Bacillus velezensis and application thereof

Abstract

The invention provides bacillus velezensis and application thereof, and belongs to the technical field of microorganisms. The bacillus velezensis ND-J with a remarkable inhibition effect on main pathogenic bacteria of the ginger root rot is successfully obtained, and the fermentation viable count of the bacillus velezensis ND-J is greatly increased through system optimization, so that a reliable strain resource and a mature fermentation process foundation are provided for developing an efficient and stable ginger root rot biological control preparation. The application of the technology is helpful to reduce the use of chemical pesticides, promotes the green and sustainable development of the ginger industry, and has positive significance in guaranteeing the yield and quality of ginger and increasing the economic benefits of ginger farmers.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a bacillus velezensis and application thereof. BACKGROUND

[0002] Ginger is an important economic crop in China, which has a wide application in condiments, medicine and health care fields. In its cultivation process, soil-borne diseases are one of the main factors restricting its yield and quality. Among them, the ginger root rot caused by Fusarium and Neocosmospora fungi has shown a high incidence in recent years. The pathogenic bacteria can survive in the soil for a long time, and the disease caused by them usually shows yellowing and wilting of plant leaves, brown rot of underground rhizome, and finally leads to serious yield reduction.

[0003] At present, the prevention and treatment of ginger root rot still depends largely on chemical fungicides. However, long-term and single use of chemical pesticides can easily lead to drug resistance of pathogenic bacteria, making the control effect decline. At the same time, pesticide residues can also damage the soil micro-ecological environment and cause the risk of agricultural product quality and safety, which does not meet the requirements of green development of modern agriculture.

[0004] Biological control, as an environmentally friendly alternative strategy, has attracted more and more attention. The core is to use beneficial microorganisms (i.e. biocontrol bacteria) to inhibit or kill pathogenic bacteria. Among many biocontrol microorganisms, Bacillus bacteria are considered to be ideal biocontrol bacterial resources because they can produce spores with strong resistance, are easy to produce and store, and have multiple functions such as antibiosis and growth promotion. Among them, Bacillus velezensis is reported to have broad-spectrum antagonistic activity against many plant pathogenic fungi and bacteria, and has shown good application potential in agricultural disease control. Developing efficient biocontrol strains for specific crop diseases and supporting optimized fermentation production technology are the key prerequisites for its industrial application. SUMMARY

[0005] The present application aims to provide a bacillus velezensis and application thereof, which solves the technical problems of limited effect of existing chemical pesticides in preventing and treating ginger root rot, easy to cause environmental pollution and drug resistance of pathogenic bacteria, and lack of efficient and specialized biocontrol agents.

[0006] In order to achieve the above application purpose, the present application provides the following technical solutions:

[0007] The present application provides a bacillus velezensis ND-J, and the preservation number of the bacillus velezensis is CGMCC NO. 36400.

[0008] The present application also provides a microbial agent, which comprises the above-mentioned bacillus velezensis.

[0009] The application further provides a fermentation liquor prepared by fermenting and culturing the above-mentioned Bacillus velezensis.

[0010] The application further provides a biocontrol agent comprising the above-mentioned Bacillus velezensis or fermentation liquor.

[0011] The application further provides application of the above-mentioned Bacillus velezensis or fermentation liquor in prevention and treatment of plant soil-borne diseases.

[0012] Preferably, the plant soil-borne disease is caused by a Fusarium or Neocosmospora pathogenic fungus.

[0013] Preferably, the plant is ginger.

[0014] The application further provides application of the above-mentioned Bacillus velezensis or fermentation liquor in inhibition of growth of a plant pathogenic fungus or promotion of plant growth.

[0015] The application further provides a method for preparing the above-mentioned Bacillus velezensis, comprising the step of fermenting and culturing the Bacillus velezensis in a culture medium.

[0016] The culture medium comprises a carbon source, a nitrogen source and inorganic salts; the carbon source is sucrose and the addition amount is 5 g / L to 8 g / L; the nitrogen source is a mixture of bran and soybean meal and the addition amount is 18 g / L to 25 g / L; the inorganic salts comprise sodium chloride, potassium dihydrogen phosphate and manganese sulfate.

