims to provide a methylotrophic Bacillus N7, its preparation, and its application in the control of soybean cyst nematodes.

[0006] One of the objectives of this invention is to provide a methyltrophic Bacillus ( Bacillus methylotrophicusN7, characterized in that the methyltrophic Bacillus N7 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on July 22, 2024, with accession number CGMCC No. 31391.

[0007] Preferably, the method for preparing methyltrophic Bacillus N7 includes treating methyltrophic Bacillus N1 with the chemical mutagen 0.4% ethyl methanesulfonate to obtain methyltrophic Bacillus N7.

[0008] The second objective of this invention is to provide an application of methyltrophic Bacillus N7 in the control of soybean cyst nematodes.

[0009] Preferably, the activity of the methyltrophic Bacillus N7 strain is 1×10⁻⁶. 9 -1×10 10 CFU / g.

[0010] Preferably, the methyltrophic Bacillus N7 is applied in low-temperature soybean production areas, where the ambient temperature range is 5~20℃.

[0011] The third objective of this invention is to provide a biocontrol agent formulation containing methyltrophic Bacillus N7, characterized in that the formulation is a wettable powder.

[0012] Preferably, the biocontrol agent formulation further includes a carrier and adjuvants;

[0013] The carrier is kaolin, and the additives include dispersants and wetting agents;

[0014] The dispersant includes sodium lignosulfonate, and the wetting agent includes sodium dodecylbenzenesulfonate.

[0015] Preferably, the mass ratio of the carrier, the adjuvant, and methyltrophic Bacillus N7 is (50~70):(5~10):(20~45).

[0016] Preferably, the biocontrol agent is used for the control of soybean cyst nematodes; when applying the biocontrol agent, it should be diluted with sterile water 100 to 200 times before spraying, and the diluted bacterial suspension should be used within 2 hours.

[0017] The beneficial effects of this invention are:

[0018] The methyltrophic Bacillus provided by this invention ( Bacillus methylotrophicusN7 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on July 22, 2024, with accession number CGMCC No. 31391. It was obtained by mutagenesis of the original strain N1 using 0.4% ethyl methanesulfonate (EMS). It exhibits significant activity against soybean cyst nematode (SCN) and excellent low-temperature tolerance. Its anti-SCN efficacy far exceeds existing control methods; at a high concentration in the seedling stage (1×10⁻⁶), it showed significant activity. 10 The CFU / g strain showed an inhibition rate of 73.2% on rhizosphere soil cysts, 71.8% on second-instar larvae in the rhizosphere soil, and 73.7% on root cysts. This is not only superior to traditional chemical insecticides (the highest inhibition rate in the positive control group was 53.4%) and commercially available inoculants (the highest inhibition rate of inoculant 1 was 46.3% and that of inoculant 2 was 41.9%), but also significantly higher than the original strain N1 (the highest inhibition rate was 55.0%). At the same time, this strain can still maintain high activity in the main soybean producing areas of Northeast China (such as Xing'an League in Inner Mongolia) at 5 to 20℃, with a rhizosphere colonization time of ≥40 days. It effectively solves the industry pain point of activity decay and control failure of existing biocontrol bacteria in low-temperature areas, and has extremely strong regional adaptability. Attached Figure Description

[0019] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention, wherein:

[0020] Figure 1 This is a diagram of the experimental site for an example.

[0021] The methyltrophic Bacillus provided by this invention ( Bacillus methylotrophicus N7 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on July 22, 2024, with accession number CGMCC No. 31391. Detailed Implementation

[0022] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. It should also be noted that, for ease of description, only the parts relevant to the invention are shown in the drawings. Unless otherwise specified, the embodiments and features described herein can be combined with each other. The present application will now be described in detail with reference to the accompanying drawings and embodiments.

[0023] Example 1

[0024] The screening and preparation methods for microbial agents are as follows:

[0025] Take 1g of fresh soil from a soybean-producing area affected by SCN, place it in a clean Erlenmeyer flask containing glass beads and 100ml of sterile water, and incubate on a shaker at 120rpm for 1 hour. Then, take a sterile test tube and dilute the soil solution to 10^10. -3 10 -4 10 -5 10 -6 The diluted soil solution was spread onto solid selective potato dextrose agar (containing chloramphenicol) medium and incubated in a cell culture incubator in the dark for 1 day. Colonies were selected and isolated and purified on new plates until a single colony appeared on the plate without other contaminants. A single colony was picked with a sterile inoculation loop and inoculated into LB medium. The culture was carried out on a shaker at 25°C and 120 rpm for 24 hours to prepare a bacterial suspension. The strain with strong nematode resistance was screened by the cyst nematode plate test and identified by 16S rDNA. A methyltrophic Bacillus N1 strain was obtained from the suspension.

