A highly efficient antagonistic bacillus subtilis ZF539 and its biocontrol application in multiple vegetable diseases

Abstract

The application discloses a high-efficiency antagonistic bacillus subtilis ZF539 and a biocontrol application of the bacillus subtilis ZF539 in various vegetable diseases, and belongs to the field of microorganisms, in particular to a bacillus subtilis with high-efficiency antagonistic characteristics and the application of the bacillus subtilis in the prevention and treatment of various vegetable diseases. The bacillus subtilis in the application is bacillus subtilis ZF539 with a preservation number of CGMCC No.34731 in the China General Microbiological Culture Collection Center. The strain has the characteristics of high-efficiency colonization, disease prevention and treatment, seedling growth promotion and the like, and has great development and application potential as a biocontrol bacterium.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a highly efficient antagonistic Bacillus subtilis strain ZF539 and its biocontrol application in controlling various vegetable diseases. Background Technology

[0002] Plant diseases are a major bottleneck restricting the sustainable development of the global vegetable industry. Long-term reliance on chemical pesticides has led to increased pathogen resistance, soil microecological imbalance, and environmental pollution. Bacillus subtilis, as an environmentally friendly biocontrol bacterium, is widely used in agricultural disease control due to its broad-spectrum antibacterial activity and safety. However, existing strains generally suffer from low field colonization efficiency, poor field application effects, and relatively singular control targets, severely limiting their industrial application.

[0003] Based on the above background, screening biocontrol strains that have highly efficient antagonistic properties against a variety of plant pathogens and have significant effects in field application can provide important biocontrol strain resources for the development of new microbial agents that can control a variety of plant diseases. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to induce plant disease resistance and inhibit or prevent infectious plant diseases.

[0005] To solve the above-mentioned technical problems, the present invention first provides a strain of Bacillus, namely Bacillus subtilis ZF539, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No. 34731.

[0006] The present invention also provides a composition comprising the Bacillus described above.

[0007] The above composition may be a culture, which may be a substance obtained by culturing the Bacillus in a microbial culture medium. The culture may be a substance obtained by culturing the Bacillus in a microbial culture medium (i.e., a fermentation product, such as a fermentation broth containing the Bacillus and a substance secreted into a liquid culture medium, or a solid fermentation product containing the Bacillus and a substance secreted into a solid culture medium).

[0008] The composition may be a microbial agent containing *Bacillus subtilis* and / or its metabolites. The metabolites may be products obtained by removing the *Bacillus* from the culture, such as culturing the *Bacillus* in a liquid fermentation medium, collecting the fermentation broth (containing the *Bacillus* and substances secreted into the liquid medium), removing the *Bacillus* from the fermentation broth, collecting the remaining components of the fermentation broth, and obtaining the metabolites of the *Bacillus*.

[0009] The above composition can be used to prepare fertilizer, and the above microbial agent can also be used to prepare fertilizer.

[0010] The above composition may have at least one of the following properties:

[0011] A1) Antagonizes pathogens,

[0012] A2) Prevention and control of cucumber angular leaf spot,

[0013] A3) Control of cucumber soft rot

[0014] A4) Control cucumber vine blight

[0015] A5) Control of melon wilt disease

[0016] A6) Prevention and control of black rot in broccoli

[0017] A7) Prevention and control of clubroot disease in broccoli

[0018] A8) Growth-promoting effect on plants.

[0019] The above composition can also be a microbial agent or a microbial fertilizer.

[0020] The above-mentioned inoculants and microbial fertilizers can be used for soil application or for seed soaking.

[0021] The active ingredients of the above-mentioned microbial agents may also contain other biological or non-biological components. The other active ingredients of the above-mentioned microbial agents can be determined by those skilled in the art based on their inhibitory effect on diseases.

[0022] In addition to the active ingredient, the above-mentioned microbial agent also contains a carrier. The carrier can be a commonly used and biologically inert carrier in the pesticide field. The carrier can be a solid or liquid carrier; the solid carrier can be a mineral material, plant material, or polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material can be at least one of corn flour, soybean flour, and starch; the polymer compound can be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent can be decane and / or dodecane.

[0023] The above-mentioned microbial agents can be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0024] Depending on the needs, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the above-mentioned microbial agents.

[0025] In the above text, the metabolites can be obtained from the fermentation broth of *Bacillus subtilis*. The metabolites can be sterile metabolites of *Bacillus subtilis* or bacterial metabolites of *Bacillus subtilis*. Specifically, the sterile metabolites of *Bacillus subtilis* (sterile fermentation filtrate) can be prepared by culturing *Bacillus subtilis* in a liquid culture medium and filtering to remove *Bacillus subtilis* from the liquid culture (fermentation broth). Specifically, the bacterial metabolites of *Bacillus subtilis* can be prepared by culturing *Bacillus subtilis* in a liquid fermentation medium and collecting the fermentation broth, which is the bacterial metabolite of *Bacillus subtilis*.

[0026] The present invention also provides a method for preparing the composition described above, the method comprising the step of using the Bacillus as a component of the composition.

[0027] The active ingredient in the above composition may be the above-mentioned Bacillus subtilis and / or the above-mentioned Bacillus subtilis metabolites.

[0028] The present invention also provides the use of the aforementioned Bacillus or composition in the preparation of products.

[0029] In the above applications, the product has at least one of the following properties:

[0030] B1) Antagonistic to pathogens,

[0031] B2) Prevention and control of cucumber angular leaf spot,

[0032] B3) Control of cucumber soft rot

[0033] B4) Control of cucumber vine blight

[0034] B5) Control of melon wilt disease

[0035] B6) Prevention and control of black rot in broccoli

[0036] B7) Prevention and control of clubroot disease in broccoli

[0037] B8) Growth-promoting effect on plants.

[0038] In one embodiment of the present invention, the growth-promoting or biocontrolling effect of the product on plants can be achieved through any of the following steps:

[0039] 1) Spray the cultivated plant seedlings with the Bacillus subtilis or the aforementioned inoculant.

[0040] 2) Soak the cultivated plant seeds in the Bacillus subtilis or the aforementioned inoculant.

[0041] The present invention also provides a method for treating and / or preventing plant diseases caused by pathogens, the method comprising contacting plants with the Bacillus or composition described above to treat and / or prevent plant diseases caused by pathogens.

[0042] In this invention, the microbial agent may be a pathogen inhibitor or a disease inhibitor, and the pathogen may be a pathogenic fungus or a pathogenic bacterium.

[0043] The pathogenic fungi may be Ascochyta citrullina, Rhizoctonia solani, Alternaria solani, Stemphylium solani, Phytophthora capsici Leonian, Fusarium oxysporum, and Plas modiophora brassicae.

[0044] The pathogenic bacteria may be *Pseudomonas amygdalipv. lachryrnan s*, *Xanthomonas campestris pV. campestris*, and *Pectobacterium brasiliense*.

