Bacillus amyloliquefaciens and application thereof in preventing and treating olives anthracnose

Abstract

This invention belongs to the field of agricultural microbiology technology, specifically relating to a strain of *Bacillus amyloliquefaciens* and its application in controlling lenticular spot disease in olives. The *Bacillus amyloliquefaciens* disclosed in this invention (…) Bacillus amyloliquefaciens This strain was deposited on April 22, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 37475. The 16S rDNA sequence of this strain is shown in SEQ ID NO:1, and it possesses nitrogen-fixing and phosphorus-solubilizing abilities, exhibiting activity against the pathogen of olive peacock spot disease (…). Cycloconium oleaginum It exhibits significant antagonistic effects, with an inhibition rate of 94.8% in a three-point confrontation test and a field prevention effect of 64.63%. This invention also provides a microbial agent containing this strain and a method for preparing its fermentation broth. Foliar spraying can effectively reduce the disease index of olive peacock spot disease, exhibiting environmental friendliness and significant control efficacy, making it suitable for green control systems for olives.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology technology, specifically relating to a strain of Bacillus amyloliquefaciens (B. amyloliquefaciens). Bacillus starch liquefier ) and its microbial agents, and their role in the prevention and control of olive peacock spot disease ( Cycloconium oleaginum Applications in ). Background Technology

[0002] olive ( European olive tree Olive (L.) is an important woody oilseed crop in my country, and the olive oil extracted from its fruit is known as "liquid gold." Olive peacock spot disease is caused by a fungus. Cycloconium oleaginum This disease, a significant foliar infection, primarily affects leaves but can also infect fruits and branches. Currently, control of olive lenticel spot disease mainly relies on chemical pesticides, such as mancozeb, difenoconazole, carbendazim, and triadimefon. However, the long-term and excessive use of chemical pesticides has led to increasingly serious problems such as increased pathogen resistance, excessive pesticide residues, environmental pollution, and disruption of the ecological balance. Furthermore, olive cultivation is mostly located on slopes or in water source protection areas, where the use of chemical pesticides is strictly limited.

[0003] Bacillus ( Bacillus spp. Due to its ability to produce heat-resistant and stress-resistant spores, ease of large-scale production, environmental friendliness, and combined growth-promoting and control effects, *Bacillus amyloliquefaciens* has become a hot topic in biological control research. B. starch liquefier It can produce lipopeptide antibiotics (such as iturin, fengycin, and surfcatin), protein-based antibacterial substances, and chitinases, exhibiting significant antagonistic activity against various plant pathogens. However, there are currently few reports on biocontrol Bacillus strains targeting olive lenticel spot disease, and systematic field application data are lacking. Therefore, screening for... C. oleaginous The development of microbial pesticides based on Bacillus species with highly effective antagonistic effects is of great significance for promoting green prevention and control of olive diseases. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a strain of Bacillus amyloliquefaciens and its application in the prevention and control of lenticular spot disease in olives. The Bacillus amyloliquefaciens disclosed in this invention (… Bacillus amyloliquefaciens It has a high inhibitory effect on the pathogen causing olive peacock spot disease ( C. oleaginous The ability of fermentation liquid to control olive peacock spot disease can be achieved by inhibiting the growth of pathogens and reducing the disease index in the field.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0006] This invention discloses a strain of Bacillus amyloliquefaciens (BAM) Bacillus amyloliquefaciensK-1 is characterized in that it is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37475, and its 16S rDNA sequence contains the sequence shown in SEQ ID NO: 1.

[0007] The present invention also discloses a microbial inoculant, characterized in that it comprises the above-mentioned Bacillus amyloliquefaciens K-1, and an agriculturally acceptable carrier or excipient.

[0008] Furthermore, the microbial agent is a liquid agent.

[0009] The present invention also discloses a fermentation broth, characterized in that it is prepared by fermentation culture of the aforementioned Bacillus amyloliquefaciens K-1.

[0010] Furthermore, the viable bacterial concentration of Bacillus amyloliquefaciens K-1 in the fermentation broth is 1×10⁻⁶. 7 CFU / mL ~ 1×10 9 CFU / mL.

[0011] This invention also discloses the above-mentioned Bacillus amyloliquefaciens K-1, any of the above-mentioned microbial agents, or any of the above-mentioned fermentation broth in inhibiting the pathogen of olive peacock spot disease (…). Cycloconium oleaginum Applications in ).

