A bacillus altitudinis microbial inoculant, a preparation method thereof and application thereof in preventing and treating apple canker

Abstract

This invention provides a Bacillus hygroscopicus inoculant, its preparation method, and its application in the control of apple rot disease, relating to the field of microbiology. The Bacillus hygroscopicus inoculant provided by this invention contains Bacillus hygroscopicus M5 with preservation number CGMCC No. 37795. M5 is a strain possessing phenolic acid degradation, biofilm formation, and antagonistic functions against apple rot pathogens. Its phenolic acid degradation function helps degrade phenolic acid autotoxic substances such as ferulic acid and p-hydroxybenzoic acid in apple orchard soil, eliminating cofactors for pathogen infection. Its biofilm formation function enhances the strain's colonization ability on apple branch surfaces, thereby strengthening its antagonistic effect against apple rot pathogens. This strain exerts a multi-faceted effect in controlling apple rot disease and has significant application value. The Bacillus hygroscopicus inoculant can be applied to apple branches by spraying to prevent infection by apple rot pathogens.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a Bacillus hygroscopicus agent, its preparation method, and its application in the prevention and control of apple rot. Background Technology

[0002] Apple rot is caused by the fungus *Pseudomonas aeruginosa* (also known as apple black rot fungus). The waltz of evil Apple rot is a major global disease affecting apple-producing regions, primarily damaging the bark of apple tree branches and trunks. This leads to weakened trees, drastically reduced yields, and in severe cases, the death of the entire tree, causing significant economic losses to fruit growers. Currently, the control of apple rot still relies mainly on chemical pesticides. However, the long-term and excessive use of chemical pesticides can lead to increased pathogen resistance, excessive pesticide residues in soil and fruit, and ecological damage, which does not meet the requirements of green agriculture and food safety development.

[0003] Biological control, with its advantages of being environmentally friendly, leaving no residue, and being less likely to induce resistance, has become a research hotspot for the control of apple rot. Bacillus strains, due to their strong resistance, rapid reproduction rate, and ability to produce various antimicrobial substances such as antimicrobial peptides and siderophores, are widely used in the biological control of plant diseases. Highland Bacillus (… Altitude Bacillus As an important member of the Bacillus genus, it has been reported in the prior art to have functions such as promoting plant growth, remediating heavy metal contaminated soil, and purifying marine environment, but there are no reports on the application of highland Bacillus in the prevention and control of apple rot.

[0004] Long-term continuous cropping in apple orchards leads to the accumulation of large amounts of phenolic acids (such as ferulic acid and p-hydroxybenzoic acid) in the soil. These substances not only inhibit apple root growth but also promote the mycelial growth and infection of apple rot pathogens, making them a significant contributing factor to the high incidence of apple rot. Simultaneously, the colonization ability of biocontrol strains on the plant surface directly affects the control effect, and the formation of biofilms can significantly enhance the adhesion and colonization ability of strains, prolonging their effective period. Most existing biocontrol strains only possess a single antagonistic function, lacking phenolic acid degradation capabilities and efficient colonization, resulting in poor control effects and short-lasting efficacy.

[0005] Therefore, developing a microbial agent that combines phenolic acid degradation, biofilm generation, and antagonism against apple rot is of great practical significance for solving the problem of green control of apple rot. Summary of the Invention

[0006] The purpose of this invention is to provide a microbial agent that combines phenolic acid degradation, biofilm formation, and antagonistic function against apple rot, thereby providing a Bacillus hygroscopicus agent, its preparation method, and its application in the prevention and control of apple rot.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a strain of Bacillus hygroscopicus (B. hygroscopicus). Altitude Bacillus The specific Bacillus hygroscopicus mentioned is Bacillus hygroscopicus M5, with accession number CGMCC No. 37795.

[0009] The Bacillus hygroscopicus provided by this invention ( Altitude Bacillus M5 was deposited on February 28, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37795.

