Bacillus licheniformis MJ3 for the prevention and control of bacterial wilt and its application

Abstract

This invention discloses Bacillus licheniformis MJ3 for the prevention and control of bacterial wilt and its applications. The depository of Bacillus licheniformis MJ3 is the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M20242215. Applying the technical solution of this invention, Bacillus licheniformis MJ3 (CCTCC M20242215) and its active bacterial agent possess phosphorus-solubilizing properties, which can improve soil fertility and increase crop yield. Simultaneously, it exhibits significant antagonistic effects against the bacterial wilt pathogen, inhibiting its growth and effectively controlling bacterial wilt. It is a strain with great potential for biofertilizer application and has good promotional value.

Description

Technical Field

[0001] This invention relates to the field of biological control, and more specifically, to a Bacillus licheniformis MJ3 strain for controlling bacterial wilt and its applications. Background Technology

[0002] Bacterial wilt is a disease caused by Ralstonia solanacearum (Ralstonia solanacearum Ralstonia solanacearum Bacterial wilt is a serious plant disease caused by bacterial wilt. Its main harm lies in infecting the vascular bundles of plants, disrupting nutrient transport channels, and preventing the normal upward supply of nutrients absorbed by the roots. This leads to plant wilting and a 30-50% reduction in yield, and in severe cases, yield reduction by half or even total crop failure. Currently, the control of peanut bacterial wilt mainly relies on resistant varieties, lacking other effective chemical pesticides or biological control measures. However, the breeding cycle for resistant varieties is long, and there is still a mortality rate of about 10%. With the development of green agriculture, agricultural microorganisms are receiving increasing attention due to their safety and high efficiency. Biological control is considered the most promising strategy for controlling bacterial wilt. Screening for bacterial wilt-resistant bacterial strains and developing a green integrated control measure combining resistant varieties and biological control is of great significance for improving the control capacity of bacterial wilt and promoting the development of a healthy agricultural product industry.

[0003] peanut( Arachis hypogaea Peanuts are an important leguminous oilseed crop, possessing nitrogen-fixing capabilities and requiring relatively high levels of phosphorus and potassium. Bacterial wilt of peanuts is a devastating soil-borne disease, often referred to as the "cancer" of peanuts. It primarily infects the vascular bundles of the plant, typically reducing yield by 20-30% in some fields, and in severe cases, causing total crop failure. Bacterial wilt of tomato is a devastating bacterial soil-borne vascular disease, prevalent in southern regions. Infection leads to plant death, widespread outbreaks, significant yield reductions, and even crop failure. The pathogen is also highly contagious and difficult to eradicate. Bacillus licheniformis is one of the more promising species in the Bacillus genus. In recent years, research on Bacillus licheniformis has increased both domestically and internationally, including applications in plant disease control, feed processing, pharmaceutical development, and environmental remediation. In the crop farming sector, its applications mainly include rhizosphere growth promotion, plant disease control, soil microbial community regulation, and pesticide residue degradation. However, there are few reports on Bacillus licheniformis capable of antagonizing Ralstonia solanacearum.

[0004] This invention addresses the shortcomings of existing control methods by large-scale isolation and screening of beneficial microorganisms to discover Bacillus licheniformis strains that antagonize Ralstonia solanacearum, enhance fertilizer efficiency, and promote growth. Based on this, it develops microbial agents in different formulations suitable for various application scenarios. When applied to crop cultivation, these agents can inhibit Ralstonia solanacearum, improve fertilizer efficiency, increase yield, reduce the use of chemical fertilizers and pesticides, and achieve a highly efficient and ecological planting model. Summary of the Invention

[0005] The present invention aims to provide Bacillus licheniformis MJ3 for the prevention and control of bacterial wilt and its application.

[0006] According to one aspect of the present invention, a strain of Bacillus licheniformis MJ3 for the prevention and control of bacterial wilt is provided. The depository of Bacillus licheniformis MJ3 is the China Center for Type Culture Collection, with accession number CCTCC NO: M20242215.

[0007] Furthermore, the 16S rDNA sequence of Bacillus licheniformis MJ3 is shown in SEQ ID NO.1.

[0008] Furthermore, Bacillus licheniformis MJ3 contains a gene cluster of antimicrobial peptides and functional substances, the sequences of which are shown in SEQ ID NO.2, SEQ ID NO:3 and SEQ ID NO:4.

