Bacillus velezensis for preventing and controlling pear fire blight and fermentation method and application thereof

Abstract

This application discloses a strain of Bacillus belyceae DW4-5-2 for controlling pear fire blight and develops a highly efficient and stable microbial preparation fermentation method for it, clarifying its application in the control of pear fire blight. Compared with existing technologies, this application has the following advantages: the strain is highly novel; it has high antagonistic activity; it has excellent pot-plant control efficacy and a long-lasting effect; the fermentation process is thoroughly optimized; and it has broad application prospects. This strain and its optimized fermentation process provide core technical support for the development of highly efficient, stable, and environmentally friendly biological pesticides for pear fire blight, which is of great significance for ensuring the green, safe, and sustainable development of my country's pear and apple industries.

Description

Technical Field

[0001] This application relates to the field of agricultural microbiology technology, specifically to a strain of Bacillus bellis for controlling pear fire blight and its fermentation method and application. Background Technology

[0002] China ranks first in the world in both apple and pear cultivation area and yield, holding a key position in the international market. Gansu Province, with its abundant sunshine and large diurnal temperature range, has the second largest apple planting area in my country, and pear cultivation is also one of the country's major producing areas. Pear fire blight, caused by *Erwinia amylovora*, is a bacterial disease that causes devastating damage to Rosaceae plants. It is one of my country's important import quarantine targets and one of the world's ten most important plant pathogens. The disease was first discovered in apple orchards in Yili, Xinjiang, in 2015, and gradually appeared in fragrant pear orchards in Korla region in 2017, and in pear orchards in Zhangye and Wuwei cities in Gansu Province in 2020-2021. Currently, the disease has spread to 70 counties (cities, districts) in these two provinces, causing huge economic losses and posing a significant threat to the safety of my country's pear and apple industries. To reduce the risk of this dangerous organism's occurrence area continuing to move eastward and to ensure the safety of fruit production west of the Hexi Corridor in my country, close monitoring and effective control of the disease are urgently needed.

[0003] Pear fire blight pathogen has a wide host range, primarily harming cash crops such as apples, pears, and cherries. It spreads rapidly and is highly virulent, capable of destroying an entire orchard in a single growing season, rendering many existing control measures ineffective. Currently, some antibiotics, copper-containing agents, and aluminum-containing agents play an important role in suppressing the occurrence and spread of pear fire blight. However, chemical control has significant limitations: firstly, long-term use easily leads to antibiotic resistance in the pathogen, causing a gradual decline in control effectiveness, and antibiotic abuse may pose risks to agricultural product quality and safety; secondly, chemical agents are difficult to precisely target key parts of the pathogen's infection (such as flowers and young shoots), while also disrupting the orchard soil microecological balance and killing pollinating insects (such as bees), contradicting the needs of the green fruit tree industry. Therefore, exploring alternative control strategies that are natural, highly targeted, and environmentally friendly has become an urgent need for controlling pear fire blight.

[0004] Soil-based biocontrol microorganisms have undergone long-term natural selection, allowing them to adapt quickly to local soil and microecological environments compared to exogenous microorganisms. They are less likely to be rejected by native microbial communities, have longer colonization cycles, and can achieve "full-process dynamic control" of pear blight, effectively compensating for the "short-acting" and "non-targeting" defects of chemical agents. Bacillus bacteria are recognized as one of the most valuable species due to their unique biological characteristics. Their core advantage lies in their ability to form highly resilient spore structures, giving them strong tolerance to drought, high temperatures, ultraviolet radiation, and certain chemical reagents. This characteristic greatly facilitates the industrial production, long-term storage, long-distance transportation, and survival and colonization of biocontrol agents after field application, solving the technical bottlenecks of short shelf life and poor field stability found in many other microbial agents. Furthermore, Bacillus species are diverse, rich in metabolites, have a broad antibacterial spectrum, and many strains also possess plant growth-promoting functions, making them ideal candidates for achieving integrated "disease prevention-growth promotion."

[0005] Although there have been some reports on biocontrol strains for pear fire blight, there is currently a lack of highly efficient and stable domestically produced specialized biological pesticides on the market. Therefore, continuously exploring new strains with independent intellectual property rights, strong antagonistic ability, and stable control efficacy, and developing corresponding efficient fermentation processes, is of great significance for ensuring the safety of my country's pear industry. Summary of the Invention

[0006] To address the aforementioned technical limitations, this application has successfully obtained a novel *Bacillus belye* strain for controlling pear fire blight by isolating and screening soil samples from specific agricultural ecological environments. This strain exhibits strong antibacterial activity against pear fire blight pathogens. Furthermore, this application has developed an efficient and stable microbial preparation fermentation method, clarifying its application in the control of pear fire blight. This overcomes the deficiencies and defects mentioned in the background technology, providing a novel, efficient, and reliable technical solution for the green development of the fruit tree industry.