[0010] Secondly, this invention provides a Bacillus hygroscopicus inoculum containing Bacillus hygroscopicus with accession number CGMCC No. 37795 ( Altitude Bacillus M5.

[0011] Furthermore, the bacterial activity of the *Bacillus hygroscopicus* inoculant is 4.52 × 10⁻⁶. 8 ~5.51×10 8 CFU / mL.

[0012] Furthermore, the Bacillus hygroscopic agent is derived from Bacillus hygroscopicus with accession number CGMCC No. 37795 (…). Altitude Bacillus M5 was obtained by culturing in fermentation medium.

[0013] Furthermore, based on a volume of 1 L of solvent water, the fermentation medium comprises the following nutrient components: 0.2-0.8 g yeast powder, 0.2-0.5 g soybean meal powder, 0.05-0.2 g dipotassium hydrogen phosphate, 0.1-1 g sodium chloride, 0.02-0.06 g magnesium sulfate, and 0.01-0.04 g potassium chloride, with a natural pH.

[0014] Furthermore, based on a volume of 1 L of solvent water, the fermentation medium comprises the following nutrient components: 0.5 g yeast powder, 0.4 g soybean meal powder, 0.15 g dipotassium hydrogen phosphate, 0.5 g sodium chloride, 0.05 g magnesium sulfate, and 0.03 g potassium chloride, with a natural pH.

[0015] Furthermore, the culture temperature was 28–35 °C, the rotation speed was 180–200 rpm, and the time was 36–48 h; the bacterial viability was 4.52 × 10⁻⁶. 8 ~5.51×10 8 CFU / mL.

[0016] Furthermore, the culture temperature was 30 ℃, the rotation speed was 200 rpm, and the time was 48 h; the bacterial viability was 4.95 × 10⁻⁶. 8 CFU / mL.

[0017] Thirdly, the present invention provides a method for preparing a Bacillus cereus inoculum, comprising the following steps: S1. Highland Bacillus with accession number CGMCC No. 37795 ( Altitude Bacillus M5 cells were activated onto NA solid medium to obtain M5 cells for later use. S2. Pick M5 cells and put them into LB liquid medium to culture and obtain M5 seed culture for later use; S3. Inoculate the M5 seed culture into the fermentation medium and ferment to obtain the Bacillus hygroscopica inoculum.

[0018] Further, in step S1, the activation conditions are: incubation at 28~35 ℃ for 48~50 h, preferably at 30 ℃ for 48 h.

[0019] Further, in step S2, the culture conditions are: 100-150 mL / 500 mL liquid volume; 28-35 ℃, 180-200 rpm shaking until the logarithmic phase. Preferably, the liquid volume is 100 mL / 500 mL; 30 ℃, 200 rpm shaking until the logarithmic phase.

[0020] Furthermore, in step S3, the fermentation culture conditions are: 28~35 ℃, 180~200 rpm for 36~48 h, preferably 30 ℃, 200 rpm for 48 h.

[0021] Further, in step S3, with a volume of 1 L of solvent water, the fermentation culture medium includes the following nutrient components: 0.2-0.8 g yeast powder, 0.2-0.5 g soybean meal powder, 0.05-0.2 g dipotassium hydrogen phosphate, 0.1-1 g sodium chloride, 0.02-0.06 g magnesium sulfate, and 0.01-0.04 g potassium chloride, with a natural pH.

[0022] Further, in step S3, with a volume of 1 L of solvent water, the fermentation culture medium includes the following nutrient components: 0.5 g yeast powder, 0.4 g soybean meal powder, 0.15 g dipotassium hydrogen phosphate, 0.5 g sodium chloride, 0.05 g magnesium sulfate and 0.03 g potassium chloride, with a natural pH.

[0023] Furthermore, the bacterial activity of the *Bacillus hygroscopicus* inoculant is 4.52 × 10⁻⁶. 8 ~5.51×10 8 CFU / mL, preferably 4.95 × 10⁻⁶. 8 CFU / mL.