[0009] According to another aspect of the present invention, a Bacillus licheniformis inoculant is provided. This Bacillus licheniformis inoculant comprises the active cells of the aforementioned Bacillus licheniformis MJ3.

[0010] Furthermore, Bacillus licheniformis inoculants are available in liquid, powder, or granule form.

[0011] Further, the Bacillus licheniformis inoculant is prepared by the following steps: (1) Bacillus licheniformis MJ3 is activated, inoculated onto NA plate medium, and placed in a constant temperature incubator for 24-36 h at 37°C; (2) the activated strain from step (1) is picked, inoculated into NB liquid medium, and cultured at 35-37°C and 150-200 rpm for 12-24 h to obtain seed liquid; (3) the seed liquid obtained in step (2) is added to the amplification medium at a volume ratio of 1-3% and cultured for 18-24 h to obtain fermentation broth, which is the liquid formulation.

[0012] Further, in step (3), the culture medium comprises 3%-7% corn starch, 3%-7% soybean meal, 0.05%-0.15% magnesium sulfate, 0.1%-0.15% potassium dihydrogen phosphate, 0-5% yeast fermentation concentrate, and the remaining component is water, based on the total mass of the culture medium.

[0013] Further, in step (3), the culture temperature is 35-37℃; and / or the culture time is 18-24h; and / or the aeration rate is 20-50L / min; and / or the tank pressure is 0.2-0.5 standard atmospheres.

[0014] Further, the fermentation broth obtained in step (3) is dried to obtain a bacterial powder formulation.

[0015] Furthermore, the inlet air temperature for the drying process is 130-145℃, the outlet air temperature is 60-70℃, and the speed of the feed peristaltic pump is 5-15 rpm.

[0016] Further, the fermentation broth obtained in step (3) is granulated to obtain a granule dosage form.

[0017] Further, the granulation process includes: centrifuging the fermentation broth to obtain bacterial sludge, adding 8-10% soluble starch as a protective agent and 10-15% zeolite powder as a carrier to the bacterial sludge, mixing evenly, and then drying and granulating at a temperature of 50-60℃ for 30-60 minutes.

[0018] Furthermore, the effective viable count in the Bacillus licheniformis inoculant shall not be less than 10 billion / ml or 10 billion / g.

[0019] According to another aspect of the present invention, the application of the above-mentioned Bacillus licheniformis MJ3, or Bacillus licheniformis inoculant, in the control of bacterial wilt is provided; preferably, the application in the control of bacterial wilt in peanuts and tomatoes.

[0020] Furthermore, the application method is root irrigation, with a dosage of 5-20 kg per acre.

[0021] Applying the technical solution of this invention, Bacillus licheniformis MJ3, with preservation number CCTCC M20242215, can effectively antagonize Ralstonia solanacearum and also possesses phosphorus-solubilizing properties. It can not only control bacterial wilt in peanuts and tomatoes but also improve soil fertility and increase yield. Attached Figure Description

[0022] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0023] Figure 1 Image A shows the morphology of Bacillus licheniformis MJ3 on NA medium, and image B shows a schematic diagram after Gram staining.

[0024] Figure 2 The image shows a phylogenetic tree of Bacillus licheniformis MJ3 and other representative strains constructed based on the 16S rDNA gene sequence.

[0025] Figure 3 The image shows the phosphorus solubilization effect of Bacillus licheniformis MJ3 on an inorganic phosphorus culture medium plate;

[0026] Figure 4 The results show the inhibitory effects of thiamethoxam copper and Bacillus licheniformis MJ3 inoculum on Ralstonia solanacearum and Ralstonia solanacearum. Detailed Implementation

[0027] Information on strain preservation

[0028] Bacillus licheniformis MJ3 ( Bacillus licheniformis MJ3 was deposited at the China Center for Type Culture Collection (CCTCC) on October 15, 2024, with accession number CCTCC NO: M20242215. The depositary address is Wuhan University, Wuhan, China, 430072, China; telephone: 027-68754052.

