Strain of bacillus amyloliquefaciens jdf630 and application thereof in preventing and treating verticillium wilt of cotinus coggygria

Abstract

This invention discloses a strain of *Bacillus amyloliquefaciens*, JDF630, and its application in controlling *Fusarium wilt* of *Cotinus coggygria*, belonging to the field of microbial technology. The *Bacillus amyloliquefaciens* strain JDF630 described in this invention was deposited on July 13, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO. 25289. This invention demonstrates that strain JDF630 exhibits broad-spectrum inhibitory activity against various plant pathogens. Treatment with JDF630 significantly reduces the damage caused by *Fusarium wilt* to *Cotinus coggygria*, providing a new microbial strain and control strategy for the prevention and control of plant pathogens, demonstrating excellent biological control effects.

Description

[0001] This application claims priority to Chinese Patent Application No. 202211318266.1, filed on October 26, 2022, entitled "A strain of Bacillus amyloliquefaciens JDF630 and its application in the control of Fusarium wilt in Cotinus coggygria", filed by Hebei Agricultural University, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus amyloliquefaciens JDF630 and its application in the prevention and control of Fusarium wilt in Cotinus coggygria. Background Technology

[0003] The smoke tree (Cotinus coggygria) is a plant belonging to the genus Cotinus in the family Anacardiaceae. It has a rounded crown and can reach a height of 3-8 meters. Its leaves turn red when the temperature difference in autumn exceeds 10℃. The tree has a beautiful shape, and both its stems and leaves have high ornamental value. Research shows that *Verticillium dahliae* is the pathogen causing wilt disease in smoke trees. When healthy smoke tree forests are infected with *Verticillium dahliae*, the trees begin to show symptoms of leaf dehydration and wilting; in severe cases, the entire tree may wither and die.

[0004] Currently, the control of Fusarium wilt in smoke trees mainly focuses on chemical control. The advantages of chemical control are convenience, economy, and no seasonal restrictions, making it very popular in the market and the most commonly used method for Fusarium wilt control. However, long-term use of chemical agents can lead to drug resistance in pathogens and can pollute soil and water bodies, disrupting the ecological balance. Therefore, chemical agents are not a long-term solution.

[0005] Bacillus species are Gram-positive bacteria with simple nutritional requirements and rapid reproduction rates. They are widely distributed in the air, aquatic environments, plants, and animal intestines, and are closely related to human life, making them an important microbial resource. Current research indicates that Bacillus amyloliquefaciens and its metabolites can be used to control various plant diseases. For example, Bacillus amyloliquefaciens BA-KA3, disclosed in a Chinese patent, can inhibit multiple plant diseases. However, not all Bacillus amyloliquefaciens have similar effects. To broaden the selection of antibacterial agents, screening for new microorganisms is an extremely important task. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of Bacillus amyloliquefaciens JDF630 and its application in the control of Fusarium wilt in Cotinus coggygria. The strain has a broad antibacterial spectrum after fermentation and can be used for green control of Fusarium wilt and other plant diseases.

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

[0008] This invention provides a strain of Bacillus amyloliquefaciens JDF630, which was deposited at the China General Microbiological Culture Collection Center on July 13, 2022, with the accession number CGMCC NO.25289.

[0009] This strain was isolated from the rhizosphere soil of healthy smoke trees. Morphological analysis and multi-site sequence analysis of different housekeeping genes identified it as belonging to the genus *Bacillus* sp., specifically *Bacillus amyloliquefaciens*, and named it JDF630. When JDF630 was cultured on LB agar plates at 37°C for 24 hours, the colonies were off-white with irregular edges and a dry surface.

[0010] This invention demonstrates, through the determination of antagonistic activity against various plant pathogens, that strain JDF630 exhibits strong antagonistic activity against the plant pathogens *Fusarium graminearum*, *Diplodia seriata*, *Diaporthe eres*, *Alternaria alternata*, *Botryosphaeria dothidea*, and *Fusarium decemcellulare*, with inhibition rates of 84.3%, 73.41%, 61.92%, 60.69%, 57.60%, and 40.43%, respectively.

