Method for efficiently evolving plant pathogenic fungus resisting activity of bacillus subtilis

Abstract

The invention provides a method for efficiently evolving the plant pathogenic fungus resisting activity of bacillus subtilis. The method comprises the following steps: connecting an APOBEC-1 gene for coding cytosine deaminase and an ssbA gene for coding DNA single-stranded binding protein of the bacillus subtilis to a pMA5 expression vector to obtain a base editor rBE; the method comprises the following steps: converting a base editor rBE into bacillus subtilis 168 to obtain a recombinant strain BS168-ASP; the recombinant strain BS168-ASP is subjected to continuous subculture for five times in an LB culture medium containing 20-50 mg / mL of chitin; fermenting the last subculture bacterial liquid, preparing supernate, screening the bacteriostatic activity, and screening a strain with high antagonistic activity for a single colony of a sample with the highest bacteriostatic activity improvement through a plate confrontation method. Therefore, bacillus subtilis gene mutation can be efficiently promoted, and strains with obviously improved plant pathogenic fungus inhibition activity can be screened out.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for efficiently evolving the antifungal activity of Bacillus subtilis against plant pathogenic fungi. Background Technology

[0002] Bacillus subtilis, belonging to the genus Bacillus, is a thermophilic, Gram-positive rod-shaped bacterium capable of spore production, and is either aerobic or facultative anaerobic. It possesses significant antifungal activity and stress resistance. However, as pathogen tolerance increases, the inhibitory effect of Bacillus subtilis on pathogenic fungi gradually decreases. Furthermore, due to the complexity of biological systems, its antibacterial phenotype may be regulated by multiple genes, and traditional rational modification methods struggle to overcome the limitations of complex metabolic networks. At the same time, natural selection is inefficient, and targeted editing technologies that rely on known gene targets (such as the CRISPR-Cas system) cannot cover antibacterial genes with unknown functions, resulting in limited evolutionary effects of antibacterial activity.

[0003] Genome mutation is a versatile tool that can effectively promote the phenotypic evolution of microorganisms, playing a significant role in helping microorganisms acquire new traits, improve tolerance, and enhance production performance. Related technologies, such as CN 120535650 A, disclose directed evolution of specific genes (such as CodY regulatory factors) in Bacillus subtilis based on a T7 RNAP-guided base editor. However, this method requires the design of sgRNA target sequences, making it difficult to achieve genome-wide random mutations to simulate natural evolutionary pressures. Therefore, there is an urgent need for an efficient evolutionary method that combines random mutation tools with environmentally induced domestication to overcome the limitations of existing technologies. Summary of the Invention

[0004] The present invention aims to at least partially solve one of the technical problems in the above-mentioned technology, namely, to provide a method for efficiently evolving the anti-plant pathogenic fungal activity of Bacillus subtilis. This method can efficiently promote gene mutation of Bacillus subtilis and screen out strains with significantly enhanced inhibitory activity against plant pathogenic fungi.

[0005] Therefore, this invention proposes a method for efficiently enhancing the antifungal activity of Bacillus subtilis against plant pathogenic fungi, which includes the following steps:

[0006] The APOBEC-1 gene encoding cytosine deaminase and the ssbA gene encoding a DNA single-strand binding protein of Bacillus subtilis were ligated into the pMA5 expression vector to obtain the base editor rBE.

[0007] The base editor rBE was transformed into Bacillus subtilis 168 to obtain the recombinant strain BS168-ASP;

[0008] The recombinant strain BS168-ASP was passaged five times consecutively in LB medium containing 20–50 mg / mL chitin.

[0009] The antibacterial activity was screened by taking the fermentation supernatant of the last passage bacterial culture. The single colony of the sample with the highest antibacterial activity was screened for strains with high antagonistic activity by plate confrontation method.