[0017] The mass ratio of the bran to the soybean meal is 1:0.8 to 1.2; the addition amount of the sodium chloride is 4 g / L to 6 g / L; the addition amount of the potassium dihydrogen phosphate is 0.5 g / L to 1.5 g / L; and the addition amount of the manganese sulfate is 0.3 g / L to 0.5 g / L.

[0018] Preferably, the fermentation and culturing is carried out at a temperature of 30°C to 40°C, an initial pH of 7.0 to 8.0 and a rotation speed of 180 rpm to 220 rpm.

[0019] The application has the following beneficial effects:

[0020] The application successfully obtains a Bacillus velezensis ND-J that has a significant inhibitory effect on the main pathogenic fungus of ginger root rot, and greatly improves the number of viable bacteria through systematic optimization, thereby providing reliable strain resources and mature fermentation process basis for developing efficient and stable ginger root rot biological control preparations. The application of the technology will help reduce the use of chemical pesticides and promote the green and sustainable development of the ginger industry, which has a positive significance for ensuring ginger yield and quality and increasing the economic benefits of ginger farmers. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Fig. 1 is a morphological structure of a ginger pathogen and a back-connection test result diagram;

[0022] Figure 2 Fig. 2 is a phylogenetic analysis result diagram of a pathogenic strain;

[0023] Figure 3 Fig. 3 is a bacteriostatic activity analysis result diagram of an antagonistic strain;

[0024] Figure 4 Fig. 4 is a single-factor optimization test result diagram of ND-J;

[0025] Figure 5 Fig. 5 is a semi-normal probability effect diagram and a Pareto diagram of Plackett-Burman;

[0026] Figure 6 Fig. 6 is a diagram of the influence of the interaction of various factors on ND-J fermentation.

[0027] Biological preservation instructions

[0028] The Bacillus velezensis provided by the application is named Bacillus velezensis ND-J, and was preserved in the China General Microbiological Culture Collection Center (CGMCC) on October 29, 2025. The address of the CGMCC is No. 3, Beichen West Road, Chaoyang District, Beijing, China, and the postal code is 100101. The Institute of Microbiology of the Chinese Academy of Sciences is located at the address, and the preservation number is CGMCC NO. 36400. DETAILED DESCRIPTION

[0029] The present application provides a Bacillus velezensis ND-J. Bacillus velezensis is an important species of Bacillus, which belongs to gram-positive bacteria. This species is known for its ability to produce a variety of antimicrobial active substances, which usually include lipopeptides, polyketides, and antibacterial proteins, making it have broad application prospects in the field of agricultural plant disease biological control. Bacillus velezensis is usually found in soil, plant rhizosphere and other environments. Because it can form spores with strong stress resistance, it is easy to be stored for a long time at room temperature, and it is also convenient to make stable microbial preparations. The strain ND-J involved in the present application has been preserved in the prescribed microbial strain preservation institution on the prescribed date, and the preservation number thereof is CGMCC NO. 36400, which ensures the traceability and reproducibility of the strain.

[0030] The present application also provides a microbial inoculant comprising the above-mentioned Bacillus velezensis ND-J. In the field, microbial inoculant refers to a product that takes beneficial microorganisms as the core active ingredient, supplemented by suitable carriers or adjuvants. Common carriers include but are not limited to inert porous materials such as grass charcoal, diatomite, attapulgite, bran, talc, zeolite powder, etc., which can provide physical protection for microorganisms and play a dilution role. Adjuvants can include wetting agents, dispersants, binders, nutrients, etc., aimed at improving the application performance, stability and colonization effect of the inoculant. The inoculant can be prepared into various dosage forms, such as wettable powder, water dispersible granule, suspension or granule, to adapt to different application scenarios and agronomic requirements.

[0031] The present application also provides a fermentation broth prepared from the above-mentioned Bacillus velezensis ND-J. Fermentation broth is a mixture formed after microorganisms grow and reproduce in a specific liquid medium under controlled environmental conditions (such as temperature, pH, aeration). It not only contains a high concentration of live bacteria and their stress-resistant spores, but also is rich in various extracellular metabolites produced by microorganisms during metabolism, which often directly or indirectly contribute to their biocontrol function. The fermentation broth can be directly diluted and applied, or used as an intermediate product, processed into the above-mentioned solid or liquid microbial inoculant through further concentration, adsorption, drying and other post-processing technologies.