[0011] This invention provides the application of the Bacillus amyloliquefaciens strain JDF630 and / or its bacterial suspension in inhibiting plant pathogens, in preparing plant pathogen inhibitors, or in controlling plant diseases caused by plant pathogens.

[0012] In particular, the present invention shows that Bacillus amyloliquefaciens JDF630 has a good inhibitory effect on the above-mentioned plant pathogens, so the JDF630 strain also has a certain control effect on diseases caused by these plant pathogens.

[0013] This invention provides a plant pathogen inhibitor containing the *Bacillus amyloliquefaciens* strain JDF630 and / or its bacterial culture.

[0014] This invention provides a method for inhibiting plant pathogens and / or preventing agricultural diseases, wherein the Bacillus amyloliquefaciens strain JDF630 is used to treat diseased hosts.

[0015] Preferably, the Bacillus amyloliquefaciens strain JDF630 is prepared into a bacterial solution and then directly applied to the roots of the diseased host.

[0016] Preferably, the bacterial solution is prepared by inoculating JDF630 strain into LB medium, incubating at 37°C for 24 hours, and refrigerating at 180 rpm.

[0017] Preferably, the bacterial solution concentration is 1×10⁻⁶. 8 CFU / mL.

[0018] The present invention has the following beneficial effects:

[0019] The JDF630 strain provided by this invention exhibits inhibitory effects against various plant pathogens, including *Fusarium graminearum*, *Crassostrea gigas*, *Metacarpa* fungi, *Alternaria*, *Botrytis*, and *Fusarium multiseptum*. It also inhibits *Verticillium dahliae* infection of *Cotinus coggygria*. Treatment with JDF630 significantly reduces the incidence of *Cotinus coggygria* wilt. Therefore, this strain can serve as an environmentally friendly green agent to replace chemical pesticides in the control of *Cotinus coggygria* wilt and other plant diseases caused by the aforementioned pathogens, providing a new microbial strain and control strategy with excellent biological control effects.

[0020] Biological Preservation Information

[0021] Bacillus amyloliquefaciens JDF630 was deposited on July 13, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.25289, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0022] Figure 1 The image shows the initial screening results of antagonistic bacteria. In the image, A represents the confrontation culture of antagonistic bacteria A and Verticillium dahliae; B represents the confrontation culture of antagonistic bacteria B and Verticillium dahliae; C represents the confrontation culture of antagonistic bacteria C and Verticillium dahliae; and CK represents the culture of Verticillium dahliae alone.

[0023] Figure 2 The image shows the results of the secondary screening of antagonistic bacteria. In the image, A represents Verticillium dahliae cultured on sterile filtrate medium containing antagonistic bacteria A; B represents Verticillium dahliae cultured on sterile filtrate medium containing antagonistic bacteria B; and CK represents Verticillium dahliae cultured on PDA medium.

[0024] Figure 3 The colony morphology of strain JDF630 on LB medium;

[0025] Figure 4 The phylogenetic tree of strain JDF630 provided by this invention;

[0026] Figure 5This diagram illustrates the antagonistic effect of strain JDF630 against plant pathogens. In the diagram, A and A0 represent confrontation cultures and single-colony cultures of *Fusarium multiseptum* and *Bacillus amyloliquefaciens* JDF630; B and B0 represent confrontation cultures and single-colony cultures of *Alternaria alternata* and *Bacillus amyloliquefaciens* JDF630; C and C0 represent confrontation cultures and single-colony cultures of *Chromospora* and *Bacillus amyloliquefaciens* JDF630; D and D0 represent confrontation cultures and single-colony cultures of *Staphylococcus aureus* and *Bacillus amyloliquefaciens* JDF630; E and E0 represent confrontation cultures and single-colony cultures of *Fusarium graminearum* and *Bacillus amyloliquefaciens* JDF630; and F and F0 represent confrontation cultures and single-colony cultures of *Metacarpa* fungi and *Bacillus amyloliquefaciens* JDF630.

