Acid-tolerant bacillus pumilus and application thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a strain of acid-tolerant Bacillus pumilus and application thereof. The present application first screens a strain of Bacillus pumilus FNBp-1, which has antagonistic activity to main pathogenic bacteria of tobacco such as Ralstonia solanacearum, Phytophthora parasitica, and Xanthomonas campestris, and is suitable for green prevention and control of tobacco bacterial wilt in acid soil conditions, and simultaneously promotes the vegetative growth of tobacco plants, and increases yield and income. Meanwhile, the strain secretes more degrading enzymes under acid conditions to inhibit Ralstonia solanacearum and reduce the infection of the bacteria on tobacco, and has important application value in the sustainable prevention and control of tobacco bacterial wilt in continuous cropping acid soil.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to an acid-resistant short spore bacillus and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Nature contains abundant microbial resources, characterized by their diversity, wide distribution, rapid reproduction, and the ability of different microorganisms to produce different degradative enzyme systems, such as degrading oils, straw, and the cell walls of plant pathogens. These microorganisms have been widely studied and applied in industries such as industry, agriculture, and food processing.

[0004] Frequent plant diseases and pests, coupled with the long-term irrational use of pesticides, have led to severe environmental pollution, excessive residues in agricultural products, and the development of resistance in pests, diseases, and weeds, seriously threatening the stable yields of grain and cash crops, as well as the ecological security of humans and the environment. Biological control is a key technology in integrated pest management. Compared to chemical agents, beneficial agricultural microorganisms (mainly bacteria and fungi) used in biological control are environmentally friendly, pollution-free, provide sustainable disease prevention, and promote plant growth, thus providing a solid foundation for ecological protection and green development while mitigating harm to human health. Of particular interest is the crucial search for plant growth-promoting rhizobacteria (PGPRs), which are active against specific pathogens and, through efficacy evaluation, have the potential to serve as more widely used antagonists (Nakkeeran et al., 2006).

[0005] Among PGPR bacteria, Bacillus spp. is a well-known and powerful genus. Many bacteria in this genus promote plant growth and effectively control plant diseases caused by pathogens through nutrient competition, phosphorus solubilization, and non-indirect nitrogen fixation (McSpaden Gardener 2004). Bacillus spp. bacteria are mobile and possess strong survival competitiveness in the rhizosphere and soil under various environmental conditions. Under adverse conditions, these bacteria can form spores to resist harsh environments. This characteristic is very useful for commercial applications because of their long shelf life (Niazi et al. 2014). Numerous studies have been conducted on the use of Bacillus-based biocontrol agents to control bacterial diseases in plants. Examples include Pseudomonas syringae infecting Arabidopsis thaliana causing foliar diseases, Xanthomonas euvesicatoria and Xanthomonas perforans infecting tomatoes causing diseases (Roberts et al. 2008), and Ralstonia solanacearum infecting mulberry (Ji et al. 2008), tobacco (Maketon et al. 2008), and pepper (Yanti et al. 2017) causing bacterial wilt. However, there are no studies on the use of Bacillus pumilus to promote tobacco growth and effectively inhibit Ralstonia solanacearum under continuously cropped acidified soil conditions. Further research is needed to discover novel microbial resources to provide new approaches to solving the problem of controlling bacterial bacterial wilt in plants in acidified soils. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an acid-resistant Bacillus subtilis strain and its applications. Experimental verification has shown that it is suitable for the green control of tobacco bacterial wilt under acidic soil conditions, while simultaneously promoting the vegetative growth of tobacco plants, increasing yield and income. Furthermore, by secreting more degrading enzymes under acidic conditions, it inhibits *Ralstonia solanacearum*, reducing the infection of tobacco by this bacterium. This invention has significant application value in the sustainable control of tobacco bacterial wilt in continuously cropped acidified soils. Based on the above research results, this invention was completed.

[0007] To achieve the above-mentioned technical objectives, the present invention relates to the following technical solutions:

[0008] In a first aspect, the present invention provides a strain of Bacillus pumilus FNBp-1, which was deposited on November 8, 2023, at the China General Microbiological Culture Collection Center (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China), with the biological accession number CGMCC No. 28899.

[0009] A second aspect of the present invention provides a fermentation production method for the above-mentioned Bacillus pumilus, the fermentation production method comprising: inoculating the Bacillus pumilus into a fermentation medium for fermentation culture to obtain the product.

[0010] A third aspect of the present invention provides a microbial inoculant containing the aforementioned Bacillus pumilus or its fermentation product or its metabolites.

[0011] In a fourth aspect, the use of the aforementioned Bacillus pumilus FNBp-1 or microbial agents in all or part of the following a)-d) is also within the scope of protection of this invention:

[0012] a) Inhibit plant pathogens or prepare plant pathogen inhibitors;

[0013] b) Suppressing plant diseases or preparing plant disease inhibitors;

[0014] c) Promote plant growth or prepare plant growth promoters;

[0015] d) Production of extracellular antagonistic proteins and / or glucanases.

[0016] A fifth aspect of the present invention provides a method for preventing and controlling diseases in tobacco plants, the method comprising applying the above-mentioned Bacillus pumilus FNBp-1 and / or the above-mentioned microbial agents to the tobacco growing environment.

[0017] A sixth aspect of the present invention provides a method for promoting tobacco growth, the method comprising applying the above-mentioned Bacillus pumilus FNBp-1 and / or the above-mentioned microbial agent to the tobacco growth environment.