[0010] This invention discloses a method for efficiently evolving the antifungal activity of Bacillus subtilis against plant pathogenic fungi. The method involves designing a random base editor that increases the mutation rate of Bacillus subtilis genes and transforming it into Bacillus subtilis. The recombinant Bacillus subtilis is inoculated into a chitin-containing medium for continuous culture and passage, and changes in antibacterial activity are measured. Single colonies are obtained by diluting and plating the bacterial suspensions with significantly enhanced antibacterial activity. Strains with significantly enhanced antibacterial activity are screened using the plate confrontation method. Ultimately, four Bacillus subtilis strains with significantly enhanced antibacterial activity against Rhizoctonia solani were screened, with the highest inhibition rate reaching 51.11 ± 0.96%, an increase of 130% compared to the starting strain. This method does not rely on known gene targets, and only five passages are needed to increase the inhibition rate of Bacillus subtilis against Rhizoctonia solani by 130%. It has advantages such as simple operation, high evolutionary efficiency, and broad antibacterial spectrum, and can be widely applied to the development of agricultural biocontrol agents.

[0011] Optionally, single colonies are first cultured in 96-well plates for 3 days, and then 5 μL of the culture is used for plate confrontation method to determine antibacterial activity in order to screen strains with high antagonistic activity.

[0012] Optionally, the indicator bacterium in the plate confrontation method is Rhizoctonia solani.

[0013] Optionally, the chitin concentration gradient is 20 mg / mL, 30 mg / mL, 40 mg / mL, and 50 mg / mL, and the passage conditions are 37℃, 180 rpm, and 24 h per generation.

[0014] Optionally, the antibacterial activity can be screened by taking the fermentation supernatant of the last passage culture, including:

[0015] The bacterial culture from the last subculture was inoculated into Landy medium containing the corresponding chitin concentration at an inoculation rate of 9% and fermented for 3 days. The fermentation broth was centrifuged at 8000 rpm for 15 min and filtered through a 0.22 μm microporous membrane to obtain the fermentation supernatant.

[0016] The mycelial growth inhibition method was used to evaluate the antibacterial effect of fermentation supernatant on Alternaria alternata, Alternaria solani, Anthracnose spp., Rhizoctonia solani, and sweet potato vine causal agent.

[0017] Optionally, the bacterial solution with the highest antibacterial activity can be diluted, spread, and used to separate single colonies, selecting single colonies with larger diameters and better morphology.

[0018] Optionally, the base editor rBE is transferred into Bacillus subtilis 168 via electroporation.

[0019] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0020] Figure 1 The plasmid map of the Bacillus subtilis random base editor according to an embodiment of the present invention;

[0021] Figure 2 This is a graph showing the mutation efficiency of the Bacillus subtilis random base editor according to an embodiment of the present invention.

[0022] Figure 3 The mutation type of the BS168-ASP rifampicin-resistant mutant strain according to embodiments of the present invention;

[0023] Figure 4 This is a growth curve of the recombinant strain BS168-ASP according to an embodiment of the present invention;

[0024] Figure 5 This is a diagram illustrating the plasmid stability of the recombinant strain BS168-ASP according to an embodiment of the present invention.

[0025] Figure 6 To illustrate the effect of different concentrations of chitin on the growth of BS168-ASP according to embodiments of the present invention;

[0026] Figure 7 This is a graph showing the changes in the broad-spectrum antibacterial activity of BS168-ASP after passage and domestication in different concentrations of chitin according to an embodiment of the present invention.

[0027] Figure 8 To screen the strain with the best antibacterial activity of BS168-ASP using the plate confrontation method according to an embodiment of the present invention. Detailed Implementation

[0028] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0029] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0030] The principle behind this application's screening of Bacillus subtilis strains with significantly enhanced inhibitory activity against plant pathogenic fungi is as follows:

[0031] Cytosine deaminase (APOBEC) is an RNA deaminase that frequently performs C-to-U modifications on ssDNA. During genome replication, ssDNA is mainly formed and stabilized by single-stranded DNA-binding proteins. When APOBEC binds to specific single-stranded DNA-binding proteins, these proteins guide APOBEC to randomly locate on single-stranded DNA, facilitating the deamination of cytosine and randomly introducing C-to-T mutations across the entire genome. When exogenous inducing substances (such as chitin, a major component of the cell wall of plant pathogenic fungi) are added, the expression of genes related to antimicrobial activity in chassis cells may be stimulated. Simultaneously, the continuous accumulation of base mutations under rBE (rBehavior-Blocking Immunosorbent Assay) ultimately leads to the evolution of antimicrobial activity-related genes in chassis cells.