[0007] To achieve the above objectives, this application adopts the following technical solution:

[0008] The inventive point of this application is to provide a strain of Bacillus velezensis DW4-5-2 for the control of pear fire blight. This strain was deposited on October 23, 2024, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and is classified as Bacillus velezensis, with accession number CCTCC NO: M 20242308.

[0009] The second inventive point of this application is to provide a microbial inoculant, wherein the active ingredients of the microbial inoculant include the fermentation broth of Bacillus belyssus DW4-5-2, bacterial cells, metabolites or a combination thereof.

[0010] The third inventive point of this application is to provide a fermentation medium for culturing the above-mentioned Bacillus belyssus DW4-5-2, wherein the carbon source of the fermentation medium is selected as soluble starch, the nitrogen source is selected as yeast extract, and the inorganic salt is selected as potassium chloride.

[0011] Optionally, in the above-mentioned fermentation culture medium, the concentration of soluble starch is 0.7%-1.2% by mass volume, preferably 0.7%; the concentration of yeast extract is 0.25%-0.75% by mass volume, preferably 0.25%; and the concentration of potassium chloride is 0.75%-1.25% by mass volume, preferably 0.75%.

[0012] The fourth inventive point of this application is to provide a fermentation method for the above-mentioned Bacillus belye DW4-5-2, the method comprising the step of inoculating activated Bacillus belye DW4-5-2 into a fermentation medium for fermentation culture to obtain it; wherein the fermentation culture conditions are: rotation speed 180-220 r / min, temperature 30-34℃, time 60-84 h; preferably 220 r / min, 30℃, time 60 h.

[0013] Optionally, in the above fermentation method, the initial pH of the fermentation medium is 5-7, preferably 7; and the inoculum amount is 3-7% by volume, preferably 3%.

[0014] Optionally, in the above fermentation method, the carbon source of the fermentation medium is selected as soluble starch, the nitrogen source is selected as yeast extract, and the inorganic salt is selected as potassium chloride; the concentration of soluble starch is 0.7-1.2% by mass-volume percentage, preferably 0.7%; the concentration of yeast extract is 0.25%-0.75% by mass-volume percentage, preferably 0.25%; and the concentration of potassium chloride is 0.75%-1.25% by mass-volume percentage, preferably 0.75%.

[0015] The fifth inventive point of this application is to provide the application of the above-mentioned Bacillus berberis DW4-5-2, or the above-mentioned microbial agent, or the above-mentioned fermentation culture medium, or the above-mentioned fermentation method in the prevention and control of pear fire blight.

[0016] Optionally, in the above application, the crop subject to pear fire blight is a Rosaceae plant, preferably a pear tree or an apple tree.

[0017] The sixth inventive point of this application is to provide a method for preventing and controlling pear fire blight, which involves applying the fermentation broth, bacterial cells, or metabolites of the aforementioned Bacillus belye DW4-5-2 to plants or plant growth media; preferably, the application method includes foliar spraying.

[0018] Compared with the prior art, this application has the following advantages:

[0019] 1. High novelty of the strain: A new strain of Bacillus belyssus, DW4-5-2, isolated from local soil in Gansu and with completely independent intellectual property rights, was provided, enriching the microbial resource bank for the prevention and control of pear blight.

[0020] 2. High antagonistic activity: This strain exhibits strong in vitro antagonistic activity against pear fire blight pathogen, with an inhibition zone width of up to 4.06 mm.

[0021] 3. Excellent potted plant control efficacy: Potted plant experiments showed that the strain had a therapeutic control efficacy of up to 80.18% against pear fire blight after 7 days, which is comparable to the control efficacy of the chemical pesticide 6% kasugamycin. Moreover, it has a long-lasting effect and has great potential to replace or reduce the use of chemical pesticides.

[0022] 4. Thorough optimization of fermentation process: This invention not only screened the culture medium components but also systematically optimized the fermentation conditions using scientific statistical methods (Plackett-Burman and response surface methodology), identifying key influencing factors and their optimal levels. This resulted in an optimized fermentation broth with a viable cell count of 2.28 × 10⁻⁶. 9 The CFU / mL parameter provides efficient and stable technical parameters for large-scale production.