[0018] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0019] The above-mentioned technical solution has for the first time screened out a strain of Bacillus pumilus FNBp-1. This strain has antagonistic activity against major tobacco pathogens such as Ralstonia solanacearum, Phytophthora indicum, and Phytophthora indicum. It also has a tobacco growth-promoting effect and is particularly suitable for the green control of tobacco bacterial wilt under acidic soil conditions. By secreting more degradation enzymes under acidic conditions, it inhibits Ralstonia solanacearum and reduces the infection of tobacco by this fungus. It has important application value in the sustainable control of tobacco bacterial wilt in continuously cropped acidified soils. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, 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 improper limitation of the invention.

[0021] Figure 1 In Embodiment 1 of the present invention Figure 1 Inhibitory activity of fermentation broth (left) and sterile filtrate (right) of strain FNBp-1 against Ralstonia solanacearum.

[0022] Figure 2 The left and right images show the inhibitory activity of strain FNBp-1 cells and fermentation broth of Example 1 of this invention against Phytophthora tobaccoii.

[0023] Figure 3 The cell lysis of Ralstonia solanacearum by strain FNBP-1 in Example 2 of the present invention is shown in the figure, wherein a, NB treatment; b, sterile supernatant of FNBP-1.

[0024] Figure 4 This invention provides a qualitative analysis of the extracellular proteins and glucanase secreted by strain FNBp-1 in Example 3 of this invention; wherein, a, glucanase activity; b, extracellular protein activity.

[0025] Figure 5 The antagonistic activity of the extracellular protein and crude dextranase extract of strain FNBp-1 in Example 3 of the present invention against Ralstonia solanacearum; wherein, a, crude dextranase extract; b, sterile supernatant (positive control); c, crude extracellular protein extract.

[0026] Figure 6 This invention demonstrates the antagonistic activity of the crude extract of FNBp-1 extracellular dextranase in Example 3 against Erythrozoa rubra (left) and black shank (right).

[0027] Figure 7 This is the tyrosine standard curve in Example 3 of the present invention.

[0028] Figure 8 This is the standard curve of dextran in Example 3 of the present invention.

[0029] Figure 9 The control effect of FNBp-1 strain on tobacco bacterial wilt in soil with different pH values ​​in Example 5 of this invention is shown. The horizontal axis represents treatment 1 to treatment 4, and the vertical axis represents the relative control effect of each treatment. a indicates the significance of the difference compared by Duncan's new multiple range method, with p < 0.05. Detailed Implementation

[0030] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0032] In a typical embodiment of the present invention, a strain of Bacillus pumilus FNBp-1 is provided. This strain was deposited on November 8, 2023, at the China General Microbiological Culture Collection Center (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China), with the biological accession number CGMCC No. 28899.

[0033] In another specific embodiment of the present invention, a fermentation production method for the above-mentioned Bacillus pumilus FNBp-1 is provided, the fermentation production method comprising: inoculating the Bacillus pumilus FNBp-1 into a fermentation medium for fermentation culture to obtain the product.

[0034] In this invention, no specific limitation is made to the fermentation production method; any conventional bacterial fermentation culture method can be used for cultivation.

[0035] The fermentation medium can be any common bacterial culture medium, such as NB medium in one specific embodiment of the present invention. Specifically, the culture conditions include: constant temperature of 25-35℃ (preferably 30℃) in NB culture medium, and a rotation speed of 100-150 rpm·min. -1 (preferably 120 rpm·min) -1 Shaking culture for 12-72 hours (preferably 48 hours).

[0036] In another specific embodiment of the present invention, a microbial inoculant is provided, which contains the aforementioned Bacillus pumilus FNBp-1 or its fermentation product or its metabolites.

[0037] In this invention, the term "fermentation product" is used to refer to fermentation products. The corresponding fermentation product can be a liquid obtained from the above-described fermentation culture of *Bacillus pumilus* FNBp-1, and therefore can also be called fermentation broth; the liquid may contain bacteria (cells), but does not necessarily need to contain bacteria. Preferably, the liquid contains metabolites produced by the *Bacillus pumilus* FNBp-1 of this invention.

[0038] Furthermore, in embodiments of the present invention, the fermentation broth or culture medium containing bacterial cells is separated from the liquid by centrifugation, filtration, sedimentation, or other means known in the art. The liquid remaining after removing the bacterial cells is called the "supernatant." In the present invention, the supernatant contains extracellular metabolites of Bacillus pumilus FNBp-1. In embodiments of the present invention, the microbial inoculum may also contain this supernatant.

[0039] Furthermore, in embodiments of the present invention, the fermentation broth or culture medium containing bacterial cells is centrifuged, filtered, settled, or otherwise known in the art to separate the bacterial cells grown in the fermentation broth or culture medium from the liquid to obtain bacterial cells. The bacterial cells can be broken up to obtain bacterial fragments. The breaking method can be ultrasound (e.g., ice bath ultrasound to break up cells) or other methods known in the art. Alternatively, the bacterial fragments can be centrifuged to collect the supernatant, which is designated as the cell-free extract. In the present invention, the bacterial fragments or cell-free extract contain intracellular metabolites of Bacillus pumilus FNBp-1. In embodiments of the present invention, the bacterial agent may also contain the bacterial fragments or cell-free extract.