[0032] It should be noted that all test materials used in this invention are commercially available products. Specifically, Bacillus subtilis 168 was purchased from Shanghai Chunshi Biotechnology Co., Ltd.

[0033] pMA5 vector was purchased from Hunan Fenghui Biotechnology Co., Ltd.;

[0034] The LB medium consists of 10g tryptone, 5g yeast extract, and 5g sodium chloride, dissolved in 1L of distilled water, and the pH is adjusted to 7.0.

[0035] The GM medium consisted of 1 g of LB broth powder and 4.55 g of sorbitol dissolved in 50 mL of distilled water.

[0036] The ETM eluent consists of 18.22 g sorbitol and 18.22 g mannitol dissolved in 200 mL of 10% glycerol;

[0037] The RM medium consisted of 1 g LB broth powder, 4.55 g sorbitol, and 3.46 g mannitol dissolved in 50 mL distilled water.

[0038] The PDA medium consists of 22.5g of potato (from which extract powder is obtained) and 1.5g of glucose, dissolved in 1L of distilled water.

[0039] Landy medium consists of 20g glucose, 10g peptone, 5g monosodium glutamate, 0.5g magnesium sulfate heptahydrate, 0.5g potassium chloride, 1g potassium dihydrogen phosphate, 0.00015g ferrous sulfate, 0.005g manganese sulfate, and 0.00016g copper sulfate, dissolved in 1L distilled water and adjusted to pH 7.1.

[0040] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0041] Example 1: Construction of a Bacillus subtilis random base editor (rBE)

[0042] Using the rat APOBEC-1 gene (GenBank:25383) and the Bacillus subtilis 168 single-stranded binding protein ssbA gene (GenBank:937911) as the main functional elements, and the pMA5 shuttle plasmid as the vector, an rBE editor was designed with ampicillin and kanamycin as the selection criteria for resistance. Figure 1 As shown.

[0043] The APOBEC-1-ssba gene was synthesized based on the gene sequence.

[0044] Specifically, the pMA5 plasmid was extracted and double-digested with XhoI-NheI. The digestion system is shown in Table 1 below.

[0045] Table 1

[0046]

[0047] After reacting at 7°C for 5 hours, the large fragment was recovered by 1% agarose gel extraction to obtain the linearized pMA5 vector.

[0048] Primers were designed using the APOBEC-1-ssba gene sequence (AS-F / R were used for recombinant primers) to construct the gene subcloning vector. The primer sequences are shown in Table 2.

[0049] Table 2

[0050]

[0051] The synthesized primers were diluted to a final working solution of 10 μmol / L, and PCR amplification was performed using the diluted primers and template. The system is shown in Table 3 below:

[0052] Table 3

[0053]

[0054] The above materials were added to thin-walled tubes, mixed thoroughly, and then placed in a PCR instrument. The final reaction program was adjusted as follows: 98℃ pre-denaturation for 2 min, followed by 98℃ denaturation for 10 sec, 58℃ annealing for 30 sec, and 72℃ extension for 30 sec, repeated for 30 cycles. Finally, the mixture was held at 72℃ for 2 min. After the reaction, agarose gel electrophoresis was performed, and the target gene was recovered. The recovered and purified target fragment was ligated with the recovered and purified vector pMA5, and the ligation product was named APOBEC-1-ssba-pMA5(ASP). Because the primers contained homologous arm sequences with the vector, homologous recombinase was used to recombine the recovered fragment with the linearized vector, completing the recombination process of the recovered target gene and the vector.