[0023] 5. Broad application prospects: This strain and its optimized fermentation process provide core technical support for the development of efficient, stable, and environmentally friendly biological pesticides for pear fire blight, which is of great significance for ensuring the green, safe, and sustainable development of my country's pear and apple industries. Attached Figure Description

[0024] Figure 1 The image shows the plate confrontation antagonistic effect of some biocontrol strains against pear fire blight pathogens, with strain DW4-5-2 showing the largest inhibition zone.

[0025] Figure 2 The figure shows the results of the screening test for fermentation medium components.

[0026] Figure 3 The figure shows the results of a single-factor experiment on the effect of different fermentation conditions (rotation speed, inoculum size, temperature, time, and initial pH) on the viable count of the fermentation broth of strain DW4-5-2.

[0027] Figure 4The figure shows the results of a potted plant test on the control effect of the antagonistic strain DW4-5-2 on pear fire blight. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0030] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0031] Example 1

[0032] 1. Materials and Methods

[0033] 1.1 Isolation and preservation of antagonistic bacteria

[0034] Bacteria in soil samples were isolated using a serial dilution method. 10 g of soil sample was weighed and placed in an Erlenmeyer flask containing 90 mL of sterile water and glass beads. The flask was incubated at 28℃ and 200 rpm for 10 min to prepare 10 μg of the sample. -1 A soil suspension of a certain concentration was allowed to stand for 10 minutes, and the supernatant was collected to prepare a solution of 10... -2 Soil suspensions of varying concentrations were serially diluted to prepare 10... -3 10 -4 and 10 -5 Three concentrations of suspensions were prepared. 200 µL of each of the three concentrations was added to LB broth (Tryptone 10 g, Yeast extract 5 g, NaCl 10 g, Agar 18 g, H2O 1000 mL) plates and spread evenly using a spreader. Each dilution was repeated three times. The plates were incubated at 28°C for 5 days. Colonies with different morphologies were selected based on their morphology, texture, and gloss and transferred to new culture media for purification. After numbering, the colonies were stored at 4°C for later use. The bacterial strains were cultured in LB liquid medium at 28°C with shaking at 200 rpm to form a bacterial suspension. This suspension was then transferred to cryovials containing 20% ​​glycerol and stored at -80°C.

[0035] 1.2 Initial and secondary screening of the antagonistic ability of isolated bacteria against pear blight pathogen.

[0036] Preparation of pear fire blight pathogen suspension: The activated single colony of pear fire blight pathogen preserved in the laboratory was inoculated into 100 ml LB liquid medium and cultured in a constant temperature shaker at 28℃ and 180 r / min for 24 h for later use.

[0037] Activation of isolated bacteria: The preserved bacterial strains were activated on LB medium for later use.

[0038] Preliminary screening of bacterial antagonistic activity against pear blight pathogen: Take 200 µL of pear blight pathogen suspension and spread it on an LB sterile plate. Draw lines at the same distance from the center of the plate to screen bacterial strains and observe whether the bacterial strains have antagonistic activity.

[0039] Re-screening of bacterial antagonistic ability against *Pyracantha fortuneana*: The plate confrontation method was used to re-screen the bacterial strains with antagonistic ability obtained in the initial screening and to test their antibacterial stability. 200 µL of *Pyracantha fortuneana* bacterial suspension was spread onto an LB agar plate. Four equidistant spots of the initially screened antagonistic bacteria were selected and inoculated, with each bacterium in triplicate. The plates were incubated at 28°C for 48 h, and the diameter of the inhibition zone and the colony diameter were measured. The inhibition radius was represented by the difference between the diameter of the inhibition zone (D) and the colony diameter (d).

[0040] 1.3 Classification and Identification of Biocontrol Strains

[0041] The selected activated biocontrol bacteria single colonies were inoculated in LB liquid culture medium and cultured for 24 h. The genome of strain DW4-5-2 was extracted using the Fast TIANamp Bacteria DNA Kit (Tiangen, Beijing).