[0029] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0030] The inventors of this application conducted large-scale isolation and screening of beneficial microorganisms, identifying a strain of *Bacillus licheniformis* capable of controlling bacterial wilt and improving soil fertility. This *Bacillus licheniformis* is suitable for application in the field of biofertilizers. The *Bacillus licheniformis* MJ3 is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M20242215. The 16S rDNA sequence of *Bacillus licheniformis* MJ3 is shown in SEQ ID NO.1. This sequence information is used to confirm the species and characteristics of the strain. Through this information, researchers can accurately identify the strain, ensuring its stability and effectiveness under different environments, providing a scientific basis for the standardized production of microbial fertilizers. According to a typical embodiment of this application, a *Bacillus licheniformis* inoculant is provided. This *Bacillus licheniformis* inoculant includes the active bacterial cells of the aforementioned *Bacillus licheniformis* MJ3. In some embodiments of this application, the *Bacillus licheniformis* inoculant can be in different formulations, including liquid, powder, and granule forms. *Bacillus licheniformis* MJ3 has antagonistic effects against *Ralstonia solanacearum* (…). Ralstonia solanacearum It has phosphorus-solubilizing ability. It can not only control bacterial wilt in crops (such as peanuts and tomatoes), but also improve soil fertility and increase crop yield.

[0031] In one embodiment of this application, the effective viable count of Bacillus licheniformis inoculant is at least 10 billion CFU / mL (preferably at least 15 billion CFU / mL) or 10 billion / g. This concentration is precisely calculated to ensure the activity of the strain without harming the crop. According to a typical embodiment of this application, the Bacillus licheniformis inoculant is prepared by the following steps: (1) Bacillus licheniformis MJ3 is activated, inoculated onto NA plate medium, and placed in a constant temperature incubator for 24-36 h at 37°C; (2) the activated strain from step (1) is picked, inoculated into NB liquid medium, and cultured at 35-37°C and 150-200 rpm for 12-24 h to obtain seed liquid; (3) the seed liquid obtained in step (2) is added to the amplification medium at a volume ratio of 1-3% and cultured for 18-24 h to obtain fermentation broth, which is the liquid inoculant of Bacillus licheniformis. According to another typical embodiment of this application, the fermentation broth obtained in step (3) is spray-dried to obtain a bacterial powder formulation. In one embodiment, the inlet air temperature of the drying process is 130-145℃, the outlet air temperature is 60-70℃, and the speed of the feed peristaltic pump is 5-15 rpm. Under these conditions, the activity of the bacterial agent can be well maintained. According to yet another typical embodiment of this application, the fermentation broth obtained in step (3) is granulated to obtain a granular formulation. In one embodiment, the granulation process includes: centrifuging the fermentation broth to obtain bacterial mud, adding 8-10% soluble starch as a protective agent and 10-15% zeolite powder as a carrier to the bacterial mud, mixing evenly, and then drying and granulating. The drying and granulation temperature is 50-60℃, and the time is 30-60 min. This preparation method is simple, easy to industrialize, and can ensure the quantity and activity of Bacillus licheniformis MJ-3 in the bacterial agent.

[0032] Preferably, in step (3), the culture medium comprises, by total mass, 3%-7% corn starch, 3%-7% soybean meal, 0.05%-0.15% magnesium sulfate, 0.1%-0.15% potassium dihydrogen phosphate, 0-5% yeast fermentation concentrate, and the remainder being water. This culture medium is particularly suitable for the culture of Bacillus licheniformis MJ-3 screened in this application.

[0033] Preferably, in step (3), the culture temperature is 35-37℃; and / or the culture time is 18-24h; and / or the aeration rate is 20-50L / min; and / or the pressure in the container is 0.2-0.5 atmospheres. Under these conditions, Bacillus licheniformis MJ-3 can grow rapidly and maintain its viability.

[0034] According to a typical embodiment of this application, the application of the above-mentioned Bacillus licheniformis MJ3 or the above-mentioned Bacillus licheniformis inoculant in the prevention and control of bacterial wilt is provided; preferably, the application in the prevention and control of bacterial wilt in peanuts and tomatoes.

[0035] According to a typical embodiment of this application, the application method is root irrigation, and the dosage is 5-20 kg per acre.

[0036] The beneficial effects of the present invention will be further illustrated below with reference to embodiments.