[0026] The above-mentioned strains were treated with 0.4% ethyl methanesulfonate (EMS) as a chemical mutagen, and mutants with enhanced nematode resistance were screened again using the cyst nematode plate test to obtain methylotrophic Bacillus N7. The specific steps are as follows:

[0027] Pre-mutation treatment: The N1 strain was inoculated into LB liquid medium and cultured at 28°C and 180 rpm on a shaker until mid-log (OD600 = 0.6 to 0.8). The cells were collected by centrifugation (6000 rpm, 10 min), washed twice with 0.85% sterile physiological saline, and resuspended to a bacterial concentration of 1×10⁻⁶. 8 CFU / mL;

[0028] EMS treatment parameter optimization: Set EMS concentration gradients (0.2%, 0.4%, 0.6%, 0.8%) and treat the above bacterial suspensions for 30 min, 60 min, and 90 min respectively. Calculate the lethality rate by plate counting. It was found that the lethality rate reached 70% to 80% when treated with 0.4% EMS for 60 min, which had the highest mutagenicity, thus preserving enough mutants while avoiding total lethality.

[0029] Screening process quantification: Post-mutation bacterial culture diluted to 1×10 3 CFU / mL was spread onto selective plates containing 20 SCN second-instar larvae / mL and incubated at 25℃ for 48h. Single colonies with a larval mortality rate ≥60% were selected and transferred to LB medium for rescreening. After three passages, strains with stable nematode resistance and low temperature tolerance (strain activity decreased by ≤10% after 72h incubation at 5℃) were selected, namely Methyltrophic Bacillus N7.

[0030]

[0031] Example 2

[0032] Field trials in areas affected by soybean cyst nematode disease:

[0033] like Figure 1 As shown, in the SCN-affected area of ​​Xing'an League, Inner Mongolia, a small-plot experiment was conducted based on the principle of randomization to analyze the effects of the above-mentioned methylotrophic Bacillus on the growth of soybean and its rhizosphere cyst nematode.

[0034] The experiment was divided into a blank control group, a positive control group, a first commercially available bacterial agent group, a second commercially available bacterial agent group, a methyltrophic Bacillus N1 group, a low-concentration methyltrophic Bacillus N7 group, a medium-concentration methyltrophic Bacillus N7 group, and a high-concentration methyltrophic Bacillus N7 group.

[0035] The positive control group used commercially available 0.5% abamectin, with a recommended dosage of 3500g / mu according to the product instructions.

[0036] The main components of the first commercially available microbial agent are Bacillus subtilis and Bacillus licheniformis, with a product description indicating a content of 1×10⁻⁶. 9 CFU / g, dosage is 500g / mu; the second commercially available inoculant mainly consists of Beauveria bassiana and Paecilomyces lilacinus, with a product description indicating a content of 1×10⁻⁶ CFU / g. 10 CFU / g, dosage is 500g / mu;

[0037] The activity of Bacillus methyltrophic group N1 strains was 1×10 9 CFU / g, dosage 500g / mu; activity of low-concentration group of methyltrophic Bacillus N7 strain is 1×10 9 CFU / g, dosage 500g / mu; the activity of the medium concentration group of Bacillus methyltrophicus N7 strains was adjusted to 5×10 9 CFU / g, dosage 500g / mu; activity of high-concentration group of methyltrophic Bacillus N7 strain adjusted to 1×10 10 CFU / g, dosage is 500g / mu.

[0038] During the land preparation stage, apply commercially available 0.5% abamectin, commercially available inoculant 1, commercially available inoculant 2, methyltrophic Bacillus N1, low concentration methyltrophic Bacillus N7, medium concentration methyltrophic Bacillus N7 and high concentration methyltrophic Bacillus N7;

[0039] 25 days after soybean sowing, apply commercially available 0.5% abamectin, commercially available inoculant 1, commercially available inoculant 2, Bacillus methyltrophicus N1, low-concentration Bacillus methyltrophicus N7, medium-concentration Bacillus methyltrophicus N7 and high-concentration Bacillus methyltrophicus N7 once each.

[0040] Four soybean plants were set up in each group. Soybean plant and rhizosphere soil samples were collected 40 days after soybean sowing (seedling stage) to detect the number of soybean plant and root cysts, the number of rhizosphere soil cysts and the number of second instar larvae. The test results are shown in Table 1. At the soybean maturity stage, plant height, number of pods per plant, water content, 100-seed weight and yield were detected. The test results are shown in Table 2.