[0045] The diseases mentioned are: 1) cucumber angular leaf spot; 2) cucumber soft rot; 3) melon wilt; 4) broccoli black rot; 5) cucumber vine blight; 6) broccoli clubroot.

[0046] The plant mentioned above can be any of the following:

[0047] C1) Angiosperms

[0048] C2) Magnoliopsida, dicotyledons, or monocotyledons

[0049] C3) Plants of the order Cucurbitales, order Brassicales, or order Poales.

[0050] C4) Plants of the Cucurbitaceae, Brassicaceae, or Poaceae families;

[0051] C5) Plants of the genera Cucurbita, Brassica, or Maize.

[0052] C6) Cucumber, melon, broccoli, cabbage, or corn.

[0053] This invention isolated and screened a strain of Bacillus subtilis ZF539 from healthy field soil. On LB agar plates, strain ZF539 colonies are grayish-white or yellowish-white with a smooth, clean surface and strong adhesion. Biolog assays and multigene phylogenetic analysis confirmed the strain as Bacillus subtilis. Pot experiments showed that seed soaking and root drenching with the fermentation broth of strain ZF539 significantly induced resistance to cucumber angular leaf spot, with control efficacies of 61.30% and 60.25%, respectively; root drenching with the fermentation broth of strain ZF539 significantly induced resistance to melon wilt, with a control efficacy of 80.76%; and root drenching with the fermentation broth of strain ZF539 significantly induced resistance to cucumber yellowing virus (CCYV), with a control efficacy of 59.49%.

[0054] In summary, strain ZF539 is a Bacillus subtilis strain with potential biocontrol application capabilities that can induce plant disease resistance. This is the first systematic and comprehensive report of Bacillus subtilis strains that have shown significant induced resistance to various bacterial, fungal, and viral diseases of cucumber under seed treatment and root irrigation treatment, and it has broad application prospects.

[0055] Preservation Instructions

[0056] Bacterial strain name: Bacillus subtilis

[0057] Latin name: Bacillus subtilis

[0058] Strain number: ZF539

[0059] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0060] Collection institution abbreviation: CGMCC

[0061] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0062] Deposit date: May 30, 2025

[0063] Registration number at the Preservation Center: CGMCC No. 34731. Attached Figure Description

[0064] Figure 1 Analysis of the antagonistic ability of ZF539 against different bacterial pathogens.

[0065] Figure 2 Analysis of the antagonistic ability of ZF539 against different fungal pathogens.

[0066] Figure 3 Photograph (A) and scanning electron microscope (SEM) image (B) of bacterial colony of strain ZF539 on LB plate.

[0067] Figure 4 This is a multigene phylogenetic tree for ZF539.

[0068] Figure 5 Laser confocal microscopy (A) and scanning electron microscopy (B) images of strain ZF539 colonized on cucumber roots 21 days after root irrigation.

[0069] Figure 6 The number of viable colonies of strains ZF539 and 168 in cucumber roots after 7 weeks of root irrigation.

[0070] Figure 7 The study investigated the effect of foliar spraying with Bacillus subtilis ZF539 on the control of cucumber angular leaf spot (potted plants). Treatment A: ZF539 foliar spraying; Treatment B: Kasugamycin foliar spraying; Treatment C: Control.

[0071] Figure 8 The study investigated the control effect of Bacillus subtilis ZF539 foliar spray on cucumber angular leaf spot (field test). A: ZF539 foliar spray treatment; B: streptomycin foliar spray treatment; C: control treatment.

[0072] Figure 9 The study investigated the effect of foliar spraying with Bacillus subtilis ZF539 on the control of soft rot in cucumbers (potted plants). Treatment A: ZF539 foliar spraying; Treatment B: Kasugamycin foliar spraying; Treatment C: Control.

[0073] Figure 10 The study investigated the effect of foliar spraying with Bacillus subtilis ZF539 on black rot control in potted broccoli. Treatment A: ZF 539 foliar spraying; Treatment B: Kasugamycin foliar spraying; Treatment C: Control.

[0074] Figure 11 The study investigated the effect of foliar spraying with Bacillus subtilis ZF539 on the control of Fusarium wilt in melons (potted plants). Treatments included: A: ZF 539 root drenching treatment; B: Carbendazim root drenching treatment; C: control treatment.

[0075] Figure 12 The study investigated the effect of Bacillus subtilis ZF539 root drenching on clubroot disease in broccoli (potted plants). Treatment A: ZF 539 root drenching; Treatment B: Fluazinam root drenching; Treatment C: Control.

[0076] Figure 13 The study investigated the effect of Bacillus subtilis ZF539 foliar spray on cucumber vine blight (potted plants). Treatments included: A: ZF539 foliar spray; B: azoxystrobin root drenching; C: control treatment.

[0077] Figure 14 This study demonstrates the effect of Bacillus subtilis ZF539 root drenching on the growth-promoting effect of Chinese cabbage. A represents the ZF539 root drenching treatment; B represents the IAA root drenching treatment; the left side represents the control, and the right side represents the treatment.

[0078] Figure 15 The effect of Bacillus subtilis ZF539 root drenching on maize growth promotion. A: ZF539 root drenching treatment; B: IAA root drenching treatment; left side is control, right side is treatment. Detailed Implementation

[0079] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0080] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0081] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0082] The pathogenic fungi in the following examples may specifically be Ascochyta citrullina, Rhizoctonia solani, Alternaria solani, Stemphylium solanum, Phytophthora capsici Leonian, Fusarium oxysporum, and Plasmodiophora brassicae; the pathogenic bacteria may be Pseudomonas amygdali pv. lachryrnans, Xanthomonas campestris pv. campestris, and Pectobacterium brasiliense.

[0083] The watermelon citrullina mentioned in the following examples has been described in: Zhao Yanjie, Li Baoju, Shi Yanxia, ​​et al. Occurrence and control of cucurbit vine blight [J]. Chinese Vegetables, 2008(02):56-57+70.

[0084] The Rhizoctonia solani in the following examples has been described in: Li Lei, Chen Lida, Huang Yishuo, Xie Xuewen, Shi Yanxia, ​​Chai Ali, Li Baoju. Establishment and application of real-time fluorescence quantitative PCR detection system for potato black scurf. Journal of Agricultural Biotechnology, 2021, 29(07):1417-1425.

[0085] The Alternaria solani in the following examples has been described in: Guo Runting, Shi Yanxia, ​​Zhao Qian, et al. Identification of Alternaria solani leaf spot pathogen in lettuce [J]. Acta Phytopathologica Sinica, 2018, 48(03):418-422.

[0086] The *Stemphylium solani* in the following examples has been described in: Xie Xuewen, Chen Lida, Cao Jinqiang, Han Daojie, Shi Yanxia, ​​Chai Ali, Li Lei, Li Baoju. Establishment and application of real-time fluorescence quantitative PCR detection method for *Stemphylium solani*. Acta Phytopathologica Sinica, 2021, 51(04):618-625. DOI:10.13926 / j.cnki.apps.000717.