[0027] Figure 6 The graph shows the control effect of strain JDF630 on Fusarium wilt in Cotinus coggygria. In the graph, A represents the application of JDF630 bacterial solution; CK represents the control group.

[0028] Figure 7 The figure shows the results of single-factor screening of Bacillus amyloliquefaciens JDF630. In the figure, A represents the screening of the optimal culture medium; B represents the screening of the optimal temperature; C represents the screening of the optimal pH; and D represents the screening of the optimal inoculum size. Different lowercase letters indicate significant differences at the 0.05 level. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0030] Unless otherwise specified, the reagent kits used in the following examples are commercially available.

[0031] LB medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar, 1000mL distilled water, sterilized for later use.

[0032] LB liquid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1000mL distilled water, sterilized for later use.

[0033] Example 1: Screening and Isolation of Strains

[0034] Three healthy smoke trees were selected, and rhizosphere soil was collected. Before collection, tools were disinfected with 75% alcohol, and soil samples were collected in sterile bags and labeled. The soil samples were brought back to the laboratory for impurity removal and sieving. Untreated soil samples were stored in a refrigerator (4°C).

[0035] To isolate antagonistic bacteria, first weigh 5g of soil and mix it with 45mL of sterile water, then shake for 30 minutes. Prepare four 2mL centrifuge tubes. Add 900μL of sterile water to each centrifuge tube, then aspirate 100μL of the shaken soil suspension into the first centrifuge tube and mix well. Then, aspirate 100μL of the liquid from the first centrifuge tube and dilute it sequentially into the fourth centrifuge tube, obtaining 10... -5 g / mL, 10 -6 g / mL, 10 -7 A soil suspension of g / mL was prepared, and 100 μL of each suspension was transferred to a petri dish and spread evenly using a spreader. Bacterial isolation and culture were performed on LB medium. The isolated bacteria were then purified and stored for later use.

[0036] A total of 138 bacterial strains, 96 fungal strains, and 46 actinomycete strains were isolated from the rhizosphere soil of healthy *Cotinus coggygria*. Preliminary screening using the plate confrontation method yielded 3 strains exhibiting significant antagonistic activity against *Verticillium dahliae*. Figure 1 The inhibition rate of strain A against Verticillium dahliae was 88.63%, that of strain B was 88.45%, and that of strain C was 83.3% (Table 1). There was no significant difference in the inhibition rate of strain A against Verticillium dahliae between strain A and strain B (P<0.05).

[0037] Table 1 Initial screening of antagonistic bacteria

[0038]

[0039] Note: The data in the table are mean ± standard error. Different lowercase letters indicate that the difference is significant at the 0.05 level (the same applies below).

[0040] Strains A and B were selected for secondary screening using the plate method containing fermentation filtrate. Figure 2 (Table 2) The experimental results showed that bacterial strain A had the strongest antagonistic ability against Verticillium dahliae, with an inhibition rate of 86.55%, while bacterial strain B had an inhibition rate of 67.18%. Therefore, bacterial strain A was selected for further research and named JDF630.

[0041] Table 2. Antagonistic Bacterial Rescreening

[0042]

[0043] Note: The data in the table are mean ± standard error. Different lowercase letters indicate significant differences at the 0.05 level, and * indicates significant differences at the 0.05 level.

[0044] Example 2: Classification and Identification of Strains

[0045] 1. Morphological identification

[0046] The strain obtained in Example 1 was placed on LB medium and incubated upside down at 37°C for 24 hours. Single colonies were selected for observation, referring to the "Handbook of Systematic Identification of Common Bacteria". The growth morphology is as follows: Figure 3 As shown: the colonies are off-white with irregular edges, dry surface, and wrinkles.