[0012] The present invention also discloses the application of the above-mentioned Bacillus amyloliquefaciens K-1, any of the above-mentioned microbial agents, or any of the above-mentioned fermentation broth in the preparation of a formulation for preventing and treating peacock spot disease of olives.

[0013] The present invention also discloses a method for preventing and controlling peacock spot disease in olives, characterized in that it includes spraying olive plants with an effective amount of the aforementioned Bacillus amyloliquefaciens K-1, any of the aforementioned microbial agents, or any of the aforementioned fermentation broth.

[0014] Furthermore, the effective amount of the spray is 1 × 10⁻⁶ live bacteria concentration of Bacillus amyloliquefaciens K-1. 7 CFU / mL ~ 1×10 9 CFU / mL.

[0015] Preferably, the spraying is performed once every 5 to 10 days, for a total of 2 to 4 times.

[0016] The strain described in this invention was isolated from olive rhizosphere soil samples through artificial screening and purification. Based on morphological characteristics, 16S rDNA sequence alignment, and phylogenetic tree analysis, strain K-1 was identified as... B. starch liquefier .

[0017] Colony morphology characteristics of strain K-1: After culturing on nutrient agar plates for 48 hours, the colonies grew rapidly, were light yellow, had wrinkled surfaces, and were 26 mm in diameter; Gram staining was positive.

[0018] The morphological characteristics of the bacteria observed under an optical microscope are: the bacteria are rod-shaped.

[0019] The physiological and biochemical characteristics of strain K-1 are as follows: Gram-positive bacteria with phosphorus solubilization and nitrogen fixation capabilities.

[0020] Experimental research shows that... B. amyloliquefaciens K-1 is effective against the pathogen causing olive pea-spot disease. C. oilseed It has a significant inhibitory effect, with an inhibition rate of 94.8% in the three-point confrontation test, and the fermentation filtrate can lead to C. oilseed The mycelium locally swells and breaks. Field trials showed that 90 days after spraying, the control effect was 38.11% in the treatment group and 64.63% in the prevention group. It has great application potential in controlling olive peacock spot disease.

[0021] The method for preventing and treating olive peacock spot disease described in this invention uses... B. amyloliquefaciens K-1 fermentation broth was used to treat olive trees to inhibit pathogen growth and reduce disease index. The treatment was performed by foliar spraying. B. amyloliquefaciens K-1 fermentation broth. As described during spraying. B. amyloliquefaciens The K-1 fermentation broth concentration was 1×10⁻⁶. 8 CFU / mL, sprayed 3 times consecutively at 7-day intervals.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0023] This invention has found that, B. amyloliquefaciens K-1 can effectively inhibit the pathogen causing olive pea-spot disease. C. oleaginous It has a significant antibacterial effect and obvious field control effect, and has great application potential in the control of olive peacock spot disease, providing a new approach for the biological control of olive peacock spot disease. Attached Figure Description

[0024] Figure 1 This is a diagram showing the colony morphology of the strain on NA medium.

[0025] Figure 2 This is a scanned Gram staining micrograph of the strain.

[0026] Figure 3 A phylogenetic tree constructed based on the 16S rRNA gene sequence.

[0027] Figure 4The strain produced a distinct phosphate-solubilizing zone on the surface of the inorganic phosphorus medium.

[0028] Figure 5 This is a diagram showing the growth of the strain on the surface of a nitrogen-free culture medium in Assumption.

[0029] Figure 6 For strains of bacteria C. oleaginous Inhibition effect diagram.

[0030] Figure 7 For the fermentation broth of the strain to C. oleaginous Inhibitory effect on mycelial growth.

[0031] Figure 8 Scanning electron micrograph of conidia of the olive peacock spot fungus attached to the olive peltate trichomes. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0033] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0034] I. Experimental Materials.

[0035] 1.1 Strains and pathogens.

[0036] Bacillus amyloliquefaciens ( B. amyloliquefaciens Strain K-1: Isolated from the rhizosphere soil of healthy olive trees (Olea europaea L., cultivar 'FS17') in Wudu District, Longnan City, Gansu Province (104°24′~105°09′E, 33°04′~33°42′N, altitude 965~1200 m). The pathogen causing olive peacock spot disease: also known as *Olea europaea* (…). Cycloconium oleaginum Cast The sexual stage is... Spilocaea oleaginea The extract was isolated and purified from diseased olive leaf tissue in Longnan City, Gansu Province, and stored on potato dextrose agar (PDA) slant at 4°C for later use.