[0024] Fourthly, the present invention provides the application of the aforementioned Bacillus hygroscopicus, the aforementioned Bacillus hygroscopicus inoculum, and the Bacillus hygroscopicus inoculum obtained by the aforementioned preparation method in producing biofilms, degrading phenolic acid autotoxic substances, inhibiting pathogens, or preventing and controlling plant diseases.

[0025] Furthermore, the phenolic acid autotoxic substances include at least one of ferulic acid, p-hydroxybenzoic acid, benzoic acid, amygdalin, and limonene.

[0026] Furthermore, the pathogens include Fusarium oxysporum (… Fusarium oxysporum ), Apple black rot fungus ( The waltz of evil ) and Alternaria appleii ( Alternaria blight At least one of the following.

[0027] Furthermore, the plant diseases include at least one of Fusarium wilt, apple rot, and apple spot disease caused by Fusarium oxysporum.

[0028] Fifthly, the present invention provides the application of the Bacillus hygroscopicus agent obtained by the preparation method described above in the prevention and control of apple branch rot disease.

[0029] Furthermore, the Bacillus hygroscopicus agent is diluted 100 to 300 times and then sprayed onto the surface of apple branches.

[0030] The technical solution provided by this invention has the following advantages: 1. Bacillus hygroscopicus M5 can degrade phenolic substances such as ferulic acid, p-hydroxybenzoic acid, benzoic acid, amygdalin, and limonene, inhibiting the promoting effect of these substances on the mycelial growth and infection of the pathogen of apple rot.

[0031] 2. Bacillus hygroscopicus M5 has the ability to produce biofilms. The production of biofilms can significantly enhance the adhesion and colonization ability of the strain and prolong its action cycle.

[0032] 3. The inhibitory effects of *Bacillus hygroscopicus* M5 and its volatile antibacterial substances on *Fusarium oxysporum* (… Fusarium oxysporum ), Apple black rot fungus ( The waltz of evil ) and Alternaria appleii ( Alternaria blight It has a certain antagonistic ability and can inhibit the growth of pathogens.

[0033] 4. The inoculum of Bacillus hygroscopicus M5 can be applied to apple branches by spraying, allowing the strain to colonize the branches and thus prevent infection by apple rot pathogens. Verification has shown that the inoculum of Bacillus hygroscopicus M5 achieves a 72.2% preventive effect against apple rot disease.

[0034] In summary, the Bacillus hygroscopicus M5 strain provided by this invention is a strain that combines phenolic acid degradation, biofilm formation, and antagonistic activity against the pathogen of apple rot. The phenolic acid degradation function helps degrade phenolic acid autotoxic substances such as ferulic acid and p-hydroxybenzoic acid in apple orchard soil, eliminating cofactors for pathogen infection. The biofilm formation function enhances the strain's colonization ability on apple branch surfaces, thereby strengthening its antagonistic effect against the pathogen of apple rot. This strain exerts a multi-faceted effect in preventing and controlling apple rot, and has significant application value. Attached Figure Description

[0035] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 This is a plate culture diagram of Bacillus hygroscopicus M5 provided by the present invention; Figure 2 This is a microscope image of Bacillus hygroscopicus M5 provided by the present invention; Figure 3 The temperature tolerance range detection diagram of Bacillus hygroscopicus M5 provided by this invention; Figure 4 This is a biofilm detection diagram of Bacillus hygroscopicus M5 provided by the present invention; Figure 5 This is a diagram showing the growth of Bacillus hygroscopicus M5 provided by the present invention on a qualitative detection medium containing ferulic acid, p-hydroxybenzoic acid, benzoic acid, amygdalin and limonene. Figure 6 This is a plate confrontation diagram of Bacillus hygroscopicus M5 with Fusarium oxysporum, Alternaria macrantha, and Alternaria macrantha provided by the present invention. Figure 7 The diagram shows the antibacterial effect of the volatile antibacterial substance of Bacillus hygroscopicus M5 provided by this invention on Fusarium oxysporum, Alternaria alternata, and Alternaria macrantha. Figure 8 This is a graph showing the in vivo control effect of the Bacillus hygroscopicus agent provided by this invention against apple rot.