[0040] Furthermore, in embodiments of the present invention, for ease of storage and transportation, and to improve the survival rate of the bacterial strain, the bacterial agent may also be a solid, and more preferably a lyophilized powder. That is, it is obtained by further lyophilizing the aforementioned Bacillus pumilus FNBp-1 or its fermentation product or its metabolites. The lyophilization technology (including vacuum lyophilization technology) can be carried out using conventional methods, and will not be elaborated further here.

[0041] Specifically, this invention demonstrates through experiments that strain FNBp-1, its fermentation broth, and its sterile supernatant all exhibit strong inhibitory effects against *Ralstonia solanacearum*. Simultaneously, strain FNBp-1 cells and fermentation broth also show good inhibitory effects against *Phytophthora blackshank*. Furthermore, the strain thrives in acidic environments (5.5 ≤ pH ≤ 6.5), which promotes the secretion of more antagonistic substances and maximizes its antibacterial activity. Moreover, its growth-promoting effect on tobacco is even better in acidic environments.

[0042] In another specific embodiment of the present invention, the microbial agent may further include excipients acceptable to the agent.

[0043] In another specific embodiment of the present invention, the excipients are selected from one or more of dispersants, wetting agents, disintegrants, binders, defoamers, antifreeze agents, thickeners, fillers, and solvents. The present invention does not impose any special restrictions on the sources of acceptable excipients for the bacterial agent; commercially available products are generally sufficient.

[0044] In another specific embodiment of the present invention, the application of the above-mentioned Bacillus pumilus FNBp-1 or microbial agent in all or part of the following a)-d) is also within the scope of protection of the present invention:

[0045] a) Inhibit plant pathogens or prepare plant pathogen inhibitors;

[0046] b) Suppressing plant diseases or preparing plant disease inhibitors;

[0047] c) Promote plant growth or prepare plant growth promoters;

[0048] d) Production of extracellular antagonistic proteins and / or glucanases.

[0049] Wherein, in a), plant pathogens include, but are not limited to, Ralstonia solanacearum, Phytophthora indicum, and Ralstonia solanacearum.

[0050] In b), plant diseases include, but are not limited to, diseases caused by Ralstonia solanacearum, such as tobacco bacterial wilt; diseases caused by Phytophthora indicum, such as tobacco black shank; and diseases caused by Phytophthora indicum, such as tobacco red spot.

[0051] In statement c), the plant can be a crop, specifically a food crop or a cash crop, preferably a cash crop such as tobacco.

[0052] The promotion of plant growth is specifically manifested in increasing the plant height, fresh weight, dry weight, root dry weight, and total chlorophyll content of tobacco plants.

[0053] In step d), the specific method for producing the extracellular antagonistic protein includes: obtaining the sterile supernatant of the fermentation of strain FNBp-1, adding ammonium sulfate to the sterile supernatant and obtaining the extracellular antagonistic protein by ammonium sulfate salting-out precipitation.

[0054] The specific method for producing glucanase includes: obtaining the sterile supernatant of the fermentation of strain FNBp-1, adding an equal volume of chloroform, shaking and mixing, and then allowing it to stand to obtain the organic phase and the milky white substance between the organic phase and the aqueous phase. After extracting the aqueous phase with an equal volume of ethyl acetate, the organic phase and the milky white substance are combined, and then the glucanase is obtained by ammonium sulfate precipitation.

[0055] In another specific embodiment of the present invention, a method for preventing and controlling tobacco plant diseases is provided, the method comprising applying the above-mentioned Bacillus simulans FNBp-1 and / or the above-mentioned microbial agents to the surface of tobacco plants and / or the tobacco growing environment.

[0056] In another specific embodiment of the present invention, a method for promoting tobacco growth is provided, the method comprising applying the above-mentioned Bacillus simulans FNBp-1 and / or the above-mentioned microbial agent to the surface of tobacco plants and / or the tobacco growth environment.

[0057] The tobacco growing environment is acidic, and specifically, the pH of the acidic conditions is 5.5 to 6.5.

[0058] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. The pathogens tested in the examples were all isolated and preserved by the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences.

[0059] Example 1: Inhibitory activity of B. pumilus FNBp-1 cells, culture medium, and sterile filtrate against major tobacco pathogens.