[0055] Transform 10 μL of the ligation product into 100 μL of DH5α competent cells: After mixing the product with the competent cells, incubate on ice for 30 min, heat shock at 42℃ for 90 s, immediately place on ice for 2 min, add 500 μL of LB medium preheated to room temperature, and culture on a shaker at 37℃ for 1 h at 180 rpm. Centrifuge at 5000 rpm for 3 min, discard 500 μL of culture supernatant, mix the remaining 100 μL with a pipette, spread evenly on an LB plate containing 50 μg / mL ampicillin, invert, and incubate overnight at 37℃.

[0056] Four single colonies were picked and inoculated into LB medium containing 5 mL of 50 μg / mL ampicillin-resistant culture medium, and incubated in a shaker at 220 rpm and 37 °C for 5 h. The cultured bacterial solutions were then used for PCR identification.

[0057] Table 4 PCR System

[0058]

[0059] PCR reaction conditions:

[0060] 95℃5min; 95℃30sec, 50℃30sec, 72℃45sec, 30cycles; 72℃10min, 4℃4min.

[0061] One mL of E. coli DH5a culture containing the ASP plasmid was added to 50 mL of LB broth with ampicillin at a final concentration of 100 μg / mL, and activated overnight at 37°C and 180 rpm. The plasmid was then extracted using a mini-plasmid extraction kit.

[0062] Bacillus subtilis seed culture was inoculated at 1% volume into 50 mL of GM medium to achieve an initial OD value of approximately 0.1. The culture was then incubated at 180 rpm and 37°C in a shaker until the logarithmic growth phase (OD = 0.6–0.7). After incubation, the culture was placed on ice for 10 min, centrifuged at 6000 rpm and 4°C for 5 min, the supernatant was discarded, and the cells were washed with 40 mL of pre-chilled ETM. This washing process was repeated three times. The cells were then resuspended in 0.5 mL of ETM, and the culture was aliquoted into 80 μL tubes to obtain competent cells. 3 μg of plasmid was added to the competent cells, and after incubating on ice for 5 min, the cells were transferred to pre-chilled 1 mm electroporation cuvettes. Electroporation was then performed once at 2000 V, 25 μF, and 200 Ω (4.5 ms–5 ms). Immediately after electroporation, add 1 mL of preheated RM (Regenerative Meridian) at 37°C and incubate at 37°C and 200 rpm for 3 hours. Then, plate the culture onto LB agar plates containing 50 μg / mL Km and incubate for 12-24 hours to observe the results. Pick several single colonies from the transformation plates and transfer them to 3 mL of LB liquid medium containing 50 μg / mL Km, and incubate for 10 hours. Perform PCR amplification of the target gene on LB medium containing 50 μg / mL Km. Primers for upstream and downstream amplification were designed by snapgene based on the APOBEC-1-ssba gene on the ASP plasmid; primer sequences were synthesized by Qingke Biotechnology.

[0063] Table 5

[0064]

[0065] PCR reaction conditions: 94℃ for 2 min; 94℃ for 30 sec, 60℃ for 30 sec, 72℃ for 36 sec, 30 cycles; 72℃ for 2 min, 4℃ for 4 min.

[0066] Table 6 PCR amplification system

[0067]

[0068] The PCR products were subjected to agarose gel electrophoresis. The size of the bands in the gel imaging results was used to determine whether the selected single colony was a positive clone. The positive clones were inoculated into fresh LB broth containing 50 μg / mL kanamycin for expansion culture. The bacterial culture was preserved after reaching the logarithmic growth phase to obtain the recombinant strain BS168-ASP.

[0069] Example 2: Determination of rBE's efficiency in inhibiting Bacillus subtilis genome mutation.

[0070] The recombinant strain BS168-ASP obtained in Example 1 was inoculated into 10 mL of LB broth containing 50 μg / mL kanamycin and incubated overnight at 37°C and 180 r / min for activation.