[0087] The *Phytophthora capsici* Leonian in the following examples has been described in: Jiang Houchun, Lü Guohua, Shi Yanxia, ​​et al. Detection of *Phytophthora capsici* in pepper seeds using selective culture medium [J]. Chinese Vegetables, 2011(06):58-61.

[0088] The *Pseudomonas amygdalipv. lach ryrnans* species causing lacrimal lesions in the following examples has been described in: Yuan Baojie, Li Lei, Zhang Hongjie, Shi Yanxia, ​​Chai Ali, Xie Xuewen, Li Baoju. Screening of antagonistic bacteria against bacterial angular leaf spot of cucumber and their control effect. Chinese Journal of Biological Control, 2022, 38(02):421-427. DOI:10.16409 / j.cnki.2095-039x.2021.06.016.

[0089] The pathogenic tomato species *Pseudomonas syringae* pv. tomato described in the following examples has been documented in: Chai Ali, Patiguli, Guo Weitao, Shi Yanxia, ​​Xie Xuewen, Xi Xianmei, Li Baoju. Establishment and application of real-time fluorescence quantitative PCR detection method for bacterial spot pathogens of tomato. Journal of Horticulture, 2019, 46(01):182-192.

[0090] The Plasmodiophora brassicae in the following examples has been described in: Chai Ali, Zhang Siyu, Li Xiaojing, et al. Identification and efficacy of Bacillus paralichrysiforme ZF480, a biocontrol bacterium for clubroot disease in cruciferous plants [J]. Journal of Plant Protection, 2022, 49(03):938-945. DOI:10.13802 / j.cnki.zwbhxb.2022.2020208.

[0091] The above-mentioned pathogens are all available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. This biological material is only for the purpose of replicating the experiments of this invention and should not be used for any other purpose.

[0092] The data in the following examples were processed using SPSS 11.5 statistical software. The experimental results are expressed as mean ± standard deviation and were tested using a one-way ANOVA test. Different letters indicate differences.

[0093] Example 1: Isolation and screening of highly efficient biocontrol strains

[0094] 228 bacterial strains were isolated from healthy rhizosphere soil of cucumbers infected with Fusarium wilt in Shouguang, Shandong Province. The isolation method is as follows:

[0095] 1. Isolation and preservation of bacterial strains

[0096] Weigh 10g of field rhizosphere soil, dissolve it in 90mL of sterile water, and treat with a shaker at 28℃ for 30min to ensure thorough mixing. Divide the mixed soil sample into 10... -1 Up to 10 -5 Perform serial dilutions, taking 100 μL of each diluted solution to a concentration of 10... -3 10 -4 10 -5 The suspension was spread onto NA plates (10.0 g peptone, 3.0 g / L beef meal, 5.0 g / L sodium chloride, 15.0 g / L agar powder, water as solute, pH 7.2 ± 0.2), with three plates for each concentration. After incubation at 28°C for 24 hours, single colonies of different morphologies were picked and streaked onto sterile NA plates for purification until single colonies with the same morphology grew. The purified single colonies were stored in glycerol tubes for later use.

[0097] 2. Plate screening of highly efficient antagonistic strains

[0098] The filter paper disc culture method was used to analyze the antagonistic ability of 228 bacterial strains isolated from cucumber rhizosphere soil against 3 pathogenic bacteria and 7 pathogenic fungi.

[0099] Plant pathogens were selected as either fungi or bacteria. Specific fungi included *Ascochyta ci trullina*, *Rhizoctonia solani*, *Alternaria solani*, *Stemphylium solani*, *Phytophthora capsici Leonian*, and *Fusarium oxysporum*. Pathogenic bacteria included *Pseudomonas amygdalipv. lachryrnans*, *Xanthomonas campestrispV. campestris*, and *Pectobacterium brasiliense*.

[0100] The specific methods for antagonistic ability analysis are as follows:

[0101] Analysis of bacterial antagonistic ability: The fermentation broth of the above-mentioned different pathogenic bacteria cultured for 48 hours was diluted to 10. 8 CFU / mL, take 100 μL and spread it on a NA plate, then let it air dry. Place a filter paper disc in the center of the plate, add 5 μL of the test bacterial solution to the filter paper disc, and incubate at 28°C for 48 hours. Observe the diameter of the inhibition zone. Each treatment is repeated 3 times. Inhibition rate (%) = inhibition diameter / control colony diameter × 100.

[0102] Analysis of fungal antagonism: Fungal mycelium with a diameter of 8 mm was inoculated in the center of the PDA plate in the experimental group. After incubation at 28℃ for 24 h, 5 μL of the culture medium of the test strain was dropped at 4 opposite points 3 cm away from the center of the plate using the cross method. The fungal mycelium with only fungal culture was used as the control group (CK). The plate was incubated at 28℃ for 5 days. Each treatment was repeated 3 times, with 3 plates per treatment.

[0103] Of the 228 bacterial strains isolated from cucumber rhizosphere soil, one highly effective biocontrol bacterium, ZF539, was screened, exhibiting antagonistic effects against three pathogenic bacteria and seven pathogenic fungi. Its antibacterial spectrum is as follows: Figure 1 and Figure 2 As shown.

[0104] Example 2: Classification, Identification and Preservation of Bacillus subtilis ZF539

[0105] 1. Morphological observation of strain ZF539

[0106] The strain ZF539, which showed the highest preventive efficacy as selected in Example 1, was cultured on LB plates for 36 hours, and the colony morphology was observed. The results are as follows: Figure 3 As shown in Figure A, the colonies are opaque, milky white or light yellow, regularly shaped, with neat edges and occasionally slightly wavy margins, approximately 2-5 mm in diameter. The colony surface is dry, often exhibiting a rough granular texture or distinct wrinkled patterns, with a slightly raised central area. Scanning electron microscopy reveals that strain ZF539 is rod-shaped, approximately 2-3 mm long and 1-1.5 mm wide, with flagella throughout. Figure 3 (B)

[0107] 2. Physiological and biochemical assays of strain ZF539

[0108] Following the methods of Bergey's Manual of Bacterial Identification and the Manual of Systematic Identification of Common Bacteria, strain ZF539 underwent Gram staining, growth temperature test, salt tolerance test, motility test, catalase test, VP test, starch hydrolysis test, gelatin liquefaction test, citrate utilization test, nitrate reduction test, and other physiological and biochemical tests.

[0109] The test results are shown in Table 1. The results indicate that strain ZF539 is a Gram-positive bacterium, with an optimal pH of 5-6 and an optimal NaCl content of 1%, and is motile. It showed positive results for catalase, VP, and gelatin liquefaction reactions, but negative results for citrate utilization, starch hydrolysis, and nitrate reduction reactions.

[0110] Table 1. Physiological and biochemical reactions of strain ZF539

[0111]

[0112] Note: + indicates that the experimental result is positive; - indicates that the experimental result is negative.