[0060] The antagonistic activity of *F. nigra*, the pathogen of tobacco bacterial wilt and black shank, isolated from Jianyang, Nanping, Fujian Province, was verified. NA medium was mixed with an appropriate amount of acidic buffer (0.3 mol / L sodium acetate and 0.2 mol / L acetic acid at a volume ratio of 1:9) to prepare NA medium plates with different pH values ​​of 5.5, 6.0, 6.5, 7.0, and 7.5. The differences in antibacterial activity of FNBp-1 strain, fermentation broth, and sterile filtrate under different pH conditions were determined using the inoculation method and the Oxford cup method. FNBp-1 was inoculated on NA plates and cultured at 28℃ for 48 h. Then, the plate confrontation method was used: *F. nigra* mycelial discs (5 mm in diameter) were inoculated on oat medium (YA) plates, and FNBp-1 colonies were picked up with an inoculation loop and streaked 1.5 cm apart on both sides of the *F. nigra* mycelial disc. The plates were then incubated at 28℃ for 7 days, and the inhibitory activity of FNBp-1 against *F. nigra* was observed. Inhibition of Ralstonia solanacearum was determined using the Oxford cup agar diffusion method. FNBp-1 was inoculated into beef extract peptone broth (NB) and cultured at 28°C with shaking at 150 rpm for 2 days. 150 μL of fermentation broth and sterile supernatant (after bacterial filtration) were then placed in Oxford cups on the surface of NA plates pre-contaminated with Ralstonia solanacearum. The mixture was incubated statically for 2 days, and the inhibitory activity of strain FNBp-1 against Ralstonia solanacearum was observed. The experiment was performed in triplicate. The results of the plate confrontation method for determining antagonistic activity showed that, firstly, in terms of indoor plate antibacterial effects, the fermentation broth and sterile supernatant of strain FNBp-1 had strong inhibitory effects on *Ralstonia solanacearum*, both exhibiting wide and transparent inhibition zones. The width and diameter of the inhibition zone in the fermentation broth were 8.00±0.63 mm and 24.04±1.04 mm, respectively, while the width and diameter of the inhibition zone in the sterile filtrate were 7.53±1.58 mm and 21.71±1.05 mm, respectively. Both showed strong antibacterial activity and comparable effects. Figure 1 (Table 1); Secondly, strain FNBp-1 cells and fermentation broth also showed good inhibitory effects on Phytophthora blackshank, mainly manifested in the inability of Phytophthora hyphae around the cells and fermentation broth to grow and reproduce normally, indicating that the inhibitory activity of this strain was significant. Figure 2 ).

[0061] Table 2 shows that strain FNBp-1 exhibits stronger inhibitory effects against *Ralstonia solanacearum* within an acidic range (5.5 ≤ pH ≤ 6.5), exceeding those under neutral or alkaline conditions. The strongest antibacterial activity was observed at pH 5.5, with an inhibition zone width of 10.67 mm, significantly different from other pH treatments. As the pH of the culture environment increased to neutral and alkaline levels, the antagonistic activity decreased, but the antibacterial activity remained strong; at pH 7.5, the inhibition zone width was 7.33 mm. These results indicate that this strain is well-suited to acidic environments and thrives in these environments by secreting more antagonistic substances to achieve optimal antibacterial activity. Therefore, applying this strain to the biocontrol of *Ralstonia solanacearum* under acidic soil conditions holds great potential.

[0062] Table 1. Antagonistic activity of B. pumilus FNBp-1 against Ralstonia solanacearum

[0063]

[0064] Note: p < 0.05 significance level.

[0065] Table 2. Differences in antagonistic activity of FNBp-1 strains under different acidic and neutral conditions (unit: mm)

[0066]

[0067] Note: p < 0.01 significance level.

[0068] Example 2: Strain B. pumilus FNBp-1 exhibits lytic activity against Ralstonia solanacearum cells.

[0069] Single colonies of pre-activated antagonistic bacteria FNBp-1 and *Ralstonia solanacearum* were picked using a sterile inoculation loop and inoculated into NB culture medium. After incubation at 30℃ and 120 rpm for 48 h with shaking, the concentrations of FNBp-1 and *Ralstonia solanacearum* fermentation broth were adjusted to 1 × 10⁻⁶ using a sterile water gradient dilution method combined with a spectrophotometer. 8 cfu / mL (OD) 600=0.1), and then the bacterial cells were removed through a bacterial filter (pore diameter of 0.22 μm) to obtain an appropriate amount of sterile supernatant of FNBp-1 in a sterile centrifuge tube for later use. Take 5 mL of supernatant and mix it with 5 mL of Ralstonia solanacearum suspension. The blank (CK) treatment was to mix an equal volume of NB liquid with Ralstonia solanacearum suspension. Each treatment was cultured at 30℃ and 120 r / min for 48 h with shaking. After centrifugation at 8000 r / min and 4℃ for 10 min, the bacterial cell pellet was collected, washed 3 times with phosphate buffer (PBS), resuspended again in 200 μL of PBS buffer, fixed with 500 μL of 2.5% glutaraldehyde solution for 24 h, centrifuged at 8000 r / min, and the pellet was dehydrated with tert-butanol gradient for 15 min and vacuum dried for 1.5 h. Then, the effect of extracellular metabolites secreted by FNBp-1 on Ralstonia solanacearum cells was observed by scanning electron microscopy (SEM).

[0070] Electron microscopy results showed that after 48 hours of treatment with a mixture of FNBp-1 supernatant and bacterial culture, the *Ralstonia solanacearum* rod-shaped bacterial cells in the control treatment appeared normal, smooth, and showed normal growth. Figure 3 a); After treatment with FNBp-1 sterile supernatant, the rod-shaped cells of Ralstonia solanacearum lysed, causing their contents to be exposed and diffused around the ruptured cells, and the cells broke apart from the middle. Figure 3 b) The bacteria cannot grow normally and eventually die.

[0071] Example 3B. extracellular protein antagonistic activity of pumilus FNBp-1 and the effect of different pH levels on its enzyme production

[0072] 1. Detection of extracellular antagonistic protease secreted by strain FNBp-1

[0073] 1.1 Qualitative detection of selective culture media

[0074] Extracellular antagonistic proteins were detected using protease-selective medium (NA medium with the nitrogen source replaced by an equal volume of skim milk powder, while other components remained unchanged), and the detection method was the same as the confrontation method in Example 1. Dextranase was detected qualitatively using selective medium (YPD medium supplemented with 0.1% carboxymethyl cellulose, containing 10g yeast extract, 15g peptone, and 15g glucose per liter, at natural pH). Under aseptic conditions, pre-activated FNBp-1 single colonies were randomly picked and inoculated onto the two selective medium plates mentioned above. After incubation at 30℃ for 72 to 96 hours, the presence and diameter of the transparent hydrolysis zone at the colony edge were observed and recorded.