[0047] 2. Identification by Multilocus Sequence Analysis (MLSA) of Different Housekeeping Genes

[0048] DNA was extracted from the selected antagonistic strains using a fecal DNA extraction kit (TIANamp Stool DNA Kit). The DNA was extracted based on internal fragment sequences of seven housekeeping genes: glpF, ilvD, ptA, purH, pycA, rpoD, and tpiA.

[0049] Table 3 Housekeeper gene sequences

[0050]

[0051]

[0052] The total PCR reaction volume was 50 μL, specifically consisting of: 25 μL Taq enzyme, 1 μL template DNA, 2 μL forward primer, 2 μL reverse primer, and 20 μL ddH2O. Amplification of the seven genes was performed using a single-cycle program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s; 54℃ annealing for 30 s; 72℃ extension for 50 s; and a final extension at 72℃ for 5 min, for a total of 40 cycles. The reaction was terminated at 4℃.

[0053] BLAST analysis of the obtained DNA sequences showed that most of the sequences similar to this strain belonged to the genus *Bacillus*. Using PhyloSuite software, the obtained sequences were tandemly linked to form the sequence of a single strain. A phylogenetic tree based on proximity connections was constructed using MEGA software to determine the taxonomic position of the species. Strain JDF630 showed the highest homology with *Bacillus amyloliquefaciens*. Figure 4 ).

[0054] Based on the above morphological identification and MLSA identification results, the strain was identified as Bacillus amyloliquefaciens and named Bacillus amyloliquefaciens JDF630 strain. This strain was deposited at the China General Microbiological Culture Collection Center on July 13, 2022, with the accession number CGMCCNO.25289.

[0055] Example 3: Broad-spectrum antibacterial activity of the strain

[0056] 1. Determination of the antagonistic activity of JDF630 strain against plant pathogens

[0057] The tested pathogens were: *Alternaria alternata* isolated from diseased branches of infected cypress; *Fusarium decemcellulare*, *Diplodia seriata*, and *Diaportheeres* isolated from the phloem of ash trees; *Botryosphaeria dothidea* isolated from the trunk of infected Fuji apple trees; and *Fusarium graminearum* isolated from the ears of infected wheat. All pathogens were preserved at 4°C and were provided by the Forest Pathology Laboratory of Hebei Agricultural University.

[0058] The tested plant pathogens were transferred to PDA plates for activation culture. After 5 days, holes were punched at the edge of the colonies using a 6 mm diameter punch and inoculated into the center of the PDA medium. JD630 bacterial suspension, cultured overnight, was spot-inoculated at a position 2.5 cm from the center of the plate (in all four directions). A control was used without JD630 inoculation. Each treatment was performed in triplicate, and the plates were incubated upside down in a dark incubator at 25°C. After 7 days, the diameter of the pathogen colonies was measured, and the inhibition rate was calculated.

[0059] Inhibition rate (%) = (Control colony diameter - Treated colony diameter) / Control colony diameter × 100, results as follows Figure 5 As shown, distinct isolation zones were observed at the confrontation cultures of Bacillus amyloliquefaciens JDF630 and six pathogens. The colonies of Fusarium graminearum and *Metacarpa* fungi in the confrontation cultures were also observed. Figure 5 The F and F0 strains have essentially ceased growth. In terms of inhibitory effect, *Bacillus amyloliquefaciens* JDF630 showed the best inhibitory effect against *Fusarium graminearum*, with an inhibition rate of 84.3%. Figure 5 The inhibitory rate was 74.31% (E, E0); followed by Chromospora, with an inhibition rate of 74.31%. Figure 5 The inhibition rate against *Metacarpa* fungi was 61.92% (C, C0). Figure 5 The inhibition rate against Alternaria was 60.69% (F0, F0); Figure 5 (B, B0); the inhibition rate against Staphylococcus aureus was 57.6% ( Figure 5 The inhibition rate against Fusarium multifiliis was 40.43% (D, D0); Figure 5 (A, A0).