[0042] PCR amplification was performed using universal primers 27F (SEQ ID No. 1: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID No. 2: 5'-GGTTACCTTGTTACGACTT-3') for the bacterial 16S rRNA gene. The gyrA gene of the strain was amplified by PCR using primers gyrA-F (SEQ ID No. 3: 5'-CAGTCAGGAAATGCGTACGTCCTT-3') and gyrA-R (SEQ ID No. 4: 5'-CAAGGTAATGCTCCAGGCATTGCT-3'). The gyrB gene of the strain was amplified by PCR using primers UP1 (SEQ ID No. 5: 5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3') and UP2r (SEQ ID No. 6: 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3'). A 25 μL reaction mixture was amplified using a PCR thermal cycler. The PCR product was purified and sent to Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) for sequencing. Sequencing results were compared with the NCBI database. The 16S rRNA, gyrA, and gyrB gene sequences of relevant species were downloaded from the NCBI Gene Bank database and phylogenetic studies were performed using MEGA 10.0. The 16S rRNA, gyrA, and gyrB gene sequences of strain DW4-5-2 were submitted to the NCBI database.

[0043] 1.4 Determination of the potted control effect of antagonistic strain DW4-5-2 on pear fire blight

[0044] 1.4.1 Preparation of bacterial culture

[0045] The selected antagonistic strains and pear fire blight pathogens were activated by inoculating them onto LB medium. Single activated colonies were then picked and inoculated into 100 ml of LB liquid medium, and cultured at 28°C with shaking at 180 rpm for 24 h as seed culture. This seed culture was then inoculated into LB liquid medium at a 1% inoculation rate and cultured for another 48 h. The concentrations of the pathogen and biocontrol bacteria were then diluted to 10% with sterile water. 8 CFU / mL, ready for use.

[0046] 1.4.2 Determination of potted plant efficacy

[0047] The efficacy of biocontrol strains against pear blight pathogens was determined by foliar spraying. Two-year-old potted early-ripening pear seedlings were used as inoculation material. Five treatments were set up: (I) Diluted biocontrol strain culture solution was sprayed onto the pear seedlings until the surface was moistened to dripping moisture. The seedlings were then placed in an inoculation shed to maintain humidity. After 72 hours, the pathogen solution was sprayed on top of the seedlings, and humidity was maintained. (II) The pathogen solution was sprayed first, followed by the biocontrol strain culture solution 72 hours later. (III) 6% kasugamycin was sprayed first, followed by the pathogen solution 72 hours later. (VI) The pathogen solution was sprayed first, followed by 6% kasugamycin 72 hours later. (V) Only pear blight pathogen solution was sprayed as a control, with sterile water spraying as a blank control. Each treatment consisted of 3 pots, repeated 3 times. The temperature in the inoculation shed was 28℃~32℃, and the relative humidity was ≥75%. Disease incidence was observed daily, the disease index was investigated, and the control effect was calculated. The grading standards for pear fire blight are as follows: Grade 0: No lesions on branches; Grade 1: Lesions on branches account for 1 / 3 of the length of the inoculated branch; Grade 3: Lesions on branches account for 1 / 3 to 2 / 3 of the length of the inoculated branch; Grade 5: Lesions on branches account for 2 / 3 of the length of the inoculated branch. The control efficacy of each treatment was calculated based on the disease index, and the results are as follows: Figure 4 As shown.

[0048] 1.5 Optimization of fermentation medium composition and conditions for inoculant DW4-5-2

[0049] 1.5.1 Preparation of Seed Liquid of Inoculant DW4-5-2

[0050] Strain strain DW4-5-2 was inoculated onto LB medium and incubated at 28°C for 48 h. A single colony was picked and added to 100 mL of LB medium (25% of the total volume). The culture was then shaken at 180 r / min for 24 h to prepare the seed culture.

[0051] 1.5.2 Viable cell count of fermentation broth for strain DW4-5-2

[0052] The viable cell count in the fermentation broth was determined using the dilution plating method. The specific method is as follows: Prepare sterilized test tubes containing 9 ml of distilled water. In a quiet workbench, use a pipette to add 1 mL of fermentation broth to the prepared test tube and mix well. Add 1 mL of the diluted solution to the next test tube, and so on, performing a serial dilution of the fermentation broth until a final concentration of 10⁻⁶ is achieved. -7 100 μL of bacterial suspension was added to LB plates and spread evenly using a spreader. The process was repeated three times. After incubation at 28°C for 24 h, the viable bacteria count was determined using a Xunshu MF3 series microcolony counting instrument.

[0053] 1.5.3 Single-factor experiment on fermentation medium components

[0054] LB medium formula: 5 g yeast extract, 10 g tryptone, 10 g NaCl, 17 g agar, 1000 mL distilled water.

[0055] Carbon source screening was conducted using LB broth as the basal medium. The carbon source in the medium was replaced with sucrose, glucose, lactose, fructose, corn starch, yeast extract, maltose, and soluble starch at concentrations of 5 g / L, respectively. LB broth without any added carbon source served as a control. The viable cell count in the fermentation broth of each treatment was determined. The initial conditions for shake-in fermentation were: 5% seed culture added, shake-incubated at 180 r / min and 28 ℃ for 36 h, with each treatment replicated in triplicate.