[0075] 1.2 Extraction and Activity Verification

[0076] 1.2.1 Crude Extraction of Extracellular Antagonistic Proteins

[0077] The pre-activated strain FNBp-1 was inoculated into NB culture medium and kept at 30℃ for 120 rpm·min. -1 After shaking culture for 48 h, the cells were removed by centrifugation at 4 °C and 8000 rpm for 10 min to obtain a sterile supernatant containing extracellular secondary metabolites. An appropriate amount of (NH4)2SO4 was slowly added to the sterile supernatant to obtain a 75% saturated solution, which was stirred thoroughly and incubated overnight at 4 °C. The sulfate precipitation solution was then centrifuged at low temperature (4 °C) for 10 min (10,000 rpm·min). -1 The supernatant was removed, and the precipitate was resuspended in 1 / 15 volume of PBS buffer (pH 7.2). The suspension was then placed in a dialysis bag (molecular weight cutoff of 5 kD), clamped at both ends with dialysis clamps, and placed in 100 volumes of PBS buffer (pH 7.2). Dialysis was performed at 4°C for 24 hours on a magnetic stirrer, followed by changing the dialysis buffer and dialyzing for another 24 hours. After dialysis, the crude extract was freeze-dried and reconstituted in an appropriate amount of PBS buffer. The extract was then filtered through a 0.22 μm microporous membrane for sterilization. The antibacterial activity of the secreted extracellular proteins was tested on agar plates. An equal volume of sterile supernatant was used as the control (CK) treatment.

[0078] 1.2.2 Crude extraction of glucanase

[0079] Following the extraction method of Bacillus subtilis glucanase by Jin Zhixiong (2011), firstly, 200 mL of sterile supernatant was prepared (method as in 1.2.1). Chloroform was added at a volume ratio of 1:1, and the mixture was shaken thoroughly and allowed to stand for 1 h. The organic phase and the milky white substance between the organic and aqueous phases were aspirated. An equal volume of ethyl acetate was added to the aqueous phase for extraction, and the organic and milky white substances were combined. Salting out, dialysis, and lyophilization were performed using the same sterilization method as in 1.2.1. 0.05 g / mL of the crude enzyme extract was mixed into PDA and oat medium at 55℃. After cooling, mycelial cakes (6 mm in diameter) of *Aureobasidium aizoon* and *Aureobasidium nigra* were placed in the center of the plates. The antibacterial activity of the crude glucan-degrading enzyme extract against these two pathogens was detected using the confrontation method. An equal volume of sterile supernatant served as the control (CK) treatment.

[0080] 1.3 Comparison of FNBp-1 glucanase activity differences under different pH conditions

[0081] Dextranase activity assay: A glucose standard curve was prepared according to the DNS reagent instructions: 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 ml of 1.0% standard glucose solution were taken and diluted to 100 ml, respectively, to obtain standard solutions of 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg / ml. Each treatment was repeated 3 times. Seven colorimetric tubes were used to add the reagents according to Table 1.

[0082] Table 3. Plotting the Standard Curve

[0083]

[0084] After shaking all the above reagents well, boil them at 100℃ for 5 minutes, remove them and let them cool at room temperature, then dilute to 5.0 ml with distilled water. Use tube No. 1 as a control and measure the absorbance at a wavelength of 520 nm using a spectrophotometer. Plot a standard curve with the absorbance at 520 nm as the ordinate and the corresponding glucose solution concentration as the abscissa.

[0085] The activity of glucanase in the sterile supernatant of acid-resistant antagonistic strain FNBp-1 under pH 5.5-pH 7.0 acidic to neutral culture conditions was determined using the DNS colorimetric method with slight modifications. The specific method is as follows: FNBp-1 standard bacterial solution was aseptically inoculated at a 1% (v / v) ratio into NB solutions at pH 5.5 and 7.0. The solution was shaken at 28℃ and 150 rpm for 3 days, then centrifuged at 10000 rpm for 10 min. 1 ml of the supernatant was collected and diluted to an appropriate concentration using sodium acetate buffer. 1.0 ml of diluted crude enzyme solution was added to the colorimetric tube, followed by 1.0 ml of 1% β-glucan solution preheated to 55℃. After shaking, the mixture was reacted in a 55℃ water bath for 10 min. After the reaction was complete, 3 ml of DNS solution was added, and the mixture was thoroughly mixed. The mixture was then boiled at 100℃ for 5 min, cooled to room temperature, and the absorbance was measured at 520 nm. The glucose content was obtained according to the standard curve. Under the conditions of this experiment, the amount of enzyme protein required per minute to break down β-glucan to produce the equivalent of 1 μmol of glucose was defined as one enzyme activity unit (U / ml). Each treatment was repeated three times.

[0086] Calculation formula: Enzyme activity units [U / ml] = (R×D) / (180×T)

[0087] In the formula, R is the standard glucose concentration (μg / ml) corresponding to the absorbance value measured on the standard curve;

[0088] D represents the dilution factor of the enzyme solution to be tested; T represents the reaction time (min); 180 represents the molecular weight of glucose.