[0041] Table 1 Seedling stage monitoring data

[0042]

[0043] Table 2. Data from soybean maturity testing

[0044]

[0045] Table 1 shows the soybean seedling monitoring data based on the soybean production area in Xing'an League, Inner Mongolia, where temperatures ranged from 5 to 20℃. Regarding nematode resistance, the blank control group had 194±14.3 years of rhizosphere soil cysts and 22.8±3.8 years of root cysts, while the high-concentration group of methyltrophic Bacillus N7 had corresponding values ​​of only 52±4.0 days and 6.0±1.2 days, respectively. The rhizosphere soil cyst inhibition rate was 73.2%, and the root cyst inhibition rate was 73.7%, significantly higher than the positive control (chemical insecticide, highest inhibition rate 53.4%), commercially available inoculant (highest inhibition rate 46.3%), and the original strain N1 (highest inhibition rate 55.0%). This demonstrates that the nematode resistance of methyltrophic Bacillus N7 significantly improved after mutagenesis with 0.4% ethyl methanesulfonate, surpassing traditional chemical insecticides, commercially available inoculants, and the original strain. In terms of strain activity adaptability, the inhibition rates of the low-concentration group, medium-concentration group and high-concentration group of Bacillus methyltrophicus N7 showed a gradient increase (57.7% to 73.7%), and the low-concentration group of Bacillus methyltrophicus N7 was superior to the first and second commercially available bacterial agents, balancing the control effect and application cost.

[0046] In terms of low-temperature applicability, the low-concentration, medium-concentration, and high-concentration groups of Bacillus methyltrophicus N7 maintained high activity at temperatures ranging from 5 to 20°C, while the inhibition rates of the first and second commercially available inoculants were only 35.1% to 46.3%. Bacillus methyltrophicus N7 could also maintain high activity in the main soybean producing areas of Northeast China (such as Xing'an League in Inner Mongolia) at temperatures ranging from 5 to 20°C, with a rhizosphere colonization time of ≥40 days. This effectively solved the industry pain point of activity decay and control failure of existing biocontrol bacteria in low-temperature areas, demonstrating extremely strong regional adaptability.

[0047] Table 2 shows the soybean maturity test data, focusing on agronomic traits and yield, further validating the comprehensive value of the technical solution. The blank control group had a plant height of 50.5 cm, a 100-seed weight of 16.95 g, and a yield of 106.8 kg / mu. The high-concentration group of methyltrophic Bacillus N7 achieved corresponding indicators of 61.1 cm, 19.5 g, and 133.1 kg / mu, representing increases of 21.0% in plant height, 15.2% in 100-seed weight, and 24.6% in yield compared to the blank control. These figures were also higher than those of commercially available 0.5% abamectin, the first commercially available inoculant, and the second commercially available inoculant, demonstrating that methyltrophic Bacillus N7 not only controls nematodes but also improves the soybean growing environment, achieving both yield and quality improvement, thus possessing both control efficacy and yield-enhancing effects. Furthermore, in the experiment, methyltrophic Bacillus N7 was applied during land preparation and supplemented with an additional application 25 days after sowing, resulting in a stable increase in maturity indicators. In addition, as a biological strain, Bacillus methyltrophicus N7 does not have the high toxicity and high residue problems of chemical pesticides, and has not had any adverse effects on soybean growth. It is non-toxic, residue-free, and environmentally friendly to the soil.

[0048] In summary, Table 1 demonstrates the effectiveness of the invention in controlling nematodes in the early stage, the adaptability of the strain activity, and the suitability for low temperatures, while Table 2 demonstrates the effectiveness of the formulation in increasing yield and quality at maturity. Furthermore, through comparison with the blank control group, the positive control group, the first commercially available inoculant group, and the second commercially available inoculant group, the core advantages of this invention—strong nematode resistance, broad low-temperature adaptability, eco-friendliness, and significant yield increase—are highlighted.

Claims

1. A methyltrophic Bacillus ( Bacillus methylotrophicus N7, characterized in that, The methyltrophic Bacillus N7 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on July 22, 2024, with accession number CGMCCNo.31391.

2. The application of the methylotrophic Bacillus N7 as described in claim 1 in the control of soybean cyst nematodes.

3. The application as described in claim 2, characterized in that, The activity of the methyltrophic Bacillus N7 strain is 1×10⁻⁶. 9 -1×10 10 CFU / g.

4. The application as described in claim 2, characterized in that, The methyltrophic Bacillus N7 is applied in low-temperature soybean production areas, where the ambient temperature range is 5~20℃.

5. A biocontrol agent formulation containing the methyltrophic Bacillus N7 as described in claim 1, characterized in that, The formulation is a wettable powder.

6. The biocontrol agent formulation as described in claim 5, characterized in that, The biocontrol agent formulation also includes a carrier and adjuvants; The carrier is kaolin, and the additives include dispersants and wetting agents; The dispersant includes sodium lignosulfonate, and the wetting agent includes sodium dodecylbenzenesulfonate.

7. The biocontrol agent formulation as described in claim 6, characterized in that, The mass ratio of the carrier, adjuvant and methyltrophic Bacillus N7 is (50~70):(5~10):(20~45).

8. The biocontrol agent formulation as described in claim 5, characterized in that, The biocontrol agent is used for the control of soybean cyst nematodes. When applying the biocontrol agent, it should be diluted 100 to 200 times with sterile water before spraying, and the diluted bacterial suspension should be used within 2 hours.

Images