[0037] 1.2 Main culture medium.

[0038] NA medium (nutrient agar): beef extract 3 g / L, peptone 10 g / L, sodium chloride 5 g / L, agar 18 g / L, distilled water 1000 mL, pH 7.0–7.2, sterilized at 121℃ for 20 min. Used for bacterial isolation, purification, and routine culture.

[0039] YPD liquid culture medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, distilled water 1000 mL, pH natural, sterilized at 121℃ for 20 min. Used for strain activation and seed culture preparation.

[0040] PDA medium: 200 g / L potato (boiled and filtered), 20 g / L glucose, 18 g / L agar, 1000 mL distilled water, natural pH, sterilized at 121℃ for 20 min. Used for pathogen culture and confrontation experiments.

[0041] Inorganic phosphorus medium: glucose 10 g / L, calcium phosphate 5 g / L, ammonium sulfate 0.5 g / L, potassium chloride 0.2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.0001 g / L, ferrous sulfate 0.0001 g / L, agar 18 g / L, pH 7.0–7.5, sterilized at 121℃ for 20 min. Used for phosphorus solubilization determination.

[0042] Ashube nitrogen-free medium: mannitol 10 g / L, potassium dihydrogen phosphate 0.2 g / L, magnesium sulfate 0.2 g / L, sodium chloride 0.2 g / L, calcium sulfate 0.1 g / L, calcium carbonate 5 g / L, agar 18 g / L, pH 7.0–7.5, sterilized at 121℃ for 20 min. Used for nitrogen fixation capacity determination.

[0043] Fermentation medium: 20 g / L white sugar, 10 g / L soybean meal, 10 g / L peptone, 10 g / L yeast extract, 1000 mL distilled water, natural pH, sterilized at 121℃ for 20 min. Used for liquid fermentation production.

[0044] 1.3 Main reagents.

[0045] DNA extraction kit (bacterial genomic DNA extraction kit, centrifuge column type); PCR primers: universal primers for 16S rRNA gene 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'); Ex Taq DNA polymerase, dNTPs, 10×PCR Buffer, DL2000 DNA Marker; Gram staining kit (crystal violet staining solution, iodine solution, 95% ethanol, safranin counterstaining solution).

[0046] 1.4 Main instruments and equipment.

[0047] Constant temperature incubator, constant temperature shaking incubator, autoclave, clean bench, biosafety cabinet, PCR instrument, gel imaging system, ultraviolet spectrophotometer, optical microscope (with oil immersion, 1000×), scanning electron microscope (SEM), 20L fermenter (with stirring, aeration, and temperature control system); backpack sprayer.

[0048] II. Experimental Methods.

[0049] 2.1 Isolation and purification of strains.

[0050] Rhizosphere soil samples were collected from healthy olive trees (2–5 mm from the root surface, 10–20 cm deep). After passing through a 2 mm sieve, 10 g of soil sample was weighed and placed in a 250 mL Erlenmeyer flask containing 90 mL of sterile water. The flask was shaken thoroughly for 30 min to disperse the soil particles, thus preparing a soil suspension. The soil suspension was then heated in a 90°C water bath for 2 h to kill non-spore-forming vegetative cells.

[0051] The heat-treated soil suspension was gradient diluted to 10. -2 ~10 -6 200 µL of each culture was spread onto NA plates and incubated at 28°C for 3 days. Single colonies of varying morphology and color (milky white or pale yellow) were picked and purified three times on NA medium using the streak plate method until pure cultures were obtained. The purified strains were inoculated onto NA slant agar and stored at 4°C for short-term storage; for long-term storage, glycerol tubes (30% glycerol) were cryopreserved at -80°C.

[0052] 2.2 Strain identification.

[0053] 2.2.1 Morphological identification.

[0054] Strain K-1 was inoculated onto NA plates and cultured at 28°C for 48 h. Colony morphology characteristics (color, gloss, surface texture, edge features, diameter, etc.) were observed and recorded. Gram staining was performed: smears of bacterial cells cultured for 48 h were flame-fixed and then subjected to crystal violet primary staining (1 min), iodine mordant staining (1 min), 95% ethanol decolorization (30 s), and safranin counterstaining (1 min). After rinsing with distilled water and air drying, the bacterial morphology and staining reaction were observed under a 1000× oil immersion microscope.