[0089] 2. Experimental Results and Analysis

[0090] 2.1 FNBp-1 secretes extracellular proteins and glucanase substances with antagonistic activity.

[0091] Strain FNBp-1 grew on both selective media, and distinct transparent hydrolysis zones of varying sizes appeared around the colonies, indicating that strain FNBp-1 has the ability to produce protease and glucanase degradation enzymes. Furthermore, the sizes of the hydrolysis zones produced on skim milk powder and YPD medium supplemented with carboxymethyl cellulose were 18.3 mm and 8.0 mm, respectively. Figure 4Preliminary findings indicate that strain FNBp-1 has a strong ability to secrete extracellular proteins.

[0092] Table 3. Ability of acid-resistant antagonistic bacteria to produce extracellular enzymes

[0093]

[0094] like Figure 5 As shown, the crude extracts of extracellular proteins obtained by salting out the sterile fermentation broth, and the crude extracts of dextranase obtained by organic solvent extraction, exhibited different degrees of antagonistic activity against *Ralstonia solanacearum*. Compared with the antibacterial activity (inhibition zone size) of the sterile supernatant of strain FNBp-1, the antibacterial activity of the crude extracellular protein extract was comparable, while the antibacterial activity of the crude dextranase extract was relatively weak, as evidenced by a significantly smaller inhibition zone. These results indicate that the extracellular antibacterial substances secreted by strain FNBp-1 are mainly antibacterial proteins. In addition, strain FNBp-1 showed relatively strong antagonistic activity against *Ralstonia solanacearum*. On day 8, it was found that *Ralstonia solanacearum* mycelia hardly grew and proliferated on PDA agar plates mixed with 1 mL of crude dextranase extract, while the mycelia on PDA agar plates treated with CK (equal amounts of sterile water) had completely covered the plates, indicating that the crude dextranase extract had stronger antibacterial activity against *Ralstonia solanacearum*.

[0095] 2.2 Acidic culture environment promotes increased production of antimicrobial proteins and glucanase in strain FNBp-1.

[0096] In this experiment, the protease activity was quantitatively determined using the Folin-Ciocalteu method. A tyrosine standard curve was constructed in the fermentation broth of the acid-resistant antagonistic strain *B. pumilus* FNBp-1 under acidic conditions (pH 5.5) and neutral conditions (pH 7.0). Figure 7 As shown, the equation is y = 0.006x + 0.0072(R). 2 =0.9903).

[0097] The results of the protease activity test showed that the protease activity yield of FNBp-1 fermentation broth cultured under acidic conditions of pH 5.5 and neutral conditions of pH 7.0 differed to 49.89 U / ml and 44.28 U / ml, respectively. This indicates that FNBp-1 produces relatively strong antimicrobial protein in an acidic growth environment, but the difference between treatments was not significant.

[0098] The extracellular dextranase activity of acid-resistant antagonistic strains under acidic (pH 5.5) and neutral (pH 7.0) culture conditions was quantitatively determined using the DNS method. The dextran standard curve is shown below. Figure 8 As shown, the standard curve equation is y = 1.7194x + 0.0051(R). 2 =0.9901).

[0099] The results of the dextranase activity assay showed that the dextranase activity in the FNBp-1 fermentation broth cultured under acidic conditions (pH 5.5) and neutral conditions (pH 7.0) differed, being 131.12 ± 7.16 a U / ml and 124.18 ± 9.46 a U / ml, respectively. This indicates that the FNBp-1 strain's extracellular dextranase production capacity was stronger under weakly acidic conditions (pH 5.5) than under neutral conditions (pH 7.0), although the difference between the treatments was not significant. Therefore, an acidic environment is conducive to the secretion of extracellular dextranase by the FNBp-1 strain to inhibit *Ralstonia solanacearum*, *Ralstonia solanacearum*, and *Ralstonia solanacearum*.

[0100] Example 4: Growth-promoting capacity in soils with different pH levels

[0101] The growth-promoting experiment was conducted under greenhouse pot conditions. The tested tobacco seedlings were the main production variety K326, with a growth period of 35 days. First, different pH levels were prepared for tobacco cultivation: pH 5.5 (acidic) and pH 7.0 (neutral). The tested soil was moist heat-sterilized soil provided by the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences. 5g of soil was weighed and placed in a 50mL Erlenmeyer flask. Deionized water was added at a water:soil ratio of 2.5:1. The mixture was shaken at 200 rpm for 20 minutes at room temperature, then allowed to stand for 30 minutes before pH measurement. The pH value was recorded using a pH meter (pH 6.8). Soil that passed through a 20-mesh sieve (0.9mm aperture) was thoroughly mixed with an appropriate amount of acid-base buffer solution. The soil pH was measured using the same method as above, and the two soil samples were adjusted to pH 5.5 and 7.0, respectively. The required amount of soil for pot cultivation was prepared in large quantities according to the above method. The growth-promoting experiment included two treatments: Treatment 1 was a FNBp-1 bacterial suspension treatment (concentration 10...). 8 Treatment 1 was a sterile water treatment (CK), with the control group receiving 10 mL of FNBp-1 bacterial solution (CFU / mL). Tobacco seedlings (cross-shaped stage) were transplanted into pots (90 mm in diameter) containing sterilized soil at pH 5.5 and 7.0, respectively. After transplanting and allowing the seedlings to recover (day 3), the roots were drenched with an equal volume of sterile water (CK). Each treatment consisted of 15 seedlings, with three replicates. All tested seedlings were cultured in an artificial climate chamber with a photoperiod of 14L:10D, a day / night temperature of 30 / 26℃, and a humidity of 60%. Thirty days later, all tobacco seedlings for each treatment were surveyed. The height of the tobacco plants was measured and recorded according to the tobacco agronomic trait survey method (YC / T142-2010). After the height measurement was completed, the tobacco seedlings were pulled out of their respective flower pots, and the soil attached to the roots was carefully washed away with running water. After being naturally dried, the fresh weight, dry weight and root dry weight of the whole plant were weighed (the tobacco plants were placed in an oven at 70℃ for 1.5 hours to constant weight).