[0056] Based on the selection of soluble starch as the carbon source, nitrogen sources were screened. The nitrogen source in the fermentation medium was replaced with NH4HCO3, NH4Cl, (NH4)2SO4, yeast extract, beef extract, and peptone at concentrations of 10 g / L, respectively. LB broth without any nitrogen source was used as a control. The viable cell count in the fermentation broth of each treatment was determined. Shake fermentation conditions were consistent with those for the carbon source, and each treatment was replicated in triplicate.

[0057] Based on the screening of soluble starch and yeast extract as carbon and nitrogen sources, inorganic salts were screened. The inorganic salts in the fermentation medium were replaced with MgSO4, CuSO4, FeSO4, NaCl, CaCl2, ZnCl2, and KCl at concentrations of 5 g / L, respectively. LB medium without any added inorganic salts was used as a control. The viable cell count in the fermentation broth of each treatment was determined. The shaker fermentation conditions were consistent with those for carbon source screening, and each treatment was replicated in triplicate.

[0058] 1.5.4 Single-factor experiment on the optimal amount of fermentation medium

[0059] Based on the screening of soluble starch, yeast extract, and KCl as the carbon and nitrogen sources and inorganic salts, their dosages were further screened, with five gradients established. The concentrations were: soluble starch 0.1%, 0.3%, 0.5%, 0.7%, and 0.9%; yeast extract 0.5%, 0.75%, 1%, 1.25%, and 1.5%; and KCl 0.5%, 0.75%, 1%, 1.25%, and 1.5%.

[0060] 1.5.5 Plackett-Burman Test

[0061] Based on the results of the single-factor shake-flask fermentation optimization experiment, the Plackett-Burman experiment was used to screen out the factors that have a significant impact on the viable number of the strain from eight factors: rotation speed, inoculum size, temperature, time, initial pH, yeast extract, soluble starch and KCl. Each factor was set to two levels: the lowest (-1) and the highest (1). The experimental factors and level values ​​are shown in Table 1.

[0062] Table 1 Factor levels and codes in the Plackett-Burman experimental design

[0063]

[0064] 1.5.6 Response Surface Method for Optimizing Experimental Design

[0065] Based on the Plackett-Burman experimental design results, three factors—rotation speed, temperature, and time—were selected, with the viable cell count in the fermentation broth as the response value, to conduct a three-factor, three-level Box-Behnken experimental design (Table 2). A fitted regression model was established and analyzed based on the Box-Behnken experimental design results.

[0066] Table 2. Box-Behnken Experimental Design Factors and Level Coding

[0067]

[0068] 2 Results and Analysis

[0069] 2.1 Isolation and screening of biocontrol bacteria

[0070] A total of 109 bacterial strains were isolated from soil samples using a serial dilution method. A preliminary screening using the streak plate confrontation method identified 37 strains with antagonistic effects against *Pyrus pyrifolia*. Further screening using the plate confrontation method revealed that 8 strains showed good antagonistic effects against *Pyrus pyrifolia*, with DW4-5-2 exhibiting the best antagonistic effect, reaching an inhibition zone width of 4.06 mm. The results are shown in Table 3. Figure 1 . Figure 1 middle, Figure 1 A: DW4-5-2, Figure 1 B: MJ16-12, Figure 1 C:LX17-01 Figure 1 D: YCT16-16(1), Figure 1 E: DX1709, Figure 1 F: QS1808 Figure 1 G: GN-8-20, Figure 1 F: HZ-2-2.

[0071] Table 3. Inhibition zone width of biocontrol strains against pear fire blight pathogen.

[0072]

[0073] 2.2 Gene sequence analysis and identification of strain DW4-5-2

[0074] The genome of strain DW4-5-2 was extracted, and PCR amplification of the 16S rRNA, gyrA, and gyrB genes was performed using primer combinations 27F and 1492R, and gyrA-F and gyrA-R, respectively. The target fragments were obtained and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The 16S rRNA, gyrA, and gyrB gene sequences of strain DW4-5-2 were found to be 1435 bp, 961 bp, and 1183 bp in length, respectively. Homology comparison analysis was performed between the obtained sequences and the 16S rRNA, gyrA, and gyrB gene sequences of some strains in the NCBI database. The results showed that the similarity of the 16S rRNA, gyrA, and gyrB gene sequences of strain DW4-5-2 with those of Bacillus velezensis was 100.00%, 99.89%, and 99.75%, respectively. Based on homology comparison analysis, strain DW4-5-2 was identified as Bacillus swezeyi, with accession numbers PX908872, PX926403, and PX926404 for its 16S rRNA, gyrA, and gyrB gene sequences, respectively. It was deposited on October 23, 2024, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCM 20242308.