[0037] The Bacillus hygroscopicus provided by this invention ( Altitude Bacillus M5 was deposited on February 28, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37795. Detailed Implementation

[0038] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0039] The culture media and their composition or preparation methods involved in the examples are as follows: NA solid medium: 10 g peptone, 3 g beef meal, 5 g NaCl, 20 g agar, 1 L distilled water, pH 7.2.

[0040] LB liquid medium: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 20 g agar, 1 L water, pH 7.

[0041] Qualitative detection medium containing p-hydroxybenzoic acid: 1000 mg p-hydroxybenzoic acid, 5 g peptone, 0.5 g ammonium sulfate, 20 g compound inorganic salt (NaCl∶KCl∶MgCl2=10∶1∶1, w / w / w), 1000 mL water, pH 7.0.

[0042] Qualitative detection medium containing ferulic acid: 1000 mg ferulic acid, 5 g peptone, 0.5 g ammonium sulfate, 20 g compound inorganic salt (NaCl∶KCl∶MgCl2=10∶1∶1, w / w / w), 1000 mL water, pH 7.0.

[0043] Qualitative detection medium containing benzoic acid: 1000 mg benzoic acid, 5 g peptone, 0.5 g ammonium sulfate, 20 g compound inorganic salt (NaCl∶KCl∶MgCl2=10∶1∶1, w / w / w), 1000 mL water, pH 7.0.

[0044] Qualitative detection medium for amygdalin: 1000 mg amygdalin, 5 g peptone, 0.5 g ammonium sulfate, 20 g compound inorganic salt (NaCl∶KCl∶MgCl2=10∶1∶1, w / w / w), 1000 mL water, pH 7.0.

[0045] Qualitative detection medium containing limonene: 1000 mg limonene, 5 g peptone, 0.5 g ammonium sulfate, 20 g compound inorganic salt (NaCl∶KCl∶MgCl2=10∶1∶1, w / w / w), 1000 mL water, pH 7.0.

[0046] PDA medium: 200 g potato (peeled and diced), 20 g glucose, 20 g agar, 1000 mL distilled water.

[0047] Fermentation medium: 0.5 g yeast powder, 0.4 g soybean meal powder, 0.15 g dipotassium hydrogen phosphate, 0.5 g sodium chloride, 0.05 g magnesium sulfate, 0.03 g potassium chloride, natural pH value.

[0048] Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. All raw materials or instruments used are commercially available conventional products, including but not limited to those used in the embodiments of this application.

[0049] Example 1: Screening of strains Rhizosphere soil samples were taken from apple trees in an orchard in Shanhaiguan District, Qinhuangdao City, Hebei Province, and bacterial strains were screened and isolated using the following method: 10 g of soil sample was weighed and added to 100 mL of water, shaken at 200 rpm for 30 min, and 5 mL of the sample solution was added to 45 mL of sterile water to obtain 10 strains. -2 The diluted solution was diluted stepwise to obtain 10. -4 10 -5 10 -6 The diluted solution was spread evenly on NA solid medium plates and incubated at 30 °C for 2 days. Single colonies were picked and cultured on NA solid medium for another 2 days at 30 °C. Colony morphology was observed, and bacterial morphology was examined using an optical microscope. The strain obtained during this screening process was named M5, and another strain obtained during this screening process was named M1.

[0050] like Figure 1 As shown, the colonies of strain M5 are milky white, irregularly round, with wavy edges and a rough surface; Figure 2 As shown, strain M5 has rod-shaped cells that can form round spores, which are terminal or proximal to the apex.