[0037] The Bacillus licheniformis MJ3 strain provided by this invention is a high-quality strain that combines phosphorus solubilization, effective prevention and control of bacterial wilt, and increased peanut yield, obtained through large-scale and rigorous screening and identification of peanut rhizosphere microorganisms.

[0038] Table 1. Information on the sources of instruments and reagents used in the embodiments of the present invention.

[0039]

[0040] Example 1: Screening, isolation and identification of lichen spore MJ3.

[0041] I. Sample Collection

[0042] In April 2023, soil samples were collected from peanut fields in Zhengyang, Henan Province, where bacterial wilt had occurred. Samples were collected from the rhizosphere tissue of healthy peanut plants and the soil, and were quickly brought back to the laboratory for preservation.

[0043] II. Separation and Purification

[0044] Sterilized NA (nutrient agar) medium containing 0.2% skim milk powder was poured into sterilized petri dishes and cooled to prepare NA plates. The formula of this NA medium was 10g peptone, 3g beef extract, 5g NaCl, 15g agar, and 1000mL distilled water.

[0045] Weigh 10g of rhizosphere sample, cut it into small pieces, add it to 100mL of cooled sterile water, and incubate in a shake flask at 37℃ for 1 hour. Then, incubate in a water bath at 80℃ for 10 minutes. Dilute with sterile physiological saline containing 0.1% Tween 80 at a concentration of 10 g each time. -1 10 -2 10 -3 Shake and mix well. Take 100 μL of suspension at different dilutions and drop it onto NA plate culture medium prepared 24 h in advance. Spread it evenly with a triangular scraper. After the solution is absorbed, invert the culture medium and incubate at 37℃ for 48 h. Pick single colonies that produce transparent hydrolysis zones and have similar morphology, color and size to Bacillus for purification and preservation.

[0046] III. Strain Identification

[0047] Morphological characteristics: Strains that produced hydrolysis zones after initial screening were cultured on NA medium plates at 37°C for 2 days, and their colony morphology and color were observed. Among them, strain MJ3 grew well on NA medium, producing white, moist colonies with serrated edges. Figure 1 As shown in Figure A, the Gram staining is purple, indicating a Gram-positive bacillus. The spores are oval-shaped, mesophyllary, or submesophyllary. Figure 1 As shown in B.

[0048] Physiological and biochemical indicators: Some physiological and biochemical indicators were measured and observed in accordance with the methods of Bergey's Manual of Bacterial Identification and Common Bacterial Identification.

[0049] Twenty-two physiological and biochemical indicators of strain MJ3 were tested, and the results are shown in Table 2.

[0050] Table 2: Physiological and Biochemical Indicators

[0051]

[0052] Note: +: positive; -: negative.

[0053] According to Bergey's Manual of Bacterial Identification and the Handbook of Common Bacteria, the morphological and physiological-biochemical characteristics of MJ3 conform to the classification criteria of Bacillus licheniformis.

[0054] Molecular biological identification

[0055] The whole genome DNA of the target strain was extracted using a bacterial genomic DNA kit. PCR amplification was performed using universal primers 27F and 1492R for the 16S rDNA gene. The amplified products were sequenced by BGI Genomics in Shenzhen and then analyzed using bioinformatics.

[0056] The 16S rDNA gene sequence of strain MJ3 obtained by sequencing is shown in SEQ ID NO: 1.

[0057] BLAST analysis of sequences in the NCBI database showed that the 16S rDNA sequence of strain MJ3 was related to... Bacillus licheniformis The strains showed 100% similarity. A phylogenetic tree constructed using MEGA 11.0 is shown below. Figure 2 As shown, the strain MJ3 obtained in this invention belongs to the same branch as Bacillus licheniformis.

[0058] Therefore, based on the above morphological, physiological and biochemical characteristics, as well as the homology analysis results of the 16S rDNA sequences of MJ3 and related strains, strain MJ3 was identified as Bacillus licheniformis.

[0059] IV. Preservation of Microbial Strains

[0060] The MJ3 strain obtained through screening was named Bacillus licheniformis. Bacillus licheniformi The MJ3 strain was deposited at the China Center for Type Culture Collection (CCTCC) on October 15, 2024, with accession number CCTCC NO:20242215.