[0102] The total chlorophyll content of tobacco leaves in each treatment was determined using the 95% ethanol extraction method. The specific procedure is as follows: Take fresh plant leaves (or other green tissues) or dry material, wipe off surface dirt, remove the midrib, and cut into small pieces. Weigh 2g of the chopped fresh sample, place it in a mortar, add a small amount of quartz sand and calcium carbonate powder, and 3mL of 95% ethanol. Grind into a homogeneous paste, then add another 10mL of ethanol and continue grinding until the tissue turns white. Let stand for 3-5 minutes. Place a sheet of filter paper in a funnel, moisten it with ethanol, and pour the extract into the funnel along a glass rod. Transfer the filtrate to a 100mL brown volumetric flask. Rinse the mortar, rod, and residue several times with a small amount of ethanol, and finally pour the filtrate and residue into the funnel. Use a pipette to draw an appropriate amount of 95% ethanol to wash all the chloroplast pigments off the filter paper into the volumetric flask. Continue until there is no green color in the filter paper and residue. Finally, dilute to 100mL with ethanol and shake well. The absorbance of chloroplast pigment extract was measured at wavelengths of 665 nm and 645 nm, with 95% ethanol as a blank control. The calculation formula is: Total chlorophyll a = 20.21A645 + 8.02A663.

[0103] The experimental results are shown in Tables 5 and 6. The acid-resistant antagonistic bacteria FNBp-1 suspension (concentration 10) was used... 8 When FNBp-4 strains (cfu / mL) were inoculated around the roots of tobacco seedlings in an acidic soil environment with a pH of 5.5, after 30 days of treatment, compared with the control (CK) treatment, the FNBp-4 strain treatment promoted the growth of tobacco plants in terms of plant height, whole plant fresh weight, dry weight, root dry weight, and chlorophyll content. However, the promotion effect on whole plant fresh weight, dry weight, and root dry weight was more significant, increasing by 21.0%, 30.5%, and 42.0%, respectively. Plant height and total chlorophyll content of leaves increased by 15.9% and 3.0%, respectively.

[0104] A suspension of acid-resistant antagonistic bacteria FNBp-1 (concentration 10) 8 The bacterial solution (10 mL / mL) was inoculated around the roots of tobacco seedlings in a neutral soil environment with a pH of 7.0. After 30 days of treatment, the results of the investigation and measurement are shown in Table 6. Compared with the CK treatment, the FNBp-1 strain also promoted the growth of tobacco plants, including plant height, whole plant fresh weight, dry weight, root dry weight, and total chlorophyll content of leaves. However, the growth-promoting effect was significantly lower than that of the strain in the acidic environment. Among them, the growth rate of whole plant dry weight was the highest, at 13.4%, while the changes in other growth-related indicators were not significant.

[0105] The above results indicate that B. pumilus FNBp-1 has a certain degree of acid tolerance, is suitable for growth in acidic environments, and can fully exert its growth-promoting effect on host plants.

[0106] Table 4. Growth-promoting effect of acid-tolerant bacteria FNBp-1 on acidic soil at pH (5.5)

[0107]

[0108] Table 5. Growth-promoting effect of acid-tolerant bacteria FNBp-1 on neutral soil pH (7.0)

[0109]

[0110] Example 5: Disease prevention effect in soils with different pH levels

[0111] The greenhouse potted plant disease prevention experiment consisted of six treatments: Treatment 1 was a suspension of strain FNBp-1 + Ralstonia solanacearum + soil at pH 5.5; Treatment 2 was 3% kasugamycin wettable powder + Ralstonia solanacearum + soil at pH 5.5; Treatment 3 was a suspension of strain FNBp-1 + Ralstonia solanacearum + soil at pH 7.0; Treatment 4 was 3% kasugamycin wettable powder + Ralstonia solanacearum + soil at pH 7.0; Treatment 5 was sterile water + Ralstonia solanacearum + soil at pH 5.5 (CK1); and Treatment 6 was sterile water + Ralstonia solanacearum + soil at pH 7.0 (CK2). Each treatment had 10 tobacco seedlings per replicate, with three replicates. After gently washing away the soil adhering to the roots, the tobacco seedlings (variety and growth stage were the same as those in Example 4) were disinfected with anhydrous ethanol and rinsed with sterile water. The solution was then added to each treatment (the concentration of FNBp-1 suspension in Treatments 1 and 3 was 10%). 8 The roots were soaked in a 1500-fold dilution of 3% kasugamycin WP for treatments 2 and 4 (CFU / mL), and an equal volume of sterile water for treatments 5 and 6 for 5 min. Then, the roots were transplanted into 90 mm pots filled with soil at pH 5.5 and 7.0. After one week of acclimatization, the roots were treated with a standard inoculum solution of Ralstonia solanacearum (concentration 10). 8 The root irrigation was performed with 10 mL of cfu / mL solution per pot. The tested tobacco seedlings were placed in an artificial climate chamber with a photoperiod of 14L:10D, a day / night temperature of 32 / 30℃, and a humidity of approximately 70%. Each treatment consisted of 15 seedlings, with three replicates. After the tobacco plants showed signs of bacterial wilt and wilting, the disease incidence was observed every other day. When the disease incidence rate in the control group (CK) reached 80%, the disease incidence rate and disease index of each treatment were investigated, and the control effect was statistically analyzed. Disease classification and disease index calculations were based on the classification and investigation methods for tobacco diseases and pests (GB / T 23222-2008).