[0032] The present application also provides a biocontrol agent comprising the above-mentioned Bacillus velezensis ND-J or the fermentation broth prepared therefrom. Biocontrol agent, i.e. biological control agent, is a type of environmentally friendly pesticide that uses living microorganisms or their metabolites to inhibit, reduce or control plant diseases. Compared with traditional chemical pesticides, biocontrol agents usually have the advantages of environmental friendliness, non-resistance, safety to non-target organisms, etc. The biocontrol agent of the present application can be further compounded with agriculturally acceptable adjuvants, including but not limited to carriers, surfactants, preservatives, antifreeze agents (such as for liquid formulations), and appropriate amounts of chemical or biological fertilizers, to form a multifunctional composite product.

[0033] The present application also provides the use of the above-mentioned Bacillus velezensis ND-J or its fermentation broth in the prevention and treatment of plant soil-borne diseases. Plant soil-borne diseases refer to diseases caused by pathogens (such as fungi, bacteria, nematodes, etc.) surviving in the soil and invading from the plant roots or stem base when conditions are suitable. This type of disease is difficult to control and is a major challenge in agricultural production.

[0034] Preferably, the plant soil-borne disease is caused by a pathogenic fungus of Fusarium spp. or Neocosmospora spp. Fusarium spp. is a group of important plant pathogenic fungi with worldwide distribution, which can cause wilt, root rot and basal stem rot in various crops. Neocosmospora spp., some members of which were originally classified as Fusarium solani species complex, can also cause severe root and stem rot symptoms.

[0035] Preferably, the plant is Zingiber officinale Roscoe. Zingiber officinale Roscoe is a perennial herb of Zingiberaceae, and its rhizome has important edible and medicinal value, and is easily infected by the above-mentioned soil-borne pathogenic fungi during growth.

[0036] In addition, the application also provides the use of the above-mentioned Bacillus velezensis or its fermentation liquor in inhibiting the growth of plant pathogenic fungi or promoting the growth of plants. Inhibiting the growth of plant pathogenic fungi can be achieved by various mechanisms, such as competition for nutrients and space, production of antibacterial substances, induction of plant systemic resistance, etc. Promoting the growth of plants can be achieved by producing plant hormones, releasing phosphorus, releasing potassium, fixing nitrogen or improving rhizosphere microecology, etc.

[0037] The application also provides a method for culturing the above-mentioned Bacillus velezensis ND-J, which comprises the step of fermenting the Bacillus velezensis in a suitable culture medium.

[0038] The culture medium comprises a carbon source, a nitrogen source and inorganic salts. The carbon source is a nutrient that provides energy and carbon skeleton for the synthesis of cell material for microbial growth. In the present application, the carbon source is sucrose, and the addition amount is 5 g / L to 8 g / L. Sucrose is a common disaccharide and is easy to be utilized by microorganisms. The nitrogen source is a nutrient that provides elements required for the synthesis of nitrogen-containing substances such as proteins, nucleic acids, etc. In the present application, the nitrogen source is a mixture of bran and soybean meal, and the addition amount is 18 g / L to 25 g / L. Bran is a byproduct of wheat processing and is rich in polysaccharides and proteins; soybean meal is the residue after extracting oil from soybeans and is high in protein content. The combination of the two can provide comprehensive and balanced nitrogen and growth factors. Preferably, the mass ratio of the bran to the soybean meal is 1:0.8~1.2, for example, close to 1:1. Inorganic salts are used to maintain the osmotic pressure of microbial cells and act as co-factors or activators for certain enzyme systems. In the present application, the inorganic salts comprise sodium chloride, potassium dihydrogen phosphate and manganese sulfate. Among them, the addition amount of sodium chloride is 4 g / L to 6 g / L, mainly for adjusting the osmotic pressure; the addition amount of potassium dihydrogen phosphate is 0.5 g / L to 1.5 g / L, mainly providing phosphorus and potassium sources and acting as a buffer; the addition amount of manganese sulfate is 0.3 g / L to 0.5 g / L, and manganese ions (Mn²⁺) are important components of many enzymes, especially important for spore formation.