[0061] Example 2 Phosphorus solubility potential test

[0062] Bacillus licheniformis MJ3 was activated by streaking on nutrient agar plates using inorganic phosphorus bacterial culture medium (Table 1). After 24 hours of activation, the strain was inoculated onto inorganic phosphorus culture medium plates and incubated at 37°C for 2-5 days. The growth of the strain was then observed.

[0063] like Figure 3 As shown, Bacillus licheniformis strain MJ3 can grow normally on inorganic phosphorus agar plates, and the phosphorus solubility coefficient reaches 2.2 (diameter of phosphorus solubility zone / colony diameter) after 3 days, indicating that Bacillus licheniformis strain MJ3 has phosphorus solubility.

[0064] Example 3: Preparation method of liquid inoculum of Bacillus licheniformis strain MJ3

[0065] Step 1: Seed liquid preparation

[0066] Bacillus licheniformis strain MJ3 was transferred to NA plates prepared 24 hours in advance and cultured at 37°C for 24 hours. Fresh seeds were then transferred to two sterile Erlenmeyer flasks containing 400 ml / L NB liquid medium. The flasks were shaken at 37°C and 200 rpm for 12 hours to obtain the seed culture.

[0067] Step 2: Liquid formulation preparation. 60L of liquid culture medium is added to a 100L fermenter. The preparation method of the liquid culture medium includes: adding 3%~7% corn starch, 3%~7% soybean meal, 0.05%~0.15% magnesium sulfate, 0.05%~0.15% potassium dihydrogen phosphate, and 0-5% yeast fermentation concentrate in sequence.

[0068] The culture medium, by total mass, consisted of 4% corn starch (2.4 kg), 4% soybean meal (2.4 kg), 0.1% magnesium sulfate (0.06 kg), 0.1% potassium dihydrogen phosphate (0.06 kg), and 2.5% yeast fermentation concentrate (1.5 kg). Tap water was then added to a volume of 60 L, and the pH was adjusted to 7. The mixture was then autoclaved at 121°C for 30 min. 800 mL of the seed culture was then transferred to the fermenter for fermentation. The culture conditions were: aeration rate of 20-50 L / min, pressure of 0.4 atmospheres, and fermentation at 37°C with ventilation for 19 h. Fermentation was stopped after most of the bacteria in the field of view had produced spores, yielding 60 L of Bacillus licheniformis MJ3 liquid inoculum. Testing showed that the inoculum had ≥10 billion CFU / mL of viable cells and a spore rate >90%.

[0069] Step 3: Preparation of powdered formulation. 30L of the fermentation broth obtained in Step 2 was dried using a spray drying tower. The speed of the peristaltic pump in the spray tower was adjusted to 12 rpm, the inlet air temperature to 130℃, and the outlet air temperature to 65℃. The resulting powdered product is a bacterial powder formulation. Testing showed that the effective viable bacteria count of this agent is ≥100 billion / g, and the spore rate is ≥95%.

[0070] Step 4: Preparation of granule dosage form

[0071] The 20L fermentation broth obtained in step 2 was separated, and 6kg of bacterial sludge was collected. Soluble starch (0.6kg) and zeolite powder (0.6kg) were added as a preservative and mixed thoroughly. The mixture was then extruded through a 2mm sieve in a rotary drum, and the drum temperature was adjusted to 55℃. After drying for 3 hours, granules were obtained. Testing showed that the particle size range was 2-4mm, the effective viable bacteria count was ≥12 billion / g, and the spore rate was ≥90%.

[0072] Example 4 Plate Inhibition Test

[0073] Using the inhibition zone method, sterilized NA plates were prepared in advance, and a highly pathogenic Ralstonia solanacearum strain activated for 24 hours was placed on the plates. Pseudomonas solanacearum (Smith) Smith, HA8-53, purchased Ralstonia solanacearum (tomato) Ralstonia solanacearum Inoculate into sterilized NB liquid medium and incubate in a shake flask at 28°C for 24 hours to 10 hours. 8 100 μL of bacterial culture was evenly spread onto NA agar plates using a sterile triangular spreader. Holes were then punched in the plates, and *Bacillus licheniformis* MJ3 was inoculated into NB agar plates and incubated at 37°C for 24 h. 100 μL of the culture was then evenly spread at 100, 500, and 1000 times dilution. Positive controls were obtained by diluting 20% ​​thiamethoxam copper at 100, 500, and 1000 times. The results showed a clear inhibition zone after 48 h, as shown in Table 3. Figure 4 As shown.