[0113] 3. Biolog measurement

[0114] Single colonies of strain ZF539 were picked and inoculated onto LB agar slants and incubated at 28°C for 24 h. The unique carbon source utilization of strain ZF539 was determined by the China Agricultural Microbial Culture Collection Center using the BIOLOG GENIII kit (following the kit instructions).

[0115] The results are shown in Table 2: Strain ZF539 is a Gram-positive bacterium with some motility. The suitable growth environment is pH 5-6 and NaCl content of 1%-8%. Biolog results show that strain ZF517 can utilize dextran, D-maltose, sucrose, D-cellobiose, and D-trehalose, etc.

[0116] Table 2. Determination of the unique carbon source utilization of strain ZF539 using BIOLOG GENIII reagent strips

[0117]

[0118] Note: "+" indicates positive, "-" indicates negative, and "w" indicates weak positive.

[0119] 4. Construction of a multi-gene phylogenetic tree

[0120] Genomic DNA of strain ZF539 was extracted using a bacterial genomic DNA extraction kit (DP302, Tiangen Biotech (Beijing) Co., Ltd.). PCR amplification was then performed on the sequences of the bacterial 16S rDNA gene, gyrA, atpD, and rpoA genes. PCR reaction conditions were: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 45 s, 35 cycles; 72℃ extension for 10 min. The obtained PCR products were sent to Biomed Biotechnology Co., Ltd. for sequencing. A phylogenetic tree was constructed using the maximum likelihood method after alignment with MEGA 6.0 to determine the taxonomic position of strain ZF539 and analyze its genetic relationships. The multigene phylogenetic tree of ZF539 is as follows: Figure 4 .

[0121] Table 3. Primer gene sequences required for multi-gene identification

[0122]

[0123]

[0124] Strain ZF539 possesses a 16S rRNA gene with the nucleotide sequence of sequence 1 (SEQ ID NO: 1, 1510 bp) in the sequence listing, a gyrA gene with the nucleotide sequence of sequence 2 (SEQ ID NO: 2, 1025 bp) in the sequence listing, an atpD gene with the nucleotide sequence of sequence 3 (SEQ ID NO: 3, 1053 bp) in the sequence listing, and an rpoD gene with the nucleotide sequence of sequence 4 (SEQ ID NO: 4, 898 bp) in the sequence listing. A multigene phylogenetic tree was constructed based on the 16S rRNA gene, gyrA, atpD, and rpoD genes, identifying strain ZF539 as *Bacillus subtilis*.

[0125] Based on the above taxonomic characteristics, strain ZF539 was identified as Bacillus subtilis.

[0126] 5. Preservation of strain ZF539

[0127] Bacillus subtilis ZF539 was deposited on May 30, 2025, at the China General Microbiological Culture Collection Center (CGMCC; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Postcode: 100101), with accession number CGMCC No. 34731. It will be referred to as Bacillus subtilis ZF539 or strain ZF539.

[0128] Example 3: Analysis of the colonization ability of ZF539 on cucumber roots

[0129] 1. Preparation of bacterial strains

[0130] Bacillus subtilis ZF539 carrying tetracycline resistance and expressing green fluorescent protein (GFP) was used;

[0131] Bacillus subtilis standard strain 168 (ATCC 23857, kindly provided by the Institute of Biotechnology, Chinese Academy of Agricultural Sciences).

[0132] 2. Plant materials

[0133] Cucumber seedlings (Zhongnong 6) at the two-leaf-one-heart stage.

[0134] 3. Inoculation treatment

[0135] Preparation of bacterial suspension: GFP-labeled Bacillus subtilis ZF539 was inoculated into LB liquid medium containing tetracycline (50 μg / mL) and cultured at 37°C with shaking until the logarithmic growth phase (OD200). 600 ≈0.8), centrifuged, and resuspended in sterile PBS to a final concentration of 1×10⁻⁸. 8 CFU / mL was used to obtain the fermentation broth of strain ZF539.

[0136] Inoculation method: Root irrigation treatment was used, with 10 mL inoculated per cucumber seedling, followed by continued cultivation.

[0137] Sampling time points: 7, 14, 21, 28, 35, 42, 49, 56, and 63 days after inoculation, plant root samples were collected (10 plants per group).

[0138] 4. Detection Method

[0139] Take 1g of sterilized roots, grind them, suspend them in 1mL PBS, serially dilute them, spread them on LB plates containing tetracycline (50μg / mL), incubate at 37℃ for 24-48 hours, count the number of colonies (CFU / g fresh root weight), and calculate the colony density.

[0140] 5. Scanning electron microscopy (SEM) observation

[0141] Plant samples taken from the highest detection point were examined using scanning electron microscopy.

[0142] Fixation and dehydration: Root samples were fixed with 2.5% glutaraldehyde (pH 7.2) for 24 hours, dehydrated by ethanol gradient (30%-100%), and critical point drying.

[0143] Gold sputtering and imaging: After the samples were sputtered with gold film, the morphology of bacterial attachment on the root surface and inside was observed by field emission scanning electron microscopy (e.g., Hitachi SU8010) (accelerating voltage 5kV, secondary electron mode).

[0144] Strain ZF539 was observed to colonize extensively on the surface of cucumber roots under a scanning electron microscope. Figure 5 The colony size peaks 14-21 days after inoculation, reaching approximately 8 × 10⁻⁶. 6 CFU / g ( Figure 6 The minimum colony density should be maintained at 10 for 60 days after inoculation. 4 CFU / g or higher. Its colonization effect is significantly higher than that of the standard strain 168 (ATCC 23857), indicating that strain ZF539 is a strain with high colonization ability and has the potential for development into field biocontrol applications.

[0145] Example 4: Efficacy of foliar spray treatment of strain ZF539 against cucumber angular leaf spot (pot experiment)

[0146] The seeds of Zhongnong No. 6 cucumber were planted in 72-cell seedling pots and cultivated in a greenhouse until the cucumber seedlings grew to one true leaf.

[0147] The tested strain of *Pseudomonas amygdalina*, a pathogen causing cucumber angular leaf spot, was removed from the freezer at -80°C. It was thawed in an ice bath, activated at 28°C on LB agar plates, and then transferred to LB liquid medium for inoculation at 200 rpm. -1 The content of the pathogenic species Pal-nm002, which causes lacrimal tearing, in the bacterial suspension was 10. 6 cfu / mL, for later use.

[0148] The content of the cultured tonsillar pseudomonad lacrimal pathogen Pal-nm002 was 10. 6 Inoculate 10 mL of a bacterial suspension (cfu / mL) onto cucumbers at the one-true-leaf stage using a micro-sprayer. After inoculation, place the cucumbers in a humidifier and maintain a temperature of approximately 20°C and a relative humidity of 90% RH.

[0149] The following treatment was performed 12 hours after inoculation with the pathogen:

[0150] Cucumber angular leaf spot pathogen + ZF539 treatment group: 12 hours after inoculation with the pathogen, 12 cucumber plants were sprayed with 120 ml of a 100-fold diluted solution of ZF539 strain fermentation broth (concentration 1×10⁻⁶). 6 (CFU / mL, the same below). The method for preparing a 100-fold dilution of the ZF539 strain fermentation broth is to dilute the ZF539 strain fermentation broth with sterile water to a factor of 100, thus obtaining a 100-fold dilution of the ZF539 strain fermentation broth (the same below).