[0039] Preferably, the temperature of the fermentation culture is controlled at 30°C to 40°C, which is the suitable temperature range for the growth of most Bacillus. The initial pH value of the fermentation culture is controlled at 7.0 to 8.0, which is a slightly alkaline to neutral condition suitable for the growth of the strain. The rotation speed of the fermentation culture is controlled at 180 rpm to 220 rpm, which helps to ensure the dissolved oxygen content in the culture solution and meet the needs of aerobic growth of the bacteria.

[0040] The technical solutions provided by the present application are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0041] Example

[0042] 1. Materials and methods

[0043] 1.1 Samples

[0044] Rhizosphere soil samples were collected from plots with severe Dajiang root rot and healthy plots in the main Dajiang planting area of Shijiazhuang City, Hebei Province (114.98°E, 38.18°N) for pathogenic / antagonistic strain screening.

[0045] 1.2 Culture medium

[0046] Potato dextrose agar (PDA) medium, LB medium were purchased from Beijing Solabio Science and Technology Co., Ltd.

[0047] 1.3 Main reagents and instruments

[0048] Sucrose, corn flour, soluble starch, glucose, maltose, sodium chloride, sodium bisulfate, calcium chloride, potassium dihydrogen phosphate, magnesium sulfate, etc. were purchased from Tianjin Yongsheng Fine Chemical Co., Ltd.; constant temperature incubation shaker, Shanghai Zhi Cheng Analytical Instrument Manufacturing Co., Ltd.; high-pressure wet heat sterilization pot, Shanghai Boxin Industry Co., Ltd.

[0049] 1.4 Pathogenic bacteria and antagonistic bacteria screening

[0050] The pathogenic bacteria of Zingiber officinale Roscoe were screened by collecting diseased ginger and rhizosphere soil from a high-incidence area in Wuji County, Shijiazhuang, Hebei Province, and the antagonistic bacteria were screened by collecting healthy ginger rhizosphere soil. The ginger / soil sample was ground and gradient diluted to prepare a bacterial suspension, which was spread on a plate culture medium and incubated at 30°C until colonies appeared. Fungal mycelial tips were used for purification, and bacterial streaking separation method was used for purification. The pathogenic bacteria were screened by back-plate to fresh ginger, and the antagonistic bacteria were screened by plate confrontation culture method and the inhibition rate was calculated.

[0051] 1.5 Strain identification

[0052] The strain was cultured to form a single colony, and then observed under a microscope after staining. The CTAB method was used to extract the genomic DNA of the strain (bacteria / fungi). The 16S rRNA gene of bacteria was amplified using primers 27F / 1492R, and the ITS region of fungi was amplified using primers ITS1 / ITS4. The PCR products were purified and sequenced for analysis, and BLAST comparison was performed on NCBI (http: / / www.ncbi.nlm.nih.gov).

[0053] 1.6 Single factor optimization

[0054] The initial medium was: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L; carbon source, nitrogen source, inorganic salt, culture temperature, initial pH, rotation speed, liquid volume, and gradient settings are shown in Table 1.

[0055] Table 1. Factor and gradient settings

[0056]

[0057] 1.7 Plackett-Burman test

[0058] On the basis of 1.2.3 test, Plackett-Burman test was designed by Design-expert software to investigate 7 factors including temperature (A, ℃), rotation speed (B, rpm), liquid volume (C, mL / 500 mL), initial pH (D), carbon source addition (E, g / L), nitrogen source addition (F, g / L), and inoculum size (G, %), and to screen the three most significant factors affecting the growth of ND-J. A dummy factor was set to investigate the test error. Each factor was set to two levels: high (+1) and low (-1). The test factor and coding level table is shown in Table 2.

[0059] Table 2 Plackett-Burman test factor and coding level table

[0060]

[0061] 1.8 Steepest ascent test

[0062] On the basis of PB test, steepest ascent test was performed on the factors that had a significant effect on the number of viable bacteria. The gradient direction of the response value change was used as the climbing direction, and the size of the effect value of each significant factor determined the step size, so that the factor approached the optimal response interval.

[0063] 1.9 Response surface optimization of fermentation conditions

[0064] The climbing results were used as the center point of the response surface test. The Box-Behnken model of Design-expert software was used to design a 3-factor 3-level test with N=17 and the number of viable bacteria as the response value to determine the optimal fermentation conditions of the biocontrol strain.