[0074] Table 3 Antibacterial zone test

[0075]

[0076] Example 5: Identification of genes producing antimicrobial peptides and functional substances from Bacillus licheniformis MJ3.

[0077] The whole genome of Bacillus licheniformis MJ3 was sequenced using third-generation Nanopore sequencing. The results of the third-generation sequencing sequence assembly and analysis are shown in Table 4.

[0078] Table 4. Statistical analysis of Bacillus licheniformis MJ3 genome information.

[0079]

[0080] The whole genome bioinformatics analysis of the strain was performed using antiSMASH (antimicrobial peptide prediction software) to predict the antimicrobial peptide and functional substance gene clusters that each strain may contain. The results showed that the strain has at least 3 antimicrobial peptide and functional substance gene clusters. The types of antimicrobial peptide and functional substance synthesis gene clusters contained in the strain genome are shown in Table 5 below.

[0081] Table 5. Gene clusters for the synthesis of antimicrobial peptides and functional substances in the genome of Bacillus licheniformis MJ3

[0082]

[0083] The sequences of antimicrobial peptides and functional substances obtained from the genome of Bacillus licheniformis MJ3 are as follows.

[0084] SEQ ID NO: 2

[0085] Bacillibactin (Bacillus siderophore / bacitracin), sequence length 51745bp, see sequence listing (xml) for details.

[0086] SEQ ID NO: 3

[0087] The sequence length of Lichenicidin VK21 A1 / A2 (Bacillus licheniformis) is 26962 bp. The specific sequence can be found in the sequence listing (xml).

[0088] SEQ ID NO: 4

[0089] >lichenysin, sequence length is 65441bp, see sequence listing (xml) for details.

[0090] The above results indicate that Bacillus licheniformis has the potential to synthesize a variety of antimicrobial peptides and functional substances.

[0091] Example 6: Peanut Field Trial

[0092] Field plot trials were conducted from May to October 2025 in Zhengyang County, Henan Province, an area with a high incidence of bacterial wilt in continuously cropped peanuts. Four treatments were set up, each with a plot size of 22 m². 2 The treatment was repeated three times. Group T1 was treated with water as a control. Treatments T2, T3, T4, and T5 were each treated with 0.32 kg, 0.64 kg, 0.16 kg, and 0.16 kg of the same solution, diluted with water and evenly applied to each peanut plant. Treatment T6 was diluted 500 times according to the instructions and evenly applied to the peanut seedlings during the seedling stage, ensuring each seedling received at least 100 ml. Treatments were repeated twice, with a 7-day interval between each treatment. Two rows of peanut plants were placed between the treatments for isolation. Note: In this example, the peanut variety used was Yuhua 37.

[0093] T1 CK (No bacterial agent applied)

[0094] T2 10kg / mu liquid Bacillus licheniformis inoculant

[0095] T3 20kg / mu liquid Bacillus licheniformis inoculant

[0096] T4 5kg / mu powdery lichen spores

[0097] T5 5kg / mu granular Bacillus licheniformis

[0098] T6 200g / mu 20% Thiamine Copper Suspension

[0099] Locally cultivated varieties were selected and sown in early May. According to local conventional fertilization standards, 40 kg of compound fertilizer (15-15-15) per mu was applied and sown together with peanut seeds. After peanut seedlings emerged in early June and before disease onset, different treatments were applied by water dilution and irrigation, and chemical pesticide thiamethoxam was applied. The treatments were repeated twice, with a one-week interval between the two treatments. The results were investigated at the peanut maturity period at the end of September.

[0100] I. Application of MJ3 liquid microbial agent to increase root nodule number and available phosphorus content in soil

[0101] In early July, the number of peanut root nodules was investigated. Compared with the control group, peanuts treated with liquid Bacillus licheniformis inoculant showed well-developed root systems and robust growth. The number of root nodules increased significantly. During the peanut seedling stage, the inoculant-treated group had more and denser root nodules, with an average of 38.4 and 43.6 root nodules per 10 kg / mu and 20 kg / mu respectively. The CK group had fewer root nodules, with an average of 30.3. Compared with the CK group, these figures represented increases of 26.7% and 43.8%, respectively.