[0071] The activated culture medium was transferred to 50 mL of fresh LB broth at a 2% inoculation rate and incubated at 37 °C and 180 rpm for 12 h. 200 μL of the culture medium was then plated onto LB agar plates supplemented with 30 μg / mL rifamycin. Simultaneously, the culture medium was diluted 10... 6 After dilution, 100 μL was spread onto LB agar plates without rifamycin. Using the original Bacillus subtilis strain (Bacillus subtilis model strain 168) as the control group, all plates were incubated upside down at 37°C for 12-16 h. The number of colonies grown was counted. The concentration of the original bacterial solution (cfu / mL) was calculated based on the number of colonies on the diluted plates. The mutation rate was calculated based on the number of colonies of the original strain and the transformed strain on plates containing rifamycin. The fold increase in mutation rate was calculated based on the ratio of the mutation rates of the original strain and the transformed strain.

[0072] Mutation rate calculation formula:

[0073]

[0074] The results are as follows Figure 2 As shown, the mutation rate of BS168 is 1.61 ± 2.57 × 10⁻⁶. -5 The mutation rate of BS168-ASP was 7.18 ± 1.09 × 10⁻⁶. -4 The editor rBE increased the rate of rifamycin resistance mutation in Bacillus subtilis by 44.49 times.

[0075] Six resistant single colonies were randomly selected for rpoB gene (GenBank: 936335) inoculation and inoculated into 5 mL LB broth, and cultured overnight at 37°C and 180 rpm. Genomic DNA was extracted from Bacillus subtilis using a DNA extraction kit, and the resulting DNA solution was stored at -20°C. Primers for upstream and downstream amplification of the rpoB gene from Bacillus subtilis 168 were designed using Snapgene, and the primer sequences were synthesized by Qingke Biotechnology.

[0076] Table 7

[0077]

[0078] Using Bacillus subtilis genomic DNA as a template, the rpoB gene on the genome was amplified. The PCR reaction conditions were set as follows: 95℃ for 5 min; 95℃ for 15 sec, 62℃ for 15 sec, 72℃ for 2 min, 30 cycles; 72℃ for 5 min, 4℃ for 4 min.

[0079] The amplification system is shown in Table 8 below.

[0080] Table 8 PCR amplification system

[0081]

[0082]

[0083] Analysis of mutation types yielded the following results: Figure 3 As shown, all mutants undergo C→T mutation at the same site, consistent with the working principle of the rBE editor.

[0084] Example 3: Effect of rBE on the growth of Bacillus subtilis

[0085] The frozen BS168-ASP strain (obtained in Example 1) was inoculated into 25 mL of LB broth containing anti-(kanamycin concentration of 50 μg / mL) and activated overnight to prepare seed culture.

[0086] The seed culture was transferred to 50 mL of antibiotic-free LB broth at an inoculum rate of 1%. Using the starting strain as a blank control, the culture was incubated at 180 rpm and 37°C for 24 h, with the absorbance (OD) of the bacterial culture measured every 2 hours. 600 ), and plot the growth curve.

[0087] The results are as follows Figure 4 As shown, the OD of BS168-ASP within 24 hours 600 The value is slightly lower than BS168, but its overall growth trend is not significantly different from BS168, indicating that the editor rBE will not have a significant impact on the growth of BS168-ASP.

[0088] Example 3: Stability of the rBE Editor

[0089] The frozen BS168-ASP strain (obtained in Example 1) was inoculated into 5 mL of LB broth for activation to prepare seed culture.

[0090] The seed culture was transferred to 10 mL of antibiotic-free LB broth at an inoculum rate of 2%. A control group was prepared using an antibiotic culture system supplemented with 50 μg / mL kanamycin. Both systems were continuously passaged at 37°C and 180 rpm for 12 hours per generation, with samples taken at every generation interval. The seed culture was used as the first generation sample. At the 10th passage, all samples were subjected to PCR and agarose gel electrophoresis to observe the amplification of the target gene.