[0065] Table 3 Box-Behnken test factor and coding level table

[0066]

[0067] 2 Results

[0068] 2.1 Isolation and identification of Zingiber officinale pathogens

[0069] The diseased Zingiber officinale plants in the Zingiber officinale planting area of Hebei Province were used as materials. After purification by tip isolation and plate streaking, the morphological characteristics and microscopic structure of the strains were identified. Twelve strains were selected, named J1-J12, of which 8 were fungi and 4 were bacteria. The isolated strains were back-inoculated into fresh Zingiber officinale rhizome sections, and after 7 days of moist culture, J4-J7 treatment groups showed obvious infection, while the other groups showed no obvious changes. Back-inoculation test showed that J-4, J-5, J-6, and J-7 were Zingiber officinale pathogens.

[0070] The ITS3 / 4 universal primer of fungi was selected to isolate the four pathogenic fungi genomes as templates for PCR amplification. The obtained sequence information was compared and analyzed in the NCBI database by BLAST. The phylogenetic results showed that J-4 and J-5 were clustered with the reference strain N. rubicola and multiple Fusarium strains (F. solani, F. tonkinense, etc.) to form a high-support monophyletic branch, indicating that J-4 and J-5 had high homology with the group. J-6 and J-7 were clustered with F. elaeagni and F. hechiense, respectively, further confirming that they were members of the Fusarium genus but located in different sub-branches from J-4 and J-5, reflecting the significant genetic differentiation and species diversity within the Fusarium genus.

[0071] 2.2 Isolation, identification and antibacterial activity analysis of antagonistic strains

[0072] Traditional soil dilution method was used to screen antagonistic bacteria in the soil of healthy plots in the sampling area. According to the colony morphology and culture characteristics, 28 different strains were isolated. The results of co-culture showed that three bacteria exhibited antagonistic phenomena, named DF-J, ND-J, and N2-10, respectively. The 16S rRNA gene sequences of the three strains were sequenced and identified as B. amyloliquefaciens, B. velezensis, and B. subtilis, respectively.

[0073] The antagonistic activity of the three antagonistic bacteria against the pathogenic bacteria of large ginger was determined by plate confrontation method, and the results are shown in Figure 3 All the three antagonistic bacteria could inhibit the growth of pathogenic bacteria, and ND-J had the best comprehensive antagonistic effect, with antagonistic rates of 51%, 52.33%, 37%, and 50% against the four pathogenic bacteria, respectively.

[0074] 2.3 Optimization of single-factor culture conditions of strain ND-J

[0075] The effects of different carbon sources, nitrogen sources, inorganic salts, temperature, pH, rotation speed, inoculum size, and liquid volume on the viable cell count of B. velezensis ND-J were analyzed, and the results are shown in Figure 4 . Figure 4 Inorganic salt combination 1 in C was sodium chloride 5 g / L, potassium dihydrogen phosphate 1 g / L, and manganese sulfate 0.4 g / L. Inorganic salt combination 2 was potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, and sodium chloride 0.5 g / L. Inorganic salt combination 3 was sodium chloride 0.5 g / L, ammonium sulfate 2 g / L, and sodium citrate 0.5 g / L.

[0076] Different carbon sources were selected, and the addition amount was fixed. Under the same conditions, the viable cell count of the sucrose group was the highest, reaching 6.0 × 109 CFU / mL, followed by the corn flour group; fixed sucrose as the sole carbon source for optimal nitrogen source screening, among the 10 nitrogen sources and combinations, organic nitrogen sources were obviously more suitable for the growth of ND-J, and the viable cell count of the bran + soybean meal combination reached 6.5 × 10 9 CFU / mL; fixed carbon source and nitrogen source type, then the best inorganic salt screening, among different types of inorganic salts and inorganic salt combinations, the inorganic salt combination 1 (sodium chloride 5 g / L, potassium dihydrogen phosphate 1 g / L, manganese sulfate 0.4 g / L) had the highest viable cell count, reaching 7.1 × 10 9 CFU / mL, among which the addition of Mn²⁺ could significantly promote the spore formation of ND-J.