[0075] 2.3 Determination of the potted control effect of antagonistic strain DW4-5-2 on pear fire blight

[0076] The pot control efficacy of this biocontrol strain was determined. The experiment showed that the protective efficacy against pear seedlings at 7 days was 70.54%, and the curative efficacy was 80.18%, which was not significantly different from the efficacy of 81.19% achieved by treating with 6% kasugamycin chemical agent. At 14 days, the protective efficacy was 71.76%, and the curative efficacy was 81.16%. At 21 days, the curative efficacy still reached 79.11%, which was not significantly different from the efficacy of 80.99% achieved by chemical agent.

[0077] 2.4 Single-factor experiment on fermentation medium components and dosage

[0078] 2.4.1 Single-factor screening experiment of fermentation medium components

[0079] Different carbon sources have a significant impact on the viable cell count of the strain. Among them, the culture medium with soluble starch as the carbon source has the highest viable cell count per unit volume, which is 13.63 × 10⁻⁶. 6The CFU / mL concentration of the culture medium was adjusted to affect viable cell count in the following order: fructose > corn starch > maltose > lactose > glucose > sucrose > yeast extract. The strains utilized polysaccharides significantly more than disaccharides and monosaccharides; therefore, soluble starch was chosen as the carbon source for subsequent experiments.

[0080] When using different nitrogen sources as culture medium components, the viable cell count per unit volume of the strain, from highest to lowest, was: yeast extract, beef extract, peptone, (NH4)2SO4, NH4Cl, and NH4HCO3. The strain did not grow in the medium using NH4HCO3 as the nitrogen source, while the viable cell count in the medium using yeast extract as the nitrogen source was significantly higher than that in the other nitrogen source media (P < 0.05). The strain utilized organic nitrogen sources more efficiently than inorganic nitrogen sources. Therefore, yeast extract was selected as the nitrogen source for subsequent experiments.

[0081] Different inorganic salt culture media had a significant effect on the viable count of the strain (P < 0.05). The strain did not grow when FeSO4, ZnCl2, and CuSO4 were used as inorganic salts; the viable count reached its maximum of 8.61 × 10⁻⁶ when KCl was used as the culture medium component. 6 The CFU / mL concentration was significantly higher than that of other inorganic salt treatments. Therefore, KCl was used as the inorganic salt in the culture medium for component ratio optimization. Figure 2 )

[0082] 2.4.2 Single-factor experiment on the amount of fermentation medium used

[0083] When the soluble starch content is between 0.1% and 0.5%, the viable count of the strain gradually decreases. When it exceeds 0.5%, the viable count gradually increases, reaching a maximum of 4.67 × 10⁻⁶ at 0.9%. 8 CFU / mL, therefore, soluble starch of 0.9% was selected for subsequent shaker tests.

[0084] As the yeast extract concentration increased from 0.5% to 1.25%, the number of viable cells per unit volume of the strain gradually decreased, indicating that within a certain range, a higher yeast extract concentration is less conducive to the growth and reproduction of the strain. Therefore, a yeast extract concentration of 0.5% was selected for subsequent experiments.

[0085] As shown in the figure below, the viable bacterial count of the strain gradually increased when the concentration of inorganic salt KCl was 0.5%-1%, and gradually decreased when the concentration was 1%-1.5%. The viable bacterial count reached its maximum value of 1.94 × 10⁸ CFU / mL at 1%. Therefore, a KCl concentration of 1% was selected for subsequent experiments. Figure 2 ).

[0086] 2.5 Optimization of shake-flask fermentation conditions

[0087] 2.5.1 Single-factor experiments on fermentation conditions

[0088] Different rotation speeds had a significant effect on the viable cell count of the strain (P < 0.05), with the viable cell count reaching its maximum at 200 r / min, which was 2.18 × 10⁻⁶. 8 CFU / mL. When the rotation speed exceeds 200 r / min, the number of viable bacteria in the fermentation broth gradually decreases. Therefore, 200 r / min is selected as the optimal fermentation speed for the strain.