[0091] The results are as follows Figure 5 As shown, the rBE editor remained stable within 6 passages, indicating that the rBE editor has good stability in Bacillus subtilis.

[0092] Example 4: Optimization of Chitin-Induced Concentration

[0093] 30 mL LB medium was prepared with chitin concentrations of 1, 5, 10, 20, and 50 g / L, respectively. Activated BS168-ASP strain was inoculated at a 3% inoculum and cultured at 180 rpm and 37°C for 24 h. Before inoculation and after culture, 1 mL of the liquid was transferred to 2 mL centrifuge tubes, dried at 60°C for 24 h, and the biomass change was determined by dry weight method.

[0094] The results are as follows Figure 6 As shown, when the chitin concentration was 20 g / L, it promoted the increase of BS168-ASP biomass, while the biomass began to decrease at 50 g / L, thus determining the optimal domestication concentration range as 20-50 g / L.

[0095] Example 5: Chitin-Induced Evolution

[0096] The acclimatization concentrations were set at 20 g / L, 30 g / L, 40 g / L, and 50 g / L. The activated BS168-ASP strain was inoculated into LB medium containing the above chitin concentrations at an inoculation rate of 3%. The strain was continuously passaged at 180 rpm and 37°C, with BS168 as a negative control. Each generation was cultured for 24 h, and the strain was passaged 5 times.

[0097] Fermentation and broad-spectrum antibacterial activity assay of evolved strains: The bacterial culture from the last subculture was inoculated into 30 mL of Landy medium containing the corresponding chitin concentration at an inoculation rate of 9% and fermented for 3 days. The fermentation broth was centrifuged at 8000 rpm for 15 min and filtered through a 0.22 μm microporous membrane to obtain sterile fermentation supernatant.

[0098] The mycelial growth inhibition method was used to evaluate the antifungal effect of fermentation supernatant on plant pathogenic fungi such as *Alternaria alternata*, *Alternaria solani*, *Anthracnose spp.*, *Rhizoctonia solani*, and *Polygonum cuspidatum*. 4 mL of fermentation supernatant was added to 36 mL of PDA medium cooled to approximately 45°C to form a 10% concentration system. After mixing, the plates were poured in triplicate. Holes were punched in the plates containing the tested plant pathogens using a 6 mm punch, and the mycelial blocks were placed in the center of the solidified PDA medium plates. Amphotericin B was used as a positive control, the original strain as a negative control, and the fermentation medium as a blank control. The plates were incubated at 28°C until the blank control mycelium reached full colony growth. The antifungal effect of the fermentation supernatant on the tested plant pathogens was observed, and the inhibition rate was calculated.

[0099] Formula for calculating antibacterial rate:

[0100]

[0101] The results are as follows Figure 7As shown, when the chitin concentration was 40 g / L, the fermentation supernatant of BS168-ASP (i.e., A-40 g / L in the figure) showed a significant increase in antibacterial activity against Rhizoctonia solani, reaching 89.80 ± 8.90%, which was 149% higher than that of the starting strain.

[0102] Example 6: Single-strain isolation and screening of strains with high inhibitory activity