[0077] After fixing the types and amounts of medium components, under different fermentation temperatures, with the increase of temperature, the viable cell count also increased, reaching a maximum of 7.2 × 10 9 CFU / mL at 35 ℃, and then began to decline; under different initial pH conditions, the viable cell count reached a maximum of 7.3 × 10 9 CFU / mL at pH 7.5; under different rotation speeds, the viable cell count was highest at 200 rpm, reaching 7.6 × 10 9 CFU / mL; among different inoculum amounts, the viable cell count had reached a high level at 2%, at which the viable cell count was 7.7 × 10 9 CFU / mL, avoiding the marginal diminishing returns and metabolic burden brought by higher inoculum; among the selected different liquid loading conditions, 100 mL / 500 mL was the best, with a viable cell count of 8.3 × 10 9 CFU / mL.

[0078] 2.4 ND-J response surface optimization results

[0079] 2.4.1 Plackett-Burman test

[0080] Based on the single factor experiment, the viable cell count of the fermentation broth was used as the response value, and the variance analysis of the experimental design results was performed. As shown in the table, the experiment model was significant (p value = 0.0445, <0.05) and well fitted (R 2 =0.9194, Adj. R 2=0.7785) showed that the selected factors together explained 77.85% of the response variation, the model was effective and had high reliability. The three factors with the smallest p values were C-liquid loading amount (p=0.0090, <0.01), E-carbon source addition amount (p=0.0329, <0.05), and F-nitrogen source addition amount (p=0.0382, <0.05), all of which reached a significant level, among which the liquid loading amount reached a highly significant level, which was consistent with the results of the semi-normal probability effect plot and the standardized effect Pareto plot. Figure 5 It was known that factors C and E were negative effects, and F was a positive effect, so the next step of the experiment was performed.

[0081] Table 4 Analysis of variance of Plackett-Burman experiment

[0082]

[0083] Note: A: temperature; B: rotation speed; C: liquid loading amount; D: initial pH; E: carbon source addition amount; F: nitrogen source addition amount; G: inoculum amount

[0084] 2.4.2 Steepest ascent experiment

[0085] According to the results of the Plackett-Burman experiment and the actual situation, the climbing direction and step length were determined, and the steepest ascent experiment was performed. The results are shown in Table 3. The viable cell count of the third group reached a maximum value of 8.9 x 10 9 CFU / mL, which was used as the center point of the response surface experiment.

[0086] Table 5 Steepest ascent experiment design and results

[0087]

[0088] 2.4.3 Response surface analysis

[0089] According to the results of the Plackett-Burman and steepest ascent experiments, the viable cell count was used as the response value, and the liquid loading amount (A) was 80 mL / 500 mL, the carbon source addition amount (B) was 6 g / L, and the nitrogen source addition amount (C) was 21 g / L as the center point. A regression model was established based on the results of the Box-Behnken experiment design and variance analysis, and the results are shown in Tables 6 and 7.

[0090] Table 6 Box-Behnken experiment design and results

[0091]

[0092] Note: A: liquid loading amount (mL / 500 mL); B: carbon source addition (g / L); C: nitrogen source addition (g / L). The same below.

[0093] Table 7 Analysis of variance of response surface experiment

[0094]

[0095] The quadratic regression model has a very high goodness of fit (R²= 0.9864, Adj-R²= 0.9689), indicating that the model can explain 98.64% of the response value variation, with excellent prediction accuracy and reliability. The analysis of variance further shows that the overall regression effect of the model is extremely significant (p<0.0001), and the one-way terms of carbon source addition amount (B) and nitrogen source addition amount (C) and all quadratic terms (A², B², C²) are extremely significant influencing factors (p<0.01), among which the influence of carbon source addition amount (coefficient +3.50) is the most prominent, and the primary and secondary order of the influence of each factor on the viable cell count is: B>C>A. It fully proves that the nonlinear relationship between the factors and the response value is highly real.

[0096] The multivariate quadratic regression equation of viable cell count (Y) to liquid loading amount (A), carbon source addition amount (B), and nitrogen source addition amount (C) is: viable cell count = 89.80 + 0.62*A + 3.50*B + 1.88*C - 0.75*A*B + 0.00*A*C + 1.75*B*C - 7.65*A² - 15.90*B² - 7.65*C².