[0089] Different inoculum sizes had significant effects on viable cell counts (P < 0.05). Inoculum sizes of 1%, 3%, 7%, and 9% had no significant effect on viable cell counts in the fermentation broth. The viable cell count reached its maximum value of 2.46 × 10⁻⁶ at an inoculum size of 5%. 8 When the inoculum concentration is below or above 5%, the number of viable bacteria in the fermentation broth gradually decreases. Therefore, 5% is selected as the optimal inoculum concentration for the strain.

[0090] Different temperatures had a significant effect on the bacterial count of the fermentation broth (P < 0.05). The bacterial count gradually increased between 28 ℃ and 32 ℃, reaching its maximum at 32 ℃ (10.72 × 10⁻⁶). 8 Therefore, 32 ℃ was chosen as the optimal fermentation temperature for the strain, based on the CFU / mL concentration. Different temperatures significantly affected the bacterial count in the fermentation broth (P < 0.05). The bacterial count gradually increased between 28 ℃ and 32 ℃, reaching its maximum at 32 ℃, at which point the viable cell count reached 10.72 × 10⁻⁶. 8 CFU / mL, therefore 32 ℃ was chosen as the optimal fermentation temperature for the strain.

[0091] The number of viable bacteria in the strain gradually increased from 36 h to 72 h, reaching a maximum of 22.52 × 10⁻⁶ at 72 h. 8 The CFU / mL value indicates a relatively long growth cycle for the strain, therefore 72 h was selected as the optimal fermentation time for the strain.

[0092] The effect of different initial pH values ​​on the viable cell count in the fermentation broth was significant (P < 0.05). The viable cell count in the fermentation broth reached its maximum value of 11.59 × 10⁻⁶ cells / mL when the initial pH was 5. 8 When the concentration of CFU / mL exceeds the initial pH of 5, the bacterial content per unit volume of the fermentation broth gradually decreases. Therefore, the initial pH of 5 was chosen as the optimal initial pH for the fermentation culture of the strain. Figure 3 ).

[0093] 2.5.2 Plackett-Burman Test

[0094] Multiple regression analysis was performed on the data in the table using Minitab 17 software. The regression equation with viable bacteria count as the response value was obtained as follows: Y = 1.837 - 0.045X1 + 0.297X2 + 0.058X3 + 0.092X4 + 0.028X5 + 0.003X6 + 0.008X7 + 0.018X8. The effects of each factor were evaluated, and the results are shown in Tables 4 and 5.

[0095] Table 4. Plackett-Burman Experimental Design and Response Values

[0096]

[0097] Table 5. Evaluation of the effects of each factor in the Plackett-Burman trial.

[0098]

[0099] Based on the magnitude of the effect values, the key factors affecting the viable cell count per unit volume of strain 7 are, in descending order: X2 (time) > X4 (rotation speed) > X3 (temperature) > X1 (soluble starch) > X5 (yeast extract) > X8 (KCl) > X7 (pH) > X6 (inoculum size). Time, rotation speed, and temperature reached significant levels (P < 0.05), while the other five factors did not. Therefore, time, rotation speed, and temperature were selected as key factors for response surface methodology (RSM). During the optimization process, considering increasing viable cell count and saving costs, and combining the results of single-factor analysis, the following experiments were conducted using 0.9% soluble starch, 0.5% yeast extract, 1% KCl, 5% inoculum size, and an initial pH of 5.

[0100] 2.5.3 Response Surface Optimization

[0101] Based on the three key factors identified in the Plackett-Burman experiment, a Box-Behnken experimental design was conducted. The analysis of variance for the experimental design factors, response values, and regression equations is shown in Tables 6 and 7. Using Design-Expert 10.0.3, a quadratic linear regression was performed on the experimental data in the tables, yielding the quadratic multinomial regression equation: Y = 1.01 - 0.43X3 - 0.25X2 - 0.35X4 + 0.28X2X3 - 0.37X3X4 + 0.24X2X4 + 0.63X3 2 -0.13X2 2 +0.24X4 2 .

[0102] Analysis of variance was performed on the regression model. As shown in the table, the model is highly significant (P < 0.0002), and the lack-of-fit term (P = 0.1013 > 0.05) is not significant. This indicates that the regression model is reliable and can be used to optimize the viable cell count of the fermentation strain. The model's coefficient of determination R0 is also shown. 2 =0.96, indicating a high degree of fit, R = Adj 2 (0.92) and R Pre 2 The difference between (0.75) and the actual value is less than 0.2, indicating that the model's predicted value has a high degree of fit with the actual value. Therefore, this model can be used for the prediction and analysis of the number of viable bacteria in the strain.