[0051] Example 2: Identification of the strain Bacterial cells of strain M5 were collected, and genomic DNA was extracted using a TRAN genomic DNA extraction kit. Primers were designed based on the most conserved sequence in bacterial 16S rDNA: primer 27F: 5'-AGA GTT TGA TCC TGG CTCA-3'; primer 1492R: 5'-GGT TAC CTT GTT ACG ACTT-3', synthesized by Beijing Liuhe BGI Genomics Co., Ltd. PCR reaction conditions: 94 ℃ for 4 min, followed by 30 cycles of 94 ℃ for 30 s, 60 ℃ for 30 s, and 72 ℃ for 30 s, with a final extension at 72 ℃ for 10 min. The PCR amplification product was ligated to a plasmid at 25 ℃ for 15 min, followed by a 45 s heat shock in a water bath and transformation into competent *E. coli* cells for cloning. Strains containing the target gene were obtained through blue-white screening and sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing.

[0052] After bidirectional splicing of the sequencing results, the obtained 16S rDNA sequence was compared on the NCBI website, and the strain M5 was identified as Bacillus hygroscopicus (B. hygroscopicus). Altitude Bacillus The homology was 100%. The identification result of strain M1 was also Bacillus hygroscopicus (Bacillus). Altitude Bacillus ).

[0053] The 16S rDNA sequence of strain M5 is shown in SEQ ID No. 1 of the sequence listing: GCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCTGCGAGACCGCAAGGTTTAGCCAATCCCACAAATCTGTTCT CAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGCAACACCCGAAGTCGGTGAGGTAACCTTTAGGAGCCAGCCG Highland Bacillus ( Altitude Bacillus M5 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 28, 2026. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC No. 37795.

[0054] Example 3: Physiological and biochemical characteristics of Bacillus hygroscopicus M5 Physiological and biochemical identification of Bacillus hygroscopicus M5 was performed with reference to the "Manual of Systematic Identification of Common Bacteria" and "Bergey's Manual of Bacterial Identification (8th Edition)". The results are shown in Table 1.

[0055] Table 1. Physiological and biochemical characteristics of Bacillus cereus M5

[0056] Note: + indicates a positive result; - indicates a negative result.

[0057] Example 4: Growth characteristics of Bacillus hygroscopicus M5 I. Experimental Methods 1. Salt tolerance test Select appropriate amounts of bacterial cells from strains M1 and M5 and place them in sterile water to prepare bacterial suspensions. Inoculate these suspensions into LB liquid medium at a 1% inoculum concentration. Adjust the salt content (NaCl concentration) of the medium to 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, and 20%, respectively. Incubate at 30 °C with shaking at 200 rpm for 1 day, and measure the OD (Organic Degradation). 600 Value (LB liquid medium without inoculation was used as a blank control and zeroed).

[0058] 2. Acid and alkali resistance test Appropriate amounts of bacterial strains M1 and M5 were respectively picked and placed into sterile water to prepare bacterial suspensions. These suspensions were then inoculated into LB liquid medium at a 1% inoculum rate. The pH values ​​of the medium were adjusted to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, respectively. The medium was incubated at 30 °C with shaking at 200 rpm for 1 day, and the OD values ​​were measured. 600 Value (LB liquid medium without inoculation was used as a blank control and zeroed).

[0059] 3. Growth temperature range detection Strawberry strain M5 was streaked onto NA solid medium and cultured at 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, and 60 ℃ for 2 days, and its growth was observed.

[0060] II. Experimental Results 1. Salt tolerance The results of the salt tolerance test of Bacillus hygroscopicus M1 and M5 are shown in Table 2.

[0061] Table 2. Results of salt tolerance tests for Bacillus hygroscopicus M1 and M5

[0062] Note: --- indicates that the strain does not grow. As shown in Table 2, strain M5 can grow in culture media with salt concentrations ranging from 1% to 20%, especially with a salt concentration tolerance of up to 20%, while strain M1 cannot grow in culture media with a salt concentration of 16%. The experimental comparison results show that strain M5 has significantly better salt tolerance than M1.

[0063] 2. Acid and alkali resistance The results of acid and alkali resistance tests for Bacillus hygroscopicus M1 and M5 are shown in Table 3.