[0055] 2.2.2 Determination of physiological and biochemical characteristics.

[0056] Gram staining: Performed according to method 2.2.1.

[0057] Phosphate solubilization capacity determination: The activated strain was inoculated onto inorganic phosphorus medium plates and cultured at 30℃ for 3 days. The colony diameter (D) and the diameter of the phosphate solubilization zone (d) were measured, and the D / d value was calculated.

[0058] Nitrogen fixation capacity test: The activated strain was inoculated onto Assumption nitrogen-free medium plates and cultured at 30℃ for 3 days. The growth was then observed.

[0059] 2.2.3 Molecular biological identification of DNA extraction.

[0060] Genomic DNA of strain K-1 was extracted using a bacterial genomic DNA extraction kit according to the instructions and stored at -20℃ for later use.

[0061] 16S rRNA gene amplification: Using extracted genomic DNA as a template, PCR amplification was performed using universal primers 27F / 1492R. PCR reaction system (50 µL): 2 µL template DNA, 2 µL each of forward and reverse primers (10 µmol / L), 25 µL 2×Taq PCRMaster Mix, and ddH2O to a final volume of 50 µL. PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 90 s, for a total of 35 cycles; final extension at 72℃ for 10 min.

[0062] Electrophoresis detection: Take 5 µL of PCR product and electrophore it in 0.8% agarose gel (containing 0.5 µg / mL EB) (voltage 80V, time 50 min). Observe and take pictures using a gel imaging system. The expected fragment size is about 1500 bp.

[0063] Sequencing and Analysis: PCR products were sent to a professional biotechnology company for sequencing. The obtained sequences were assembled using DNAman 6.0 software and then aligned to the NCBI GenBank database using BLAST homology. Sequences of highly homologous type strains were selected, and a phylogenetic tree was constructed using MEGA 7.0 software based on the Neighbor-Joining method to determine the taxonomic position of the strains.

[0064] 2.3 Antagonistic activity assay.

[0065] 2.3.1 Three-point standoff test.

[0066] pathogens C. oleaginous Activate the bacteria on PDA plates and incubate for 7 days. Collect 5 mm diameter bacterial discs from the edge of the colonies. Inoculate the pathogenic bacterial discs in the center of a fresh PDA plate, and inoculate the test strain K-1 (NA plate cultured for 48 h) at three equidistant points 25 mm from the center. Use plates inoculated only with the pathogen as a control. Repeat each treatment three times and incubate at 25°C for 7–10 days. When the control colonies have fully colonized the plate, measure the diameter of the pathogenic colonies and calculate the inhibition rate. In the formula: Dc The diameter of pathogen colonies in the control group (mm); D t The diameter (mm) of pathogen colonies in the treatment group.

[0067] 2.3.2 Determination of antibacterial activity of fermentation filtrate.

[0068] Strain K-1 was inoculated into YPD liquid medium and cultured at 30℃ and 180 r / min for 72 h with shaking. The fermentation broth was centrifuged at 8000 r / min for 15 min, and the supernatant was filtered through a 0.22 µm microporous membrane to obtain sterile fermentation filtrate. The filtrate was poured into plates, and pathogenic mycelial cakes (5 mm in diameter) were inoculated into the plates. The plates were cultured at 25℃ for 3 days. Small pieces of culture medium were cut from the edge of the colonies to prepare scanning electron microscopy samples to observe whether the hyphae showed abnormal morphology such as swelling, breakage, or deformity.

[0069] 2.4 Preparation of fermentation broth.

[0070] 2.4.1 Seed liquid preparation.

[0071] The preserved strain K-1 was streaked onto NA plates and incubated at 30°C for 48 h. A single colony was picked and inoculated into 50 mL of YPD liquid medium, and cultured at 30°C with shaking at 180 r / min for 12 h to obtain the primary seed culture. A 1% (v / v) inoculum was then transferred to 200 mL of YPD medium and cultured under the same conditions for 72 h to obtain the secondary seed culture. Cell counts were performed using a hemocytometer, and the cell concentration was adjusted to 2 × 10⁻⁶. 9 CFU / mL.

[0072] 2.4.2 Liquid fermentation.