[0103] In summary, the optimal conditions for the number of viable cells in the fermentation strain are: 220 r / min fermentation speed, 30℃ temperature, and 60.01 h fermentation time (calculated by regression equation). Considering the experimental design and actual production conditions, the optimal conditions for adjustment are 220 r / min fermentation speed, 30℃ temperature, and 60 h fermentation time. The other five factors did not reach a significant level on the number of viable cells per unit volume of fermentation broth in the Plackett-Burman experiment. Therefore, the optimal conditions for the other five factors are: 0.7%-1.2% soluble starch, 0.25%-0.75% yeast extract, 0.75%-1.25% potassium chloride, 3%-7% inoculum size, and initial pH 5.0-7.0. Considering the experimental design and actual production conditions, the optimal conditions for adjustment are: 0.7% soluble starch, 0.25% yeast extract, 0.75% potassium chloride, 3% inoculum size, initial pH 7.0, 220 r / min fermentation speed, 30℃ temperature, and 60 h fermentation time. Under these conditions, the model predicted a maximum viable cell count of 2.48 × 10⁹ CFU / mL. Validation experiments were conducted under these optimal conditions, yielding a viable cell count of 2.28 × 10⁹ CFU / mL, which is close to the model's prediction. This indicates that the model has high reliability and can be applied to the fermentation conditions of the strain.

[0104] The Box-Behnken response surface methodology and its results are shown in Table 6, and the variance analysis results of the regression model are shown in Table 7.

[0105] Table 6 Box-Behnken response surface methodology and results

[0106]

[0107] Table 7 Results of ANOVA for the Regression Model

[0108]

[0109] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A strain of Bacillus belye DW4-5-2 for controlling pear fire blight, characterized in that, The strain was deposited at the China Center for Type Culture Collection (CCTCC) on October 23, 2024, and classified as *Bacillus belyssae*. Bacillus velezensis (The accession number is CCTCC NO: M 20242308).

2. A microbial inoculant, characterized in that, The active ingredients of the microbial agent include the fermentation broth, bacterial cells, metabolites, or a combination thereof of Bacillus belyssus DW4-5-2 as described in claim 1.

3. A fermentation medium for culturing Bacillus belye DW4-5-2 as described in claim 1, characterized in that, The carbon source of the fermentation medium is soluble starch, the nitrogen source is yeast extract, and the inorganic salt is potassium chloride.

4. The fermentation medium according to claim 3, characterized in that, In the fermentation medium, the concentration of soluble starch is 0.7%-1.2% by mass volume, preferably 0.7%; the concentration of yeast extract is 0.25%-0.75% by mass volume, preferably 0.25%; and the concentration of potassium chloride is 0.75%-1.25% by mass volume, preferably 0.75%.

5. A fermentation method for Bacillus belye DW4-5-2 according to claim 1, characterized in that, The method includes the step of inoculating activated Bacillus belye DW4-5-2 into a fermentation medium for fermentation culture to obtain it; wherein the fermentation culture conditions are: rotation speed 180-220 r / min, temperature 30-34℃, time 60-84 h; preferably 220 r / min, 30℃, time 60 h.

6. The fermentation method according to claim 5, characterized in that, The initial pH of the fermentation medium is 5.0-7.0, preferably 7.0; the inoculum amount is 3-7% by volume, preferably 3%.

7. The fermentation method according to claim 5, characterized in that, The carbon source of the fermentation medium is selected as soluble starch, the nitrogen source is selected as yeast extract, and the inorganic salt is selected as potassium chloride; the concentration of soluble starch is 0.7%-1.2% by mass-volume percentage, preferably 0.7%; the concentration of yeast extract is 0.25%-0.75% by mass-volume percentage, preferably 0.25%; and the concentration of potassium chloride is 0.75%-1.25% by mass-volume percentage, preferably 0.75%.

8. The application of Bacillus belyssus DW4-5-2 as described in claim 1, or the microbial agent as described in claim 2, or the fermentation medium as described in any one of claims 3-4, or the fermentation method as described in any one of claims 5-7, in the prevention and control of pear fire blight.

9. The application according to claim 8, characterized in that, The target crop for pear fire blight is a plant of the Rosaceae family, preferably a pear or apple tree.

10. A method for preventing and controlling pear blight, characterized in that, The fermentation broth, bacterial cells, or metabolites of Bacillus belye DW4-5-2 as described in claim 1 are applied to plants or plant growth media; preferably, the application method includes foliar spraying.

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