[0064] Table 3. Results of acid and alkali resistance tests for Bacillus hygroscopicus M1 and M5

[0065] Note: --- indicates that the strain does not grow. As shown in Table 3, strain M5 can tolerate a pH range of 1 to 14, and can grow within this range, demonstrating high acid and alkali tolerance. In contrast, strain M1 can tolerate a pH range of 3 to 11. The experimental results show that strain M5 has significantly better acid and alkali tolerance than strain M1.

[0066] 3. Growth temperature range like Figure 3 As shown, the growth temperature of Bacillus hygroscopicus M5 ranges from as low as 10 ℃ to as high as 60 ℃, with the optimal growth temperature being 30 ℃, exhibiting good resistance to both low and high temperatures.

[0067] Example 5: Biofilm production capability of Bacillus cereus M5 I. Experimental Methods Two activated strains, M1 and M5, were inoculated into LB liquid medium and shaken at 30 ℃ and 200 rpm to the logarithmic phase. They were then transferred to test tubes containing 6 mL of LB liquid medium at an inoculation rate of 1% (v / v) as the experimental group. The control group was inoculated with an equal amount of LB liquid medium and incubated at 30 ℃ for 24 h. The presence of biofilm on the liquid surface was then observed.

[0068] II. Experimental Results like Figure 4 As shown (from left to right: control group, M1 experimental group, M5 experimental group), after 24 h of static culture, strain M5 produced a biofilm with a higher density on the surface of LB liquid medium than the M1 experimental group (as indicated by the red arrow in the figure). This demonstrates its stronger biofilm-producing ability, which can more effectively isolate roots from pathogens and plays a crucial role in the prevention and control of various soil-borne diseases. While strain M1 also has the ability to produce biofilm, its ability is relatively weak.

[0069] Example 6: Ability of Bacillus hygroscopicus M5 to degrade phenolic acid autotoxic substances I. Experimental Methods Strawberry strain M5 was streaked onto a qualitative detection medium containing phenolic acid autotoxins (ferulic acid, p-hydroxybenzoic acid, benzoic acid, amygdalin, and limonene), and incubated at 30 °C for 3 days to observe its growth status.

[0070] II. Experimental Results As shown in Table 4 and Figure 5 As shown, strain M5 can grow on a qualitative detection medium containing ferulic acid, p-hydroxybenzoic acid, benzoic acid, amygdalin and limonene, indicating that it can degrade and utilize phenolic acid substances, thereby reducing the harm to crop growth caused by the accumulation of autotoxic substances in the soil, and also helping to alleviate crop continuous cropping obstacles to some extent.

[0071] Table 4 Detection of Autotoxic Substances from Degraded Phenolic Acids

[0072] Note: + indicates that the strain can grow on the corresponding qualitative detection medium.

[0073] Example 7: Antagonistic ability of Bacillus cereus M5 against pathogens I. Experimental Methods 1. Flat Plate Standoff Experiment Fusarium oxysporum ( Fusarium oxysporum ), Apple black rot fungus ( The waltz of evil) and Alternaria appleii ( Alternaria blight Mycelial discs (6 mm in diameter) were inoculated into PDA agar plates. One mycelial disc was inoculated in the center of each plate. Strain strain M5 was inoculated 2.5 cm to the right of each mycelial disc as the experimental group. The control group consisted of a mycelial disc without inoculation on the right side. The plates were incubated at 28 °C for 6 days. The inhibition rate of strain M5 against the pathogen was calculated using the following formula: Inhibition rate = (Pathogen colony radius - Inhibition band length) / Pathogen colony radius × 100% 2. Experiment on the antibacterial effect of volatile antibacterial substances PDA medium was poured onto the upper plate, and the pathogen Fusarium oxysporum (…) Fusarium oxysporum ), Apple black rot fungus ( The waltz of evil ) and Alternaria appleii ( Alternaria blight Inoculate PDA culture medium plates with bacterial cakes. Pour NA solid medium into the lower plate and streak strains M1 and M5 onto each plate. Seal the interfaces of the upper and lower plates with sealing film. Perform three replicates for each strain. The NA solid medium in the lower plate, without inoculation, serves as the control group. Calculate the inhibition rate using the following formula: Inhibition rate = (Diameter of pathogen in control group - Diameter of pathogen in experimental group) / Diameter of pathogen in control group × 100% II. Experimental Results 1. Results of the flat plate confrontation experiment Strain M5 against Fusarium oxysporum ( Fusarium oxysporum ), Apple black rot fungus ( The waltz of evil ) and Alternaria appleii ( Alternaria blight The results of the flat plate confrontation experiment are as follows: Figure 6 As shown in the figure (the left column is the control group and the right column is the experimental group), the calculated inhibition rates were 56.5%, 80.8%, and 68.9%, respectively. It can be seen that strain M5 has a high inhibition rate against the above three pathogens.