[0151] Cucumber angular leaf spot pathogen + kasugamycin treatment group: 12 cucumber plants were foliar sprayed with 120 ml of 3% kasugamycin wettable powder diluted 1000 times. The 3% kasugamycin wettable powder was prepared by diluting commercially purchased 3% kasugamycin with sterile water to a concentration of 1000 times (the same applies below).

[0152] Cucumber angular leaf spot pathogen treatment (control group) ZF539: 12 hours after inoculation with the pathogen, 12 cucumber plants were taken and their leaves were sprayed with 120ml of sterile water.

[0153] After inoculation, the plants were placed in a humidifier at a temperature of 26°C until the cucumber plants developed symptoms. The relative humidity was maintained at 90% RH. The presence of symptoms in the healthy control group was observed, and the disease index was recorded. The experiment was repeated three times.

[0154] The results of this experiment were repeated three times. The disease grading criteria are as follows:

[0155] Grade 0: No lesions;

[0156] Grade 1: Lesions cover less than 5% of the leaf area;

[0157] Grade 3: Lesions cover more than 5% to 25% of the leaf area;

[0158] Level 5: Lesions cover more than 25% to 50% of the leaf area;

[0159] Level 7: Lesions cover more than 50% to 75% of the leaf area;

[0160] Level 9: Disease spots cover more than 75% of the leaf area.

[0161] Data processing: Excel 2010 was used to calculate the disease index of cucumber leaves treated with each strain.

[0162] Disease index = [∑(number of diseased leaves at each level × representative value of the relative level) / (total number of leaves × representative value of the highest level)] × 100

[0163] Prevention efficacy (%) = ((Disease index of control group - Disease index of treatment group) / Disease index of control group) × 100

[0164] The results show (Table 4 and) Figure 7 The control efficacy of cucumbers treated with ZF539 fungicide against angular leaf spot was as high as 76.19%, which was higher than that of the control agent succinic acid (67.92%).

[0165] Table 4. Control efficacy of Bacillus subtilis against angular leaf spot in potted cucumbers.

[0166] deal with Disease index Relative efficacy / % Cucumber angular leaf spot fungus + ZF539 16.4±1.11a 76.19 Cucumber angular leaf spot fungus + kasugamycin 22.1±2.13b 67.92 Cucumber angular leaf spot fungus 68.9±3.21c -

[0167] Example 5: Efficacy of foliar spray treatment of strain ZF539 against cucumber angular leaf spot (field trial)

[0168] The seeds of Zhongnong No. 6 cucumber were planted in 72-cell seedling pots and cultivated in a greenhouse. When the cucumber seedlings grew to one true leaf, they were transplanted to the experimental greenhouse of the Vegetable Research Institute of the Chinese Academy of Agricultural Sciences on April 10, 2024, and watered with drip irrigation every two days. This experiment was divided into three blocks, each measuring 3m × 3m with a planting spacing of 30cm, and each block contained 16 cucumber plants.

[0169] Twenty-one days after transplanting cucumber seedlings, the tested strain of *Pseudomonas amygdalina*, a pathogen causing cucumber angular leaf spot, Pal-nm002, was removed from a -80℃ freezer. It was thawed in an ice bath, activated at 28℃ on LB agar plates, and then transferred to LB liquid medium for inoculation at 200 rpm. -1 The content of the pathogenic species Pal-nm002, which causes lacrimal tearing, in the bacterial suspension was 10. 6 CFU / mL, spray 120 ml of bacterial suspension onto the leaves using a micro-sprayer. Twelve hours after inoculation with the pathogen, perform the following treatment:

[0170] Cucumber angular leaf spot fungus + ZF539 treatment group: Take 16 cucumber plants and spray them with 320ml of 100 times diluted fermentation broth of ZF539 strain.

[0171] Cucumber angular leaf spot fungus + streptomycin treatment group: Take 16 cucumber plants and spray the leaves with 320ml of 3% streptomycin wettable powder diluted 1000 times.

[0172] Treatment of cucumber angular leaf spot pathogen (control group): Take 16 cucumber plants and spray the leaves with 320ml of sterile water.

[0173] Once the cucumber plants develop the disease, observe whether healthy controls develop the disease at a relative humidity of 90% RH, and record the disease index.

[0174] The ZF539 disease grading criteria and data processing method are the same as in Example 4. This experiment was repeated 3 times to obtain the bioassay results.

[0175] The results show (Table 5 and) Figure 8In field trials, cucumbers treated with ZF539 fungicide showed a control efficacy of up to 70.69% against angular leaf spot, which was higher than that of the control agent sclerotin (66.32%).

[0176] Table 5. Control efficacy of Bacillus subtilis against cucumber angular leaf spot in the field.

[0177] deal with Disease index Relative efficacy / % Cucumber angular leaf spot fungus + ZF539 11.4±2.23a 70.69 Cucumber angular leaf spot fungus + kasugamycin 13.1±3.35b 66.32 Cucumber angular leaf spot fungus 38.9±3.98c -

[0178] Example 6: Determination of the control efficacy of foliar spray treatment of strain ZF539 against cucumber soft rot (pot experiment)

[0179] The seeds of Zhongnong No. 6 cucumber were planted in 72-cell seedling pots and cultivated in a greenhouse until the cucumber seedlings grew to one true leaf.

[0180] The tested cucumber soft rot pathogen, *Pectinobacillus brasiliensis* SX309, stored at -80℃, was removed from the freezer. It was thawed in an ice bath, activated at 28℃ on LB agar plates, and then transferred to LB liquid medium for inoculation at 200 rpm. -1 The bacterial suspension contained approximately 10% *Pectinobacter brasiliensis* SX309. 6 CFU / mL, inoculate 10 ml of *Pectinobacter brasiliensis* SX309 suspension onto a cucumber at the true leaf stage using a micro-sprayer. After inoculation, place the plant in a humidity chamber, maintaining a temperature of approximately 20°C and a relative humidity of 90% RH.

[0181] The following treatment was performed 12 hours after inoculation with the pathogen:

[0182] Cucumber soft rot pathogen + ZF539 treatment group: Take 12 cucumber plants and spray them with 120ml of 100 times diluted fermentation broth of ZF539 strain.

[0183] Cucumber soft rot pathogen + streptomycin treatment group: Take 12 cucumber plants and spray the leaves with 120ml of 3% streptomycin wettable powder diluted 1000 times.

[0184] Treatment of cucumber soft rot pathogen (control group): Take 12 cucumber plants and spray the leaves with 120ml of sterile water.

[0185] After inoculation, the plants were placed in a humidifier at a temperature of 26°C. Once the cucumber plants developed symptoms, the relative humidity was maintained at 90% RH. The indoor healthy controls were observed to determine if they developed symptoms, and the disease index was recorded.