[0112] A survey was conducted on day 12 post-inoculation. Based on the disease severity and incidence rate of all tested tobacco seedlings under all treatments, the disease index of each treatment and the disease control effects of the inoculum solution and control agent were statistically analyzed. The survey results are shown in Table 7. Figure 6As shown, treatments CK1 and CK2 had the highest incidence and disease index, indicating the most severe disease severity. Compared with the two blank control treatments, all four treatments exhibited varying degrees of inhibitory effects against tobacco bacterial wilt under soil conditions of pH 5.5 and pH 7.0, reducing the incidence and severity of disease in tobacco seedlings and mitigating the occurrence of tobacco bacterial wilt. Specifically, strain FNBp-1 showed disease control effects in both pH 5.5 acidic soil and pH 7.0 neutral soil. At pH 5.5, this strain showed the best resistance to bacterial wilt. The treatment showed the best efficacy, with a disease incidence rate of 56.67% and a disease index of 16.66 in tobacco seedlings, achieving a control effect of 73.18%. Under the same conditions, the control agent 3% kasugamycin WP had a disease index of 19.25 and a control effect of only 69.37% against bacterial wilt, with no significant difference between treatments. In neutral soil with a pH of 7.0, the disease control effect of strain FNBp-1 decreased slightly to 68.86%, but was still higher than that of 3% kasugamycin WP (67.32%), with no significant difference between treatments. These results indicate that Bacillus pumilus FNBp-1 has the most ideal disease control effect in acidic soil environments, exceeding that of the control agent 3% kasugamycin WP, and has the potential to improve the green control effect of tobacco bacterial wilt in continuously cropped acidified soils.

[0113] Table 6. Disease control effect of Bacillus pachycarpa FNBp-1 on tobacco bacterial wilt under different soil pH conditions.

[0114] Process 1 56.67 16.66 73.18a Process 2 60.00 19.25 69.37a Process 3 50.00 15.93 68.86a Process 4 50.00 16.67 67.32a CK1 73.30 63.70 - CK2 70.00 54.44 -

[0115] Note: The data in the table are the average values ​​of each indicator.

[0116] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above, they are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A strain of short-lived Bacillus ( Bacillus pumilus FNBp-1, characterized in that, The Bacillus pumilus FNBp-1 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 8, 2023, with the accession number CGMCC No. 28899.

2. The fermentation production method of Bacillus pumilus FNBp-1 according to claim 1, characterized in that, The fermentation production method includes: inoculating the Bacillus pumilus FNBp-1 into a fermentation medium for fermentation culture.

3. The fermentation production method as described in claim 2, characterized in that, The fermentation medium includes NB medium; the fermentation conditions include: constant temperature at 25-35℃ and shaking at 100-150 rpm for 12-72 h in NB medium.

4. A microbial inoculant, characterized in that, It contains the Bacillus pumilus FNBp-1 as described in claim 1, or its fermentation product or its metabolites.

5. The use of Bacillus pumilus FNBp-1 as described in claim 1 or the microbial agent as described in claim 4 in all or part of the following a)-d): a) Inhibit plant pathogens or prepare plant pathogen inhibitors; b) Suppressing plant diseases or preparing plant disease inhibitors; c) Promote plant growth or prepare plant growth promoters; d) Production of extracellular antagonistic proteins and / or glucanase; The plant pathogens include Ralstonia solanacearum, Phytophthora indicum, and Phytophthora indicum. The plant diseases mentioned include those caused by Ralstonia solanacearum; those caused by Phytophthora indicum; and those caused by Ralstonia solanacearum. The plant in question is tobacco.

6. The application as described in claim 5, characterized in that: The plant diseases mentioned include tobacco bacterial wilt, tobacco black shank, and tobacco red spot disease.

7. The application as described in claim 5, characterized in that: The promotion of plant growth is specifically manifested in increasing the plant height, fresh weight, dry weight, root dry weight, and total chlorophyll content of tobacco plants.

8. A method for controlling tobacco plant diseases, characterized in that, The method includes applying the Bacillus simulans FNBp-1 of claim 1 and / or the microbial agent of claim 4 to the surface of tobacco plants and / or the tobacco growing environment.

9. A method for promoting tobacco growth, characterized in that, The method includes applying the Bacillus simulans FNBp-1 of claim 1 and / or the microbial agent of claim 4 to the surface of tobacco plants and / or the tobacco growing environment.

10. The method as described in claim 8 or 9, characterized in that, The tobacco growing environment is acidic, with a pH of 5.5 to 6.5.

Images