[0074] 2. Results of the experiment on the antibacterial effect of volatile antibacterial substances The volatile antibacterial substances produced by strains M1 and M5 are effective against Fusarium oxysporum ( Fusarium oxysporum ), Apple black rot fungus ( The waltz of evil ) and Alternaria appleii ( Alternaria blight The antibacterial effect of ) is as follows Figure 7As shown (the first row targets Fusarium oxysporum, the second row targets Chlorella vulgaris, the third row targets Alternaria maculata, the first column is the control group, the second column is the experimental group of strain M1, and the third column is the experimental group of strain M5), the calculated inhibition rates of strain M5 against the three pathogens were 52.3%, 42.3%, and 43.5%, respectively, while the inhibition rates of strain M1 against the three pathogens were 40.3%, 25.6%, and 33.3%, respectively. This demonstrates that the antibacterial effect of the volatile antibacterial substances in strain M5 is significantly better than that in strain M1.

[0075] Example 8: Preparation of Bacillus hygroscopic inoculum This embodiment provides a method for preparing Bacillus hygroscopicus inoculum, the specific steps of which are as follows: S1. Activate Bacillus hygroscopicus M5 onto NA solid medium and incubate at 30 ℃ for 48 h to obtain M5 cells for later use; S2. Pick M5 cells into LB liquid medium, with a volume of 100 mL / 500 mL, shake at 30 ℃ and 200 rpm until the logarithmic phase to obtain M5 seed culture, for later use; S3. Inoculate the M5 seed culture into the fermentation medium and incubate at 30 ℃ and 200 rpm for 48 h to obtain the Bacillus hygroscopicus inoculum, with a cell viability of 4.95 × 10⁻⁶. 8 cfu / mL.

[0076] Example 9: Effect of applying Bacillus hygroscopicus inoculant to apple detached branches I. Experimental Methods The antagonistic activity of the Bacillus hygroscopicus agent prepared in Example 8 against apple rot was determined using the in vitro branch inoculation method: Dilute the Bacillus subtilis inoculant 100 times with water and set aside. Select healthy three-year-old Daphne odora branches, cut them into 10 cm twigs, disinfect them sequentially with sodium hypochlorite and alcohol, rinse with sterile water, and air dry. Seal both ends of the branches with paraffin wax. After sterilizing with a sterile punch, make a 6 mm wound behind the branching point in the middle of the apple branch. Spray the apple branches with the diluted inoculant solution (100 µL per branch), then apply the apple black rot fungus (…). The waltz of evilThe fungal cake (6 mm in diameter) was applied to the wound, wrapped with sterile absorbent cotton soaked in sterile water, and then wrapped with plastic wrap. This was the treatment group. Branches that were sprayed with blank fermentation medium after injury and then inoculated with apple rot fungal cake served as the positive control group. Branches that were sprayed with only blank fermentation medium after injury and not inoculated with apple rot fungal cake served as the negative control group. All branches from the treatment group, positive control group, and negative control group were placed in a 25 ℃ environment for 16 h light / 8 h dark humidity incubation. After 7 days of incubation, the size of the lesions (longitudinal length) was measured, and the inhibition rate was calculated using the following formula: Antibacterial rate = (Average lesion size in control group - Average lesion size in treatment group) / Average lesion size in control group × 100% II. Experimental Results like Figure 8 As shown, the negative control group branches did not develop the disease, the positive control group branches were severely affected with large lesion areas, while the treated group branches had very small lesion areas. This indicates that the Bacillus hygroscopicus agent has a significant antagonistic effect on detached branches of apple rot pathogens. The calculated inhibition rate reached 72.2%.