[0060] Table 4. Inhibitory effects of Bacillus amyloliquefaciens JDF630 on six plant pathogens.

[0061]

[0062] Example 4: Control of Fusarium wilt in Cotinus coggygria by strain JDF630

[0063] 1. Potted seedling control experiment

[0064] Verticillium dahliae was inoculated onto PDA medium in 6mm discs and incubated at 25°C for 5-6 days. Once the colonies had fully covered the plate, mycelial discs were picked from the edge of the colonies and inoculated into Erlenmeyer flasks containing 300mL of PDA liquid medium. The flasks were then incubated in the dark at 28°C and 180 rpm for 5 days. The Verticillium dahliae culture was filtered through four layers of gauze to remove mycelia. Finally, the spore concentration of the culture was diluted to 1×10⁻⁶. 8 CFU / mL available for use.

[0065] Strawberry strain JDF630 was streaked onto LB agar plates and incubated at 37°C in the dark for 24 hours. Once individual colonies appeared, a single colony was picked up using a sterile picking needle and inoculated into an Erlenmeyer flask containing 300 mL of LB liquid medium. The flask was then incubated at 37°C and 180 rpm in the dark for 24 hours. The bacterial concentration was then diluted to 1 × 10⁻⁶. 8 CFU / mL, for later use.

[0066] Sixty healthy smoke tree plants with uniform growth were selected. The root-damage method was used to inoculate the plants with *Fusarium wilt* pathogen. A small knife, approximately 15cm long, was used to pierce the soil around the plant to a depth of about 10cm to cause root damage. Each plant was treated with the same *Verticillium dahliae* culture solution (1×10⁻⁶). 8 400 mL of antagonistic bacteria culture solution (1×10⁻⁶ CFU / mL) was applied to the roots. After 7 days, 30 pots were selected and treated with 400 mL of antagonistic bacteria culture solution (1×10⁻⁶ CFU / mL). 8 The root irrigation was performed with CFU / mL, and 30 pots were treated with 400mL of clean water as a control. The incidence and disease index of smoke trees were investigated, and the control effect was calculated.

[0067] Incidence rate (%) = Number of infected plants / Total number of plants surveyed × 100

[0068] Disease index = Σ(Number of diseased plants at each level × Representative value at each level) / Total number of plants × Highest representative value

[0069] Prevention and control efficacy (%) = (Disease index of control group - Disease index of treatment group) / Disease index of control group × 100

[0070] Table 5 Grading Standards for Fusarium Wilt Disease in Potted Smoke Tree Seedlings

[0071]

[0072] The results are as follows Figure 6As shown, Bacillus amyloliquefaciens strain JDF630 exhibits broad-spectrum antibacterial activity. It demonstrates strong antagonistic activity against plant pathogens such as Fusarium graminearum, Chromosporium, *Metacarpa* fungi, *Alternaria*, *Staphylococcus*, and *Fusarium multifiliis*, with inhibition rates of 84.3%, 73.41%, 61.92%, 60.69%, 57.60%, and 40.43%, respectively. Furthermore, Bacillus amyloliquefaciens JDF630 can effectively inhibit the infection of *Verticillium dahliae* by *Cotinus coggygria*. When *Cotinus coggygria* wilt disease occurs, the leaves turn yellow, most of the leaves fall off, and the few remaining leaves become scorched. This indicates that *Verticillium dahliae* can successfully infect *Cotinus coggygria* in this experiment. Although the plants treated with JD630 bacterial solution also showed yellowing leaves, it was significantly better than the control group. This shows that JD630 bacterial solution can effectively inhibit *Verticillium dahliae* infection of *Cotinus coggygria* and reduce the occurrence of *Cotinus coggygria* wilt disease.

[0073] Table 6. Survey on the control effect of Fusarium wilt in potted seedlings of Smoke Tree.

[0074]

[0075] Note: * indicates a significant difference at the 0.05 level (the same applies below).