[0097] Image analysis shows that the interaction surface of carbon source addition amount (B) and nitrogen source addition amount (C) opens downward, and there is an obvious extreme point, indicating that the synergistic effect of the two factors on the viable cell count is significant, and there is an optimal matching area. The curvature of the interaction surface of liquid loading amount (A) and other factors is relatively flat, but the coefficient of its own quadratic term (A²) is significant, indicating that this factor still affects the growth of bacteria through its own nonlinear effect (see Figure 6 ).

[0098] Based on the model analysis and graphical results, the influence of each factor on the viable cell count has a complex surface effect. Carbon source addition amount shows the strongest positive effect within the selected level range, and is the key factor to improve the viable cell count; nitrogen source addition amount is second, and has a synergistic promoting effect with carbon source; although the linear effect of liquid loading amount is not significant, it still affects the system response through the quadratic term.

[0099] The model predicts the optimal fermentation conditions as follows: liquid loading amount 80.71 mL / 500 mL, carbon source addition amount 6.35 g / L, and nitrogen source addition amount 21.41 g / L, with a predicted viable cell count of 90.14 x 10 8 CFU / mL. The actual viable cell count reaches 91 x 10 8The CFU / mL is highly consistent with the predicted value, the above results fully prove that the model is accurate and reliable, and high-precision prediction of the viable cell count and process optimization are successfully achieved.

[0100] It can be seen from the above that the B. velezensis ND-J provided by the application can achieve the best strain antagonistic effect. Through the combination of single factor test and response surface method, the culture medium for high-density fermentation of the B. velezensis ND-J is determined as follows: sucrose 6.35 g / L, bran (water) + soybean meal powder (1:1) 21.41 g / L, NaCl 5 g / L, KH2PO4 1 g / L, MnSO4 0.4 g / L, and the key process parameters are as follows: fermentation temperature 35 ℃, initial pH 7.5, rotation speed 200 rpm, inoculation amount 2%, liquid loading amount 80.71 mL / 500 mL, and the final viable cell count reaches 9.1×10 9 The CFU / mL is highly consistent with the predicted value, the above results fully prove that the model is accurate and reliable, and high-precision prediction of the viable cell count and process optimization are successfully achieved.

[0101] The above only describes the preferred embodiments of the application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the application, and these improvements and refinements should also be considered as the protection scope of the application.

Claims

1. A type of Bacillus velezensis ND-J, characterized in that, The preservation number of the Bacillus belyssus is CGMCC NO.36400.

2. A microbial inoculant, characterized in that, The microbial agent comprises Bacillus berleis as described in claim 1.

3. A fermentation broth, characterized in that, It is prepared by fermentation culture of Bacillus berleis as described in claim 1.

4. A biocontrol agent, characterized in that, It contains Bacillus berberis as described in claim 1 or the fermentation broth as described in claim 3.

5. The application of Bacillus belye as described in claim 1 or the fermentation broth as described in claim 3 in the prevention and control of soil-borne plant diseases.

6. The application according to claim 5, characterized in that, The soil-borne plant diseases mentioned are caused by pathogenic fungi of the genus Fusarium or Neocaridium.

7. The application according to claim 5, characterized in that, The plant in question is ginger.

8. The use of Bacillus berberis as described in claim 1 or the fermentation broth as described in claim 3 in inhibiting the growth of plant pathogenic fungi or promoting plant growth.

9. A method for culturing Bacillus belye as described in claim 1, characterized in that, This includes the step of fermenting the Bacillus belesii in a culture medium; The culture medium contains a carbon source, a nitrogen source, and inorganic salts; the carbon source is sucrose and is added at a concentration of 5 g / L to 8 g / L; the nitrogen source is a mixture of wheat bran and soybean meal and is added at a concentration of 18 g / L to 25 g / L; the inorganic salts include sodium chloride, potassium dihydrogen phosphate, and manganese sulfate. The mass ratio of wheat bran to soybean meal is 1:0.8~1.2; the amount of sodium chloride added is 4 g / L~6 g / L; the amount of potassium dihydrogen phosphate added is 0.5 g / L~1.5 g / L; and the amount of manganese sulfate added is 0.3 g / L~0.5 g / L.

10. The method according to claim 9, characterized in that, The fermentation temperature is 30°C to 40°C; the initial pH of the fermentation is 7.0 to 8.0; and the fermentation speed is 180 rpm to 220 rpm.

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