[0077] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A type of highland Bacillus ( Bacillus altitudinis The bacterial agent is characterized in that, Contains Bacillus geysiensis M5 with accession number CGMCC No.37795.

2. The Bacillus cereus inoculant according to claim 1, characterized in that, The bacterial activity of the Bacillus hygroscopicus agent was 4.52 × 10⁻⁶. 8 ~5.51×10 8 CFU / mL.

3. A method for preparing a Bacillus cereus inoculum, characterized in that, Includes the following steps: S1. Activate Bacillus geysiensis M5 with preservation number CGMCC No.37795 onto NA solid medium to obtain M5 cells for later use; S2. Pick M5 cells and put them into LB liquid medium to culture and obtain M5 seed culture for later use; S3. Inoculate the M5 seed culture into the fermentation medium and ferment to obtain the Bacillus hygroscopica inoculum.

4. The method for preparing the Bacillus cereus inoculum according to claim 3, characterized in that, At least one of the following conditions must be met: (1) In step S1, the activation conditions are: 28~35 ℃ for 48~50 h; (2) In step S2, the culture conditions are: 100~150 mL / 500 mL of liquid; 28~35 ℃, 180~200 rpm shaking to the logarithmic phase. (3) In step S3, the fermentation culture conditions are: 28~35 ℃, 180~200 rpm for 36~48 h; (4) In step S3, the fermentation culture medium, with a volume of 1 L of solvent water, includes the following nutrient components: 0.2-0.8 g yeast powder, 0.2-0.5 g soybean meal powder, 0.05-0.2 g dipotassium hydrogen phosphate, 0.1-1 g sodium chloride, 0.02-0.06 g magnesium sulfate and 0.01-0.04 g potassium chloride, with a natural pH.

5. The method for preparing the Bacillus cereus inoculum according to claim 3, characterized in that, At least one of the following conditions must be met: (1) In step S1, the activation conditions are: 30 ℃ for 48 h; (2) In step S2, the culture conditions are: 100 mL / 500 mL liquid volume; shaking at 30 ℃ and 200 rpm until the logarithmic phase; (3) In step S3, the fermentation culture conditions are: 30 ℃, 200 rpm for 48 h; (4) In step S3, the fermentation culture medium, with a volume of 1 L of solvent water, includes the following nutrient components: 0.5 g yeast powder, 0.4 g soybean meal powder, 0.15 g dipotassium hydrogen phosphate, 0.5 g sodium chloride, 0.05 g magnesium sulfate and 0.03 g potassium chloride, with a natural pH.

6. The application of the Bacillus hygroscopicus agent according to claim 1 or 2, or the Bacillus hygroscopicus agent prepared by any one of claims 3 to 5, in inhibiting pathogens or preventing plant diseases.

7. The application according to claim 6, characterized in that, The pathogens include Fusarium oxysporum (… Fusarium oxysporum ), Apple black rot fungus ( Valsa mali ) and Alternaria appleii ( Alternaria mali At least one of the following.

8. The application according to claim 6, characterized in that, The plant diseases include at least one of Fusarium wilt, apple rot, and apple spot disease caused by Fusarium oxysporum.

9. The application of the Bacillus hygroscopicus agent obtained by the preparation method according to any one of claims 3 to 5 in the prevention and control of apple branch canker infection.

10. The application according to claim 9, characterized in that, The Bacillus hygroscopicus agent was diluted 100 to 300 times and then sprayed onto the surface of apple branches.

Images