[0076] 2. Field control trials

[0077] In May 2022, five smoke trees (diameter > 10cm) with similar age, vigor, and disease incidence were selected and treated with 30L of antagonistic bacteria (1×10⁻⁶). 8 Five trees were treated with 30L of water per 5 trees for root irrigation (CFU / mL). One-meter-long branches were selected from the east, south, west, and north directions of the trees, and the number and length of new shoots, as well as the tree's diameter at ground level, were measured. These measurements were repeated in August and September of the same year to calculate the increase in diameter at ground level, the increase in the length and number of new shoots. Disease incidence was also investigated on new shoots from branches in different directions, and the incidence rate, disease index, and control effect were calculated. The calculation method was the same as above.

[0078] Field surveys (Table 7) showed that after applying the antagonistic bacterium JDF630, the diameter increase of the smoke tree was 0.72 cm, compared to 0.39 cm in the control; the increase in the length of new branches was 9.26 cm, compared to 3.18 cm in the control; and the increase in the number of new branches was 37, compared to 17 in the control. All of these were significantly higher than the control group (P<0.05), and the field control effect reached 66.25%.

[0079] Table 7. Field control effect survey on Fusarium wilt of Cotinus coggygria.

[0080]

[0081] Example 5: Optimization of fermentation conditions for antagonistic bacteria

[0082] 1. Seed liquid preparation

[0083] Single colonies of the isolated and purified antagonistic bacteria were picked up using a sterile picking needle and inoculated into LB medium. The medium was then incubated for 24 hours in the dark at 37°C and 180 rpm on a shaker. The OD values ​​were measured using a spectrophotometer. 600 It is prepared as a seed liquid for later use.

[0084] 2. Single-factor screening of fermentation conditions

[0085] LB medium, PDB medium, YPG medium, TSA medium, NA medium, and soluble starch medium were separately divided into 300 mL Erlenmeyer flasks, with each flask containing 200 mL of culture. Three flasks were used for inoculation with antagonistic bacteria at a 1% inoculation rate, and three flasks served as controls without inoculation with antagonistic bacteria. The cultures were incubated on a shaker at 37°C and 180 rpm in the dark for 24 h. The OD was measured using a visible light spectrophotometer. 600 In order to determine the optimal culture medium.

[0086] The optimal culture medium was inoculated with seed culture at an inoculum size of 1%, and cultured in the dark at a shaker speed of 180 rpm. The culture temperatures were set at 28℃, 31℃, 34℃, 37℃, 40℃, and 43℃. After 24 hours of culture, the OD600 of the bacterial culture was measured to determine the optimal fermentation temperature. Each treatment was repeated three times.

[0087] Different pH values ​​were set: 5, 5.5, 6, 6.5, 7, 7.5, and 8, with other conditions remaining unchanged. Each treatment was performed in triplicate, and OD was measured after 24 hours. 600 Determine the optimal pH.

[0088] Different inoculum sizes were set at 1%, 5%, 10%, 15%, 20%, and 25%, and the culture was carried out under dark conditions at a shaker speed of 180 rpm. Each treatment was repeated in triplicate, and OD was measured after 24 hours. 600 Determine the optimal vaccination rate.

[0089] Single-factor screening results are as follows Figure 7 As shown, the growth of Bacillus amyloliquefaciens JDF630 differed significantly among the six tested culture media (P < 0.05). LB was the optimal medium, and the fermentation broth OD... 600 The absorbance was highest in soluble starch medium, followed by PDB medium. *Bacillus amyloliquefaciens* JDF630 showed the worst growth in soluble starch medium. 37℃ and 40℃ were the optimal culture temperatures for JDF630, with the worst fermentation performance observed at 28℃. The effect of pH on the fermentation of *Bacillus amyloliquefaciens* JDF60 showed an initial increase followed by a decrease; at pH 6, the OD of the fermentation broth was highest.600 The absorbance reaches its maximum at pH 5, which is the most unfavorable temperature for the growth of the strain. Different inoculum sizes also have varying effects on the fermentation of *Bacillus amyloliquefaciens* JDF630; for example, when the inoculum size reaches 10%, the OD value... 600 At its highest, as the inoculum size further increases, the absorbance of the fermentation broth begins to decrease, reaching a maximum at an inoculum size of 25%. 600 The minimum vaccination rate is 10%, therefore the optimal vaccination rate is 10%.