[0102] In early July, soil samples were collected from each treatment using a five-point sampling method (four corners + center of the field). Approximately 500g of sample was taken from each point. Stones, roots, and other impurities were removed, the sample was crushed, sieved through a 2mm sieve, mixed thoroughly, and air-dried. The available phosphorus content was then determined using a molybdenum-antimony spectrophotometric method. The results show that the available phosphorus content in the soil is as follows: Compared with the CK group, the available phosphorus content in soils T2, T3, T4, and T5 increased by 19.3%, 24%, 5.6%, and 7.2%, respectively.

[0103] Table 6 Available phosphorus content in different soils

[0104]

[0105] II. Application of MJ3 liquid inoculant to improve peanut biomass

[0106] In the experimental control group (CK), the dry weight of peanuts in T1 ranged from 5.1 to 6.4 kg, with an average of 5.6 kg. In the chemical pesticide treatment group (T6), the average dry weight of peanuts was 6.2 kg (Table 7). Compared to T1 (conventional fertilization), T2 showed a yield increase of 30.7%, T3 a 35.2%, T4 a 17.6%, T5 a 28.9%, and T6 (chemical pesticide group) a 10.9% increase. Compared to T6 (chemical pesticide group), T2 showed a yield increase of 17.8%, T3 a 22.1%, T4 a 12.7%, and T5 a 16.1%.

[0107] Table 7 Changes in peanut yield

[0108]

[0109] III. The application of MJ3 liquid inoculant effectively reduces the incidence of peanut bacterial wilt.

[0110] From the early flowering stage to the pod-setting stage of peanuts, five sampling points were used after two weeks of treatment, with 20 plants selected at each point. Based on the symptoms, the incidence rate was counted and the control efficacy was calculated. The results showed that the incidence rate of each treatment group was lower than that of the T1 conventional fertilization group, and the control efficacy of the T3, T4, and T5 treatment groups was higher than that of the chemical pesticide treatment group. The control efficacy of the T2 treatment group was close to that of the chemical treatment group (Table 8).

[0111] Table 8. Control of Bacterial Wilt Disease in Peanuts

[0112]

[0113] Example 7: Experiment on the control efficacy against bacterial wilt in tomatoes

[0114] To verify the control effect of MJ3 and its inoculant on bacterial wilt of tomatoes, a pot experiment was conducted in greenhouses around Yichang. Soil from fields with bacterial wilt in tomatoes was used, with 3 kg of diseased soil per pot and 1 seedling per pot. The tomato variety was Maofen 802. The specific treatments are as follows:

[0115] (1) Treatment 1: Tomato diseased soil + compound fertilizer 15-15-15 (1g) as the control group;

[0116] (2) Treatment 2: Tomato diseased soil + compound fertilizer 15-15-15 (1g) + MJ3 liquid bacterial agent 6ml;

[0117] (3) Treatment 3: Tomato diseased soil + compound fertilizer 15-15-15 (1g) + MJ3 powdered bacterial agent 2g;

[0118] (4) Treatment 4: Tomato diseased soil + compound fertilizer 15-15-15 (1g) + MJ3 granular inoculant 6g;

[0119] (5) Treatment 5: Tomato diseased soil + compound fertilizer 15-15-15 (1g) + 20% thiamethoxam suspension 3ml, as the chemical pesticide group.

[0120] Tomato seedlings were first cultivated to the 5-6 leaf stage and then transplanted into nutrient pots containing diseased soil with the compound fertilizer in the above proportions. Then, the above-mentioned microbial agents and pesticides were applied to the roots according to the following proportions: 3 ml of MJ3 liquid microbial agent, diluted 100 times, was applied to the root zone of the tomatoes in treatment 2; 1 g of MJ3 powdered microbial agent, diluted 500 times, was applied to the root zone of the tomatoes in treatment 3; 3 g of MJ3 granular microbial agent, diluted 100 times, was applied to the root zone of the tomatoes in treatment 4; and 1 ml of 20% thiamethoxam suspension, diluted 500 times, was applied to the root zone of the tomatoes in treatment 5. The control group (treatment 1) was treated with 300 ml of water. The treatments were repeated after a 10-day interval. Each treatment had 20 replicates. The growth cycle was 100 days. Tomato growth and disease incidence were observed and recorded regularly. After the pot experiment, the severity of disease and biomass were statistically analyzed.