[0103] The bacterial culture with the highest antibacterial activity in Example 5 (i.e., the bacterial culture sample obtained after fermenting BS168-ASP in 40 g / L chitin-containing medium in Example 5, corresponding to group A-40 g / L in the figure, which showed significantly enhanced inhibitory activity against Rhizoctonia solani) was selected and diluted 10 times. 4～5 After multiplication, 100 μL was spread onto LB agar plates to obtain several single colonies. Fifty colonies with good morphology and large diameter were selected and numbered. 1 mL of Landy medium was added to each well of a 96-well plate, and the single colonies were inoculated and cultured at 37°C and 180 rpm for 3 days. Using *Rhizoctonia solani*, which showed the strongest inhibitory effect, as an indicator bacterium, the plate confrontation method was used to screen for evolved strains of *Bacillus subtilis*. A bacterial disc was taken using a 6 mm punch and inoculated, hyphae side down, into the center of a cooled and solidified PDA plate. Then, 5 μL of *Bacillus subtilis* single-strain bacterial suspension was added at three equidistant locations on the plate surface. Plates without bacterial suspension served as negative controls, and plates with amphotericin B added served as positive controls. This was repeated three times, and the plates were incubated in the dark at 28°C for 4 days to screen for single strains with good antagonistic effects against *Rhizoctonia solani*. The magnitude of antagonistic performance is represented by the inhibition rate. The colony radius of pathogens in the blank group and the experimental group were measured respectively, and the inhibition rate was calculated.

[0104] Inhibition rate calculation formula:

[0105]

[0106] The results are as follows Figure 8 As shown, among the 50 single colonies, 33 single colonies showed significantly improved inhibition rates against Rhizoctonia solani compared to the starting strain. Among them, 4 single colonies showed inhibition rates of over 45% against Rhizoctonia solani, with the highest inhibition rate reaching 51.11±0.96%, which was 130% higher than that of the starting strain.

[0107] In summary, according to the embodiments of the present invention, the mutation rate of Bacillus subtilis can be increased by 44.49 times through random base editing, and the antibacterial activity can be significantly improved in just 5 passages by combining chitin-induced subculturing. High-throughput culture in 96-well plates combined with plate confrontation screening can efficiently obtain single strains with high inhibitory activity, and the optimal strain can be obtained quickly, making it suitable for industrial applications.

[0108] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0109] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for efficiently enhancing the antifungal activity of Bacillus subtilis against plant pathogenic fungi, characterized in that, Includes the following steps: The APOBEC-1 gene encoding cytosine deaminase and the ssbA gene encoding a DNA single-strand binding protein of Bacillus subtilis were ligated into the pMA5 expression vector to obtain the base editor rBE. The base editor rBE was transformed into Bacillus subtilis 168 to obtain the recombinant strain BS168-ASP; The recombinant strain BS168-ASP was passaged five times in LB medium containing 20-50 mg / mL chitin. The antibacterial activity was screened by taking the fermentation supernatant of the last passage bacterial culture. The single colony of the sample with the highest antibacterial activity was screened for strains with high antagonistic activity by plate confrontation method.

2. The method as described in claim 1, characterized in that, Single colonies were first cultured in 96-well plates for 3 days, and then 5 μL of the culture was used for plate confrontation method to determine antibacterial activity in order to screen strains with high antagonistic activity.

3. The method as described in claim 1, characterized in that, The indicator bacterium in the plate confrontation method is Rhizoctonia solani.

4. The method as described in claim 1, characterized in that, The chitin concentration gradients were 20 mg / mL, 30 mg / mL, 40 mg / mL, and 50 mg / mL, and the passage conditions were 37 ℃, 180 rpm, and 24 h per generation.

5. The method as described in claim 1, characterized in that, Screening for antibacterial activity using the fermentation supernatant of the last passage culture includes: The bacterial culture from the last subculture was inoculated at an inoculation rate of 9% into Landy medium containing the corresponding chitin concentration and fermented for 3 days. The fermentation broth was centrifuged at 8000 rpm for 15 min and filtered through a 0.22 μm microporous membrane to obtain the fermentation supernatant. The mycelial growth inhibition method was used to evaluate the antibacterial effect of fermentation supernatant on Alternaria alternata, Alternaria solani, Anthracnose spp., Rhizoctonia solani, and sweet potato vine causal agent.

6. The method as described in claim 1, characterized in that, The bacterial suspension with the highest antibacterial activity was diluted, spread, and single colonies were isolated. Single colonies with larger diameters and good morphology were selected.

7. The method as described in claim 1, characterized in that, The base editor rBE was transferred into Bacillus subtilis 168 via electroporation.

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