[0186] Twelve hours after inoculation with the pathogen, the leaves were sprayed with a 100-fold dilution of the fermentation broth of strain ZF539. After inoculation, the plants were placed in a humidifier at approximately 26°C. Once the cucumber plants developed symptoms, the relative humidity was maintained at 90% RH. The presence of disease in the healthy control group was observed, and the disease index was recorded. A 1000-fold dilution of 3% kasugamycin wettable powder was used as a control, and sterile water soaking was used as a negative control. The disease grading criteria and data processing methods were the same as in Example 4. This experiment was repeated three times.

[0187] The results show (Table 4 and) Figure 9 Cucumbers treated with ZF539 fungicide showed a control efficacy of up to 64.53% against soft rot, which was higher than that of the control agent, succinic acid (57.65%).

[0188] Table 6. Control efficacy of Bacillus subtilis against soft rot in potted cucumbers.

[0189] deal with Disease index Relative efficacy / % Cucumber soft rot pathogen + ZF539 29.4±2.02a 64.53 Cucumber soft rot bacteria + kasugamycin 35.1±1.43b 57.65 Cucumber soft rot bacteria 82.9±4.01c -

[0190] Example 7: Determination of the control efficacy of foliar spray treatment of strain ZF539 against black rot in broccoli (pot experiment)

[0191] The seeds of broccoli variety "Zhongqing 16" were planted in 72-cell seedling pots and cultivated in a greenhouse. When the broccoli seedlings had grown to two true leaves, the pathogen of black rot in broccoli, *Xanthomonas spp.*, stored at -80℃, was removed from the freezer. It was thawed in an ice bath, activated at 28℃ on LB agar plates, and then transferred to LB liquid medium for inoculation at 200 rpm. -1 The content of *Xanthomonas aeruginosa* strain in the bacterial suspension was approximately 10. 6 CFU / mL, inoculate 10 ml of bacterial suspension into broccoli at the two true leaf stage using a micro-sprayer. After inoculation, place in a humidifier and maintain a temperature of approximately 20°C and a relative humidity of 90% RH.

[0192] The following treatment was performed 12 hours after inoculation with the pathogen:

[0193] Black rot pathogen of broccoli + ZF539 treatment group: Take 12 broccoli plants and spray them with 120ml of 100 times diluted fermentation broth of ZF539 strain.

[0194] Treatment group for black rot of broccoli + kasugamycin: Take 12 broccoli plants and spray them with 120ml of 2% kasugamycin wettable powder.

[0195] Treatment of black rot in broccoli (control group): Take 12 broccoli plants and spray the leaves with 120ml of sterile water.

[0196] After inoculation, the plants were placed in a humidifier at a temperature of 26°C. Once the cucumber plants developed symptoms, the relative humidity was maintained at 90% RH. The indoor healthy controls were observed to determine if they developed symptoms, and the disease index was recorded.

[0197] The ZF539 disease grading criteria and data processing method are the same as in Example 4. This experiment was repeated 3 times to obtain the bioassay results.

[0198] The results show (Table 7 and) Figure 10 The control efficacy of broccoli treated with ZF539 microbial agent against black rot reached 54.69%, which was higher than that of the control agent kasugamycin (44.62%).

[0199] Table 7. Control efficacy of Bacillus subtilis against black rot in potted broccoli.

[0200] deal with Disease index Relative efficacy / % Broccoli black rot fungus + ZF539 24.44±2.31a 54.69 Broccoli black rot fungus + Kasugamycin 29.87±1.86b 44.62 Broccoli black rot fungus 53.94±4.32c -

[0201] Example 8: Determination of the control efficacy of ZF539 root irrigation treatment against Fusarium wilt in melons.

[0202] The biocontrol strain ZF539, preserved in a -80℃ storage tube, was activated on LB agar plates using the streak method. After incubation at 28℃ for 1 day, a single colony was picked and cultured in a shaker tube at 28℃ and 180 rpm for 18 hours. The bacterial culture was then diluted to OD. 600 Approximately 0.8.

[0203] Selected colonies of *Fusarium oxysporum* were placed in PD medium and incubated on a shaker at 28°C for 7 days. The culture broth was then filtered through two layers of gauze to obtain a spore suspension. The *Fusarium oxysporum* spore suspension was diluted to a spore concentration of 1×10⁻⁶ using a hemocytometer. 6 Spores / mL.

[0204] Pathogen soil was prepared according to the ratio of 1L of spore suspension to 4L of substrate. The melons were planted in 72-cell seedling trays and induced to grow in a greenhouse.

[0205] On days 1, 5, and 10 after sowing, perform the following treatments:

[0206] Melon wilt pathogen + ZF539 treatment group: Take 24 melon plants, and spray each melon plant's leaves with 20 mL of 100 times diluted fermentation broth of strain ZF539.

[0207] Melon wilt pathogen + kasugamycin treatment group: 24 melon plants were used, and each plant was drenched with 20 ml of a 500-fold dilution of 60% carbendazim wettable powder. The 500-fold dilution of 60% carbendazim wettable powder was prepared by diluting commercially purchased 60% carbendazim with sterile water to a concentration of 500 times.

[0208] Melon wilt pathogen (control group): Take 24 melon plants and irrigate the roots with 20ml of sterile water.

[0209] After inoculation, the plants were placed in a humidifier at a temperature of 26°C. Once the cucumber plants developed symptoms, the relative humidity was maintained at 90% RH. The indoor healthy controls were observed to determine if they developed symptoms, and the disease index was recorded.

[0210] The ZF539 disease grading criteria are as follows. The results of this experiment were repeated three times:

[0211] Level 0: No symptoms;

[0212] Grade 1: Cotyledons slightly wilted;

[0213] Grade 2: Cotyledons severely wilted;

[0214] Level 3: True leaves wilting;

[0215] Level 4: The entire plant is slightly wilted;

[0216] Level 5: The entire plant dies or fails to sprout.

[0217] Data processing: Excel 2010 was used to calculate the disease index of melon plants treated with each strain.

[0218] Disease index = [Number of plants at each level * Representative value of that level] * 100 / [Total number of plants surveyed * Representative value of the highest level].

[0219] The results show (Table 8 and) Figure 11 The control efficacy of melons treated with ZF539 fungicide against Fusarium wilt was as high as 64.87%, which was higher than that of the control fungicide carbendazim (54.38%).

[0220] Table 8. Control efficacy of Bacillus subtilis ZF539 against Fusarium wilt in melon.

[0221] deal with Disease index Relative efficacy / % Melon wilt pathogen + ZF539 24.34±1.89a 64.87 Melon wilt fungus + carbendazim 31.61±3.29b 54.38 Melon wilt fungus 69.29±5.21c -

[0222] Example 9: Determination of the control efficacy of ZF539 root irrigation treatment against clubroot disease in broccoli

[0223] Remove the swollen roots of the cabbage from the refrigerator, thaw at room temperature, and then place them in a blender with an appropriate amount of sterile water to blend. Filter the clubroot suspension through four layers of gauze into a new, clean container to obtain a bacterial suspension of *Plasmodiophora brassicae*, the pathogen causing clubroot disease in broccoli. Thoroughly mix the prepared bacterial suspension with the substrate to achieve a *Plasmodiophora brassicae* dormant spore concentration of 10 in the soil. 8 Spores / L substrate.