[0073] Prepare 20 L of fermentation medium according to the formula, and pour it into a 20 L fermenter. Sterilize at 121℃ for 30 min, and cool to 30℃. Inoculate with secondary seed culture (5% inoculum, v / v). Control the fermentation conditions as follows: temperature 30±1℃, stirring speed 180-200 r / min, aeration rate 1:0.5~1.0 (vvm), tank pressure 0.03~0.05 MPa, fermentation time 72 h, and determine the cell concentration. After fermentation, determine the viable cell count in the fermentation broth using a hemocytometer and adjust to 1×10⁻⁶. 8 CFU / mL available for use.

[0074] 2.5 Field efficacy trials.

[0075] 2.5.1 Overview of the test site.

[0076] The experiment was conducted from April to July 2025 in an olive orchard in Wudu District, Longnan City, Gansu Province. The experimental site was at an altitude of 965 m, with an average annual temperature of 14.9℃ and an annual precipitation of 474 mm. The tested olive trees were 15 years old, of variety 'FS17', with a spacing of 4 m × 5 m. Peacock spot disease had occurred in previous years, and no other fungicides were applied.

[0077] 2.5.2 Experimental Design.

[0078] A randomized block design was used, with 3 treatments, 3 trees per treatment, and 3 replicates, for a total of 9 plots. A protection row was included between plots. The treatments were as follows: CK (control): sprayed with an equal volume of water; Tre (treatment group): sprayed with fermentation broth of strain K-1 (1×10⁻⁶) starting at the early stage of disease (approximately 15% disease incidence). 8 CFU / mL); Pre (prevention group): Spray with strain K-1 fermentation broth (1×10⁻⁶ CFU / mL) before disease onset (early April, during the new shoot emergence period). 8 (CFU / mL).

[0079] 2.5.3 Application method.

[0080] Foliar spraying was performed using a backpack sprayer with a nozzle diameter of 0.7 mm, an operating pressure of 0.3 MPa, and a spray volume of 5 L / plant. The spray should be applied until both sides of the leaves are evenly coated with the solution without dripping. Applications were repeated three times at 7-day intervals. Application was conducted between 9:00 AM and 11:00 AM, avoiding rainy or windy days. No other fungicides were used during the trial period; routine cultivation management was maintained.

[0081] 2.5.4 Survey methods and indicators.

[0082] Each tree was randomly surveyed in four directions: east, west, south, and north. 25 leaves were surveyed in each direction, for a total of 100 leaves per tree. Disease condition was assessed before application (0 days) and 90 days after the last application.

[0083] Disease severity grading criteria (refer to GB / T 17980.100 and field symptoms of olive peacock spot disease): Grade 0: No lesions; Grade 1: Lesion area ≤10% of total leaf area; Grade 2: Lesion area 11%–25% of total leaf area; Grade 3: Lesion area 26%–40% of total leaf area; Grade 4: Lesion area 41%–55% of total leaf area; Grade 5: Lesion area >55% of total leaf area or leaf drop.

[0084] Calculation formula: In the formula: CK1 is the disease index after drug administration in the blank control area; PT1 is the disease index after drug administration in the treatment area.

[0085] Statistical analysis: SPSS 22.0 software was used for analysis of variance, and Duncan's New Multiple Range Test (DMRT) was used for multiple comparisons (P<0.05).

[0086] III. Experimental Results.

[0087] 3.1 Morphological characteristics of the strain.

[0088] When strain K-1 was cultured on NA plates at 28°C for 48 h, the colonies were pale yellow, dry, wrinkled, with irregular edges, 26–30 mm in diameter, and viscous in texture. Figure 1 Gram-positive, the bacteria are rod-shaped, occurring singly or in pairs, and approximately (0.6–0.8) µm × (2.0–3.0) µm in size. Figure 2 ).

[0089] 3.2 Physiological and biochemical characteristics.

[0090] The main physiological and biochemical characteristics of strain K-1 are shown in Table 1. Consultation of the Bacterial Identification Handbook indicates that strain K-1 shares similar physiological and biochemical characteristics with Bacillus species. Furthermore, strain K-1 is a Gram-positive bacterium. Figure 3 It has phosphorus-solubilizing ability (phosphorus-solubilizing zone diameter D / d=1.25±0.08). Figure 4 ) and nitrogen fixation ability (can grow on nitrogen-free Assumption medium, Figure 5 Based on Bergey's Manual of Systematic Bacteriology, it was preliminarily identified as belonging to the genus Bacillus. Bacillus sp. ).

[0091] Table 1. Physiological and biochemical characteristics of the strain.

[0092] Note: "+" indicates positive; "-" indicates negative; " / " indicates no reaction or weak reaction.