[0121] The results showed that the incidence of bacterial wilt in all treatment groups was lower than that in the control group (treatment 1), and the control efficacy was higher than that in the chemical pesticide group (treatment 5). Regarding plant height and fresh biomass, treatments 2, 3, and 4 were significantly higher than those in treatments 1 and 5.

[0122] Table 9: Effects of different treatments on bacterial wilt control and tomato growth in greenhouse pot experiments.

[0123]

[0124] The above application experiments show that the MJ3 and its inoculant described in this invention can inhibit bacterial wilt pathogens, significantly reduce the incidence of bacterial wilt, improve the prevention effect, and have phosphorus solubilization function, improve fertilizer efficiency, and increase yield, making it highly valuable for promotion and application.

[0125] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A strain of Bacillus licheniformis MJ3 used for the prevention and control of bacterial wilt, characterized in that, The Bacillus licheniformis MJ3 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M20242215.

2. A Bacillus licheniformis inoculant, characterized in that, It includes the active bacterial cells of Bacillus licheniformis MJ3 as described in claim 1.

3. The Bacillus licheniformis inoculant according to claim 2, characterized in that, The Bacillus licheniformis inoculant is in liquid, powder, or granule form.

4. The Bacillus licheniformis inoculant according to any one of claims 2 or 3, characterized in that, The Bacillus licheniformis inoculum is prepared by the following steps: (1) Activate the Bacillus licheniformis MJ3, inoculate it onto NA plate medium, place it in a constant temperature incubator, and incubate it at 37°C for 24-36 hours; (2) Pick the activated strains from step (1), inoculate them into NB liquid culture medium, and culture them at 35-37℃ and 150-200rpm for 12-24h to obtain seed culture; (3) Add the seed liquid obtained in step (2) to the amplification culture medium at a volume ratio of 1-3% and culture for 18-24 hours to obtain the fermentation broth, which is the liquid dosage form.

5. The Bacillus licheniformis inoculant according to claim 4, characterized in that, In step (3), the culture medium consists of 3%-7% corn starch, 3%-7% soybean meal, 0.05%-0.15% magnesium sulfate, 0.1%-0.15% potassium dihydrogen phosphate, 0-5% yeast fermentation concentrate, and the remaining components are water, based on the total mass of the culture medium.

6. The Bacillus licheniformis inoculant according to claim 5, characterized in that, In step (3), the culture temperature is 35-37℃; And / or, the incubation time is 18~24h; And / or, ventilation rate is 20~50 L / min; And / or, the tank pressure is 0.2-0.5 standard atmospheres.

7. The Bacillus licheniformis inoculant according to claim 5, characterized in that, The fermentation broth obtained in step (3) is dried to obtain a bacterial powder formulation.

8. The Bacillus licheniformis inoculant according to claim 7, characterized in that, The inlet air temperature of the drying process is 130-145℃, the outlet air temperature is 60-70℃, and the speed of the feed peristaltic pump is 5-15 rpm.

9. The Bacillus licheniformis inoculant according to claim 5, characterized in that, The fermentation broth obtained in step (3) is granulated to obtain a granule dosage form.

10. The Bacillus licheniformis inoculant according to claim 9, characterized in that, The granulation process includes: centrifuging the fermentation broth to obtain bacterial sludge, adding 8-10% soluble starch as a protective agent and 10-15% zeolite powder as a carrier to the bacterial sludge, mixing them evenly, and then drying and granulating them. The drying and granulation temperature is 50-60℃ and the time is 30-60 minutes.

11. The Bacillus licheniformis inoculant according to claim 2 or 3, characterized in that, The effective viable count of the Bacillus licheniformis inoculant shall not be less than 10 billion / ml or 10 billion / g.

12. The use of Bacillus licheniformis MJ3 as described in claim 1, or the Bacillus licheniformis inoculant as described in any one of claims 2 to 11, in the prevention and control of bacterial wilt.

13. The application as described in claim 12, characterized in that, The application is in the prevention and control of bacterial wilt in peanuts and tomatoes.

14. The application according to claim 12 or 13, characterized in that, The application method is root irrigation, with a dosage of 5-20 kg per acre.

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