[0090] 3. Orthogonal experimental design for fermentation conditions

[0091] Based on the results of single-factor experiments, an orthogonal experimental design was conducted to investigate the three factors affecting fermentation: temperature, pH, and inoculum size. Three levels were selected for each factor, and a three-factor, three-level orthogonal experimental table was generated using SPSS. Each treatment was replicated three times. The shaker speed was set to 180 rpm, and fermentation was carried out in the dark. OD was measured after 24 hours. 600 The values ​​and bacterial counts are used to determine the final fermentation conditions.

[0092] The results of the orthogonal experiment are shown in Table 8. At a temperature of 37℃, an inoculum size of 10%, and a pH of 5.5, the fermentation broth of *Bacillus amyloliquefaciens* JDF630 exhibited the highest absorbance (2.04), with a concentration of 4.65 × 10⁻⁶. 10 CFU / mL.

[0093] Table 8 Results of Orthogonal Experimental Design

[0094]

[0095] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens The application of JDF630 strain and / or its fermentation broth in the prevention and control of agricultural diseases caused by plant pathogens is characterized by, The preservation number of the Bacillus amyloliquefaciens strain JDF630 is CGMCC NO.25289; The plant pathogen is: Verticillium dahliae Verticillium dahliae Fusarium graminearum Fusarium graminearum , coloripol Diplodia seriata fungi of the genus *Synapticus* Diaporthe eres Alternaria Alternaria alternata Staphylococcus aureus B otryosphaeria dothidea and Fusarium multiseptum Fusarium decemcellulare .

2. The application according to claim 1, characterized in that, The fermentation method of the fermentation broth includes the following steps: inoculating the Bacillus amyloliquefaciens JDF630 strain into a liquid culture medium and culturing it in the dark to obtain the fermentation broth; The liquid culture medium is PDA or LB.

3. The application according to claim 2, characterized in that, The inoculation rate is 1-20%; During the dark culture, the pH value is 5.5~7.5 and the temperature is 34~40℃.

4. An inhibitor of plant pathogens, characterized in that, Contains Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens The strain JDF630 and / or its fermentation broth, wherein the preservation number of the Bacillus amyloliquefaciens JDF630 strain is CGMCC NO.25289; The plant pathogen is: Verticillium dahliae Verticillium dahliae Fusarium graminearum Fusarium graminearum , coloripol Diplodia seriata fungi of the genus *Synapticus* Diaporthe eres Alternaria Alternaria alternata Staphylococcus aureus B otryosphaeria dothidea and Fusarium multiseptum Fusarium decemcellulare .

5. A method for inhibiting plant pathogens and / or preventing diseases caused by plant pathogens, characterized in that, This includes the use of Bacillus amyloliquefaciens (BAM) Bacillus amyloliquefaciens The JDF630 strain and / or its fermentation broth were used to treat the diseased host. The preservation number of the Bacillus amyloliquefaciens JDF630 strain is CGMCC NO.25289. The plant pathogen is: Verticillium dahliae Verticillium dahliae Fusarium graminearum Fusarium graminearum , coloripol Diplodia seriata fungi of the genus *Synapticus* Diaporthe eres Alternaria Alternaria alternata Staphylococcus aureus B otryosphaeria dothidea and Fusarium multiseptum Fusarium decemcellulare .

6. The method according to claim 5, characterized in that, The treatment includes root irrigation.

7. The method according to claim 5, characterized in that, The disease in question is wilt of the smoke tree.

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