[0093] 3.3 Molecular biological identification.

[0094] The 16S rRNA gene sequencing results (SEQ ID NO:1, sequence length 1129 bp) were compared with those obtained by BLAST. Bacillus amyloliquefaciens The type strain LMG 12234T (GenBank accession number: X60613) showed 99.12% similarity. Phylogenetic analysis revealed that strain K-1 is similar to... B. amyloliquefaciens Clustered in the same branch ( Figure 3 Based on morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, strain K-1 was identified as *Bacillus amyloliquefaciens*. B. starch liquefier ).

[0095] 16S rRNA gene sequence:

[0096] 3.4 Pair C. oleaginous antagonistic activity.

[0097] The results of the three-point confrontation test showed that strain K-1 was effective against the pathogen causing olive peacock spot disease. C. oleaginous It exhibits significant inhibitory effects, with an inhibition bandwidth of 12–15 mm and an inhibition rate of 94.8%. Figure 6 The antibacterial test of the fermentation filtrate showed that fermentation filtrate containing 10% (v / v) K-1 had an antibacterial effect on... C. oleaginous The pathogen exhibits inhibitory effects, causing hyphae to break, swell, and become deformed. This indicates that strain K-1 can disrupt the cell structure of the pathogen by producing antimicrobial substances. Figure 7 ).

[0098] 3.5 Results of field efficacy trials.

[0099] The results of the field trial (Table 2) showed that 90 days after application, the disease index in the treatment group (Tre) decreased from 15.56 to 9.63, with a relative control effect of 38.11%; the disease index in the prevention group (Pre) decreased from 4.92 to 1.74, with a relative control effect of 64.63%. The disease index in the control group (CK) increased from 7.75 to 16.86, with the disease naturally progressing. The control efficacy of the prevention group was significantly higher than that of the treatment group (P<0.05), indicating that the fermentation broth of strain K-1 has a good preventive effect against olive lenticular disease.

[0100] Table 2. Field control effect of Bacillus amyloliquefaciens K-1 fermentation broth on peacock spot disease of olive.

[0101] Note: Different lowercase letters after the data in the same column indicate significant differences (P<0.05).

[0102] 3.6 Quality indicators of fermentation broth.

[0103] The number of viable Bacillus amyloliquefaciens K-1 bacteria in the fermentation broth was measured to be 1.0 × 10⁻⁶. 8 ~2.0×10 8 CFU / mL, spore rate ≥80%, pH 7.2, no obvious odor, meets the quality standards for microbial inoculants.

[0104] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens K-1, characterized in that, It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37475, and its 16S rDNA sequence contains the sequence shown in SEQ ID NO:

1.

2. A microbial inoculant, characterized in that, It includes Bacillus amyloliquefaciens K-1 as described in claim 1, and an agriculturally acceptable carrier or adjuvant.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is a liquid agent.

4. A fermentation broth, characterized in that, It is prepared by fermentation culture of Bacillus amyloliquefaciens K-1 as described in claim 1.

5. The fermentation broth according to claim 4, characterized in that, The viable bacterial concentration of Bacillus amyloliquefaciens K-1 in the fermentation broth was 1×10⁻⁶. 7 CFU / mL ~ 1×10 9 CFU / mL.

6. The Bacillus amyloliquefaciens K-1 as described in claim 1, the microbial agent as described in claim 2 or 3, or the fermentation broth as described in claim 4 or 5, in inhibiting the pathogen of olive peacock spot disease ( Cycloconium oleaginum Applications in ).

7. The use of Bacillus amyloliquefaciens K-1 as described in claim 1, the microbial agent as described in claim 2 or 3, or the fermentation broth as described in claim 4 or 5 in the preparation of a formulation for preventing and treating peacock spot disease in olives.

8. A method for preventing and controlling peacock spot disease in olive trees, characterized in that, This includes spraying olive plants with an effective amount of Bacillus amyloliquefaciens K-1 as described in claim 1, the microbial agent as described in claim 2 or 3, or the fermentation broth as described in claim 4 or 5.

9. The method according to claim 8, characterized in that, The effective amount of the spray is 1 × 10⁻⁶ live bacteria concentration of Bacillus amyloliquefaciens K-1. 7 CFU / mL ~ 1×10 9 CFU / mL.

10. The method according to claim 8 or 9, characterized in that, The spraying is carried out once every 5 to 10 days, for a total of 2 to 4 times.

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