[0224] The experiment was set up with 3 treatments, 15 seedlings in each treatment, and three replicates, as follows:

[0225] The broccoli clubroot pathogen + ZF539 treatment group: Before sowing, spray the substrate surface with a 100-fold dilution of the fermentation broth of strain ZF539, and irrigate the roots of each broccoli plant with 10 mL of the solution. Fifteen days after sowing, irrigate the roots again with a 100-fold dilution of the fermentation broth of strain ZF539, and administer 50 mL of the solution to each plant.

[0226] Treatment group of clubroot pathogen of broccoli + 500g / L fluazinam suspension: Before sowing, spray 500g / L fluazinam suspension on the substrate surface, and drench each broccoli plant with 10mL of suspension. 15 days after sowing, spray 500g / L fluazinam suspension on the substrate surface again, and administer 50mL of suspension to each plant.

[0227] The broccoli clubroot pathogen treatment group (control): Before sowing, sterile water was sprayed onto the substrate surface, and each broccoli plant was inoculated with 10 mL of sterile water by root irrigation. Fifteen days after sowing, each plant was again inoculated with 50 mL of sterile water. ZF539

[0228] After sowing, place the seedlings in a greenhouse at around 20℃. Water them for the first 14 days to maintain 100% soil moisture. After 14 days, water as needed and pay attention to the prevention and control of diseases and pests. About 30 days after sowing, after the control group in clean water showed uniform disease, investigate the disease situation and calculate the disease index.

[0229] Investigation and grading standards for clubroot disease in broccoli:

[0230] Grade 0, no swelling at the root;

[0231] Grade 1: Swollen roots are attached only to lateral roots, accounting for 1% to 25% of the entire root system;

[0232] Grade 2, with swollen roots attached to the main root and swollen roots accounting for more than 25% to 50% of the lateral roots;

[0233] Grade 3: Swollen roots are attached to the main root, and the swollen roots account for more than 50% to 75% of the root system;

[0234] Grade 4, with root swellings attached to the main root, and the swollen roots accounting for more than 75% of the root system.

[0235] Incidence rate (%) = (Number of diseased plants treated / Total number of plants treated) * 100

[0236] Disease index = [Number of plants at each level * Representative value for that level] * 100 / [Total number of plants surveyed * Representative value for the highest level]

[0237] Efficacy (%) = (Control disease index - Treatment disease index) / Control disease index × 100

[0238] The results show (Table 9 and) Figure 12 The control efficacy of ZF539 inoculant for clubroot disease in broccoli was as high as 61.49%, which was slightly lower than that of the control agent fluazinam (70.46%).

[0239] Table 9. Control efficacy of Bacillus subtilis ZF539 against clubroot disease in broccoli

[0240] deal with Disease index Relative efficacy / % Clubroot of broccoli + ZF539 28.34±2.19b 61.49 Broccoli clubroot pathogen + 500g / L fluazinam suspension 21.74±1.31a 70.46 broccoli clubroot 73.61±6.09c -

[0241] Example 10: Determination of the control efficacy of foliar spray treatment of strain ZF539 against cucumber anthracnose (pot experiment)

[0242] The seeds of Zhongnong No. 6 cucumber were planted in 72-cell seedling pots and cultivated in a greenhouse until the cucumber seedlings grew to one true leaf.

[0243] After transferring the cucumber vine blight pathogen *Diplosporium hygroscopicum* to PDA plates and culturing for 4 days, holes were punched along the edge of the colonies using a sterile punch with a diameter of 0.8 cm. Fresh, undamaged cucumbers were selected, rinsed thoroughly with tap water, wiped with 75% alcohol, and air-dried. The skin was then lightly removed from the fruit surface using a sterile punch with a diameter of 0.8 cm. Mycelial blocks of different cultured strains were placed on these blocks and placed directly under natural diffused light. The cucumbers were then cultured for 4 days at 18-22℃ (generally in a shaded indoor environment) with a light intensity <1000 lx.

[0244] Spray the bacterial suspension evenly onto the cucumber seedlings using a spray bottle, and cultivate them in a sealed greenhouse at 22-27℃ for 24-48 hours while maintaining humidity. Regularly observe the seedling disease situation, and calculate the disease incidence rate, disease index, and control effect.

[0245] 4. Inoculation treatment,

[0246] Twelve hours after inoculation with the pathogen, each treatment was sprayed with 10 mL of a 100-fold dilution of the fermentation broth of strain ZF539. After inoculation, the plants were placed in a humidifier at approximately 26°C. Once the cucumber plants developed symptoms, the relative humidity was maintained at 90% RH. The presence of disease in the healthy controls was observed, and the disease index was recorded. A 50% azoxystrobin water-dispersible granule was used as a control, and sterile water soaking was used as a negative control. The disease grading criteria are as follows. This experiment was repeated three times.

[0247] 5. Prevention efficacy survey

[0248] Disease severity grading criteria:

[0249] Diseased leaf grading criteria: Observe the plant growth status daily, record the disease occurrence, and determine the disease level.

[0250] Leaf disease severity grading criteria:

[0251] Level 0: Healthy seedlings;

[0252] Grade 1: Lesions cover less than 5% of the cotyledon area;

[0253] Grade 3: Lesions cover more than 5-10% of the cotyledon area;

[0254] Level 5: Lesions cover more than 10-25% of the cotyledon area;

[0255] Level 7: Lesions cover more than 25-50% of the cotyledon area;

[0256] Level 9: Lesions cover more than 51% of the cotyledon area.

[0257] Disease index = [∑(number of diseased leaves at each level × representative value of the relative level) / (total number of leaves × representative value of the highest level)] × 100

[0258] Prevention efficacy = [(average disease index of control - average disease index of treatment) / average disease index of control] × 100%

[0259] The results show (Table 10 and) Figure 13 Cucumbers treated with ZF539 fungicide showed a control efficacy of up to 53.96% against anthracnose, which was higher than that of the control agent azoxystrobin (45.12%).

[0260] Table 10. Control efficacy of Bacillus subtilis against vine blight in potted cucumbers.

[0261] deal with Disease index Relative efficacy / % Cucumber wilt pathogen + ZF539 22.24±1.21a 53.96 Cucumber wilt bacterium + azoxystrobin 26.51±2.67b 45.12 Cucumber vine blight 48.31±3.45c -

[0262] Example 10: Evaluation of the effect of root irrigation treatment with strain ZF539 on promoting the growth of Chinese cabbage.

[0263] Seeds of fast-growing, green-flowering Chinese cabbage were planted in 72-cell seedling pots and cultivated in a greenhouse. When the cabbage reached the two-leaf, one-heart stage, ZF539 treatment was initiated. Each treatment had three biological replicates, with 20-30 seedlings per replicate. The specific treatments are as follows:

[0264] ZF539 treatment: drench each cabbage plant with 10 mL of a 100-fold fermented dilution of ZF539; perform a second root drench on the 5th day after the first drench, drench each cabbage plant with 10 mL of the 100-fold fermented dilution of ZF539 during the second drench.

[0265] IAA treatment: 10 mL of 10 ng / ml IAA solution was applied to the roots of each cabbage plant; a second root drench was performed on the 5th day after the first root drench, with 10 mL of 10 ng / ml IAA solution applied to the roots of each cabbage plant during the second root drench.

[0266] CK treatment: 10 mL of sterile water was applied to the roots of each cabbage plant; a second root drench was performed on the 5th day after the first root drench, with 10 mL of sterile water applied to the roots of each cabbage plant during the second root drench.

[0267] The growth of the cabbage was observed daily. On the 7th day after the second treatment, the height and fresh weight of the cabbage plants were measured and photographed.

[0268] The results showed that strain ZF539 had a significant growth-promoting effect on Chinese cabbage (Table 11 and 2019). Figure 14 This drug significantly increased the plant height and above-ground fresh weight of Chinese cabbage, with growth promotion rates reaching 29.54% and 24.41% respectively, which were higher than the growth promotion effect of the IAA control agent.

[0269] Table 11. Growth-promoting effect of strain ZF539 on Chinese cabbage

[0270]

[0271]

[0272] Example 11: Evaluation of the effect of root irrigation treatment with strain ZF539 on promoting maize growth.

[0273] Nongke Yu 368 maize seeds were planted in 36-cell seedling pots and cultivated in a greenhouse. Three weeks after sowing, when the Jingkenuo 2000 maize seedlings had grown to two leaves and one bud, root irrigation treatment was initiated. Each treatment was set up with three biological replicates, with 18 seedlings per replicate. The specific treatments are as follows:

[0274] ZF539 treatment: Drench each corn plant with 10 mL of a 100-fold fermented dilution of ZF539; perform a second drench on the 5th day after the first drench, drench each corn plant with 10 mL of the 100-fold fermented dilution of ZF539 during the second drench.

[0275] IAA treatment: 10 mL of 10 ng / ml IAA solution was applied to the roots of each corn plant; a second root drench was performed on the 5th day after the first root drench, with 10 mL of 10 ng / ml IAA solution applied to the roots of each corn plant during the second root drench.

[0276] CK treatment: each corn plant was irrigated with 10 mL of sterile water; a second irrigation was performed on the 5th day after the first irrigation, with each corn plant receiving 10 mL of sterile water during the second irrigation.

[0277] The growth of the corn was observed daily, and the plant height and fresh weight were measured on the 7th day after the second treatment, and photos were taken for record-keeping.

[0278] The results showed that strain ZF539 had a significant growth-promoting effect on maize (Table 12 and 1333). Figure 15 It can significantly increase the plant height and root length of maize, with growth promotion rates reaching 6.06% and 21.77% respectively, which are higher than the growth promotion effect of the IAA control agent.

[0279] Table 12. Effects of strain ZF539 on maize growth promotion.

[0280] deal with Plant height / cm growth rate(%) Root length / / cm growth rate(%) CK 33.97±0.82c - 22.50+0.39b - ZF539 36.03+0.38b 6.06 27.40+0.26b 21.77 IAA 35.00+0.42a 3.03 26.17+0.36a 16.31

[0281] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Bacillus, characterized in that, The Bacillus species is Bacillus subtilis ( Bacillus subtilis ZF539 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34731.

2. The composition, characterized in that, The composition contains the Bacillus of claim 1.

3. The composition according to claim 2, characterized in that, The composition is a culture, which is a substance obtained by culturing the Bacillus in a microbial culture medium.

4. The composition according to claim 2, characterized in that, The composition is a microbial agent.

5. The composition according to any one of claims 2-4, characterized in that, The composition has antimicrobial properties.

6. The composition according to any one of claims 2-4, characterized in that, The composition has at least one of the following properties: A1) Prevention and control of cucumber angular leaf spot, A2) Prevention and control of cucumber soft rot. A3) Control cucumber vine blight A4) Control of melon wilt disease A5) Prevention and control of black rot in broccoli A6) Prevention and control of clubroot disease in broccoli A7) Growth-promoting effect on plants.

7. A method for preparing the composition, characterized in that, The composition is the composition according to any one of claims 2-6, and the method includes the step of using the Bacillus of claim 1 as a component of the composition.

8. The use of the Bacillus of claim 1 or the composition of claim 2 or 3 in the preparation of a product, wherein the product is a microbial agent or microbial fertilizer; The bacterial agent has the property of antagonizing pathogens, wherein the pathogens are pathogenic fungi or pathogenic bacteria; The pathogenic fungus is Diplostomum hygroscopicum (Dictyophora indica). Ascochyta citrullina Rhizoctonia solani ( ), Rhizoctonia solani ), Alternaria solani ( Alternaria solani ), eggplant stalk mold ( Stemphylium solani ), Phytophthora capsici ( Phytophthora capsici Leonian, Fusarium oxysporum ( Fusarium oxysporum ) and Plasmodium brassicae ( Plasmodiophora brassicae ); The pathogenic bacteria is *Pseudomonas tonsillar*, a species that causes lacrimal lacrimation. Pseudomonas amygdali pv lachrymans Xanthomonas aeruginosa ( Xanthomonas campestris pv. campestris ) and Brazilian pectinobacterium ( Pectobacterium brasiliense ); The microbial fertilizer has a growth-promoting effect on corn and / or cabbage.

9. A method for preventing and controlling plant diseases caused by pathogens, characterized in that, The method includes contacting plants with the Bacillus of claim 1 or the composition of any one of claims 2-6 to prevent plant diseases caused by pathogens; The pathogen is a pathogenic fungus or a pathogenic bacterium; The pathogenic fungus is Diplosporum canis (Watermelon shell dispora). Ascochyta citrullina Rhizoctonia solani ( ), Rhizoctonia solani Alternaria solanacearum ( Alternaria solani ), eggplant stalk mold ( Stemphylium solani ), Phytophthora capsici ( Phytophthora capsici Leonian Fusarium oxysporum ( Fusarium oxysporum ) and Plasmodium brassicae ( Plasmodiophora brassicae ); The pathogenic bacteria is *Pseudomonas tonsillar*, a species that causes lacrimal lacrimation. Pseudomonas amygdali pv lachrymans Xanthomonas aeruginosa ( Xanthomonas campestris pv. campestris ) and Brazilian pectinobacterium ( Pectobacterium brasiliense The plant in question is either a plant of the Cucumber genus or a plant of the Brassica genus.

10. The method according to claim 9, characterized in that, The Cucumber species are cucumbers or melons; the Brassica species are broccoli or Chinese cabbage.

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