Efficient antagonistic bacillus subtilis ZF539 and biocontrol application thereof in various vegetable diseases

Abstract

The invention discloses an efficient antagonistic bacillus subtilis ZF539 strain and a biocontrol application of the efficient antagonistic bacillus subtilis ZF539 strain in various vegetable diseases. The invention relates to the field of microorganisms, in particular to bacillus subtilis with an efficient antagonistic characteristic and application of the bacillus subtilis to prevention and treatment of various vegetable diseases. The bacillus subtilis disclosed by the invention is bacillus subtilis ZF539, and the preservation number of the bacillus subtilis in the China General Microbiological Culture Collection Center is CGMCC (China General Microbiological Culture Collection Center) No. 34731. The strain has the characteristics of efficient colonization, disease control, seedling growth promotion and the like, and has huge development and application potential as a biocontrol bacterium.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms, in particular to a high-efficiency antagonistic Bacillus subtilis ZF539 and its biocontrol application in preventing and controlling various vegetable diseases. BACKGROUND

[0002] Plant diseases are an important bottleneck restricting the sustainable development of the global vegetable industry. Long-term reliance on chemical pesticides has led to problems such as increased pathogen resistance, soil micro-ecological imbalance, and environmental pollution. Bacillus subtilis is an environmentally friendly biocontrol agent that is widely used in agricultural disease control due to its broad-spectrum antibacterial activity and safety. However, existing strains generally have low field colonization efficiency, poor field application effect, and relatively single target for prevention and control, which severely limits their industrial application effect.

[0003] Based on the above background, screening of biocontrol strains with high antagonistic properties against multiple plant pathogens and significant field application effect can provide important biocontrol strain resources for the development of new microbial agents that can prevent and control multiple plant diseases. SUMMARY

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

[0005] To solve the above technical problems, the present application first provides a Bacillus subtilis ZF539, which has a preservation number of CGMCC No. 34731 at the China General Microbiological Culture Collection Center.

[0006] The present application also provides a composition containing the Bacillus subtilis described above.

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

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

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

[0010] The above-mentioned composition can have at least one of the following properties:

[0011] A1) antagonizing pathogenic fungi,

[0012] A2) preventing and treating cucumber angular leaf spot,

[0013] A3) preventing and treating cucumber soft rot,

[0014] A4) preventing and treating cucumber gummy stem blight,

[0015] A5) preventing and treating melon fusarium wilt,

[0016] A6) preventing and treating black rot of broccoli,

[0017] A7) preventing and treating clubroot of broccoli,

[0018] A8) promoting plant growth.

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

[0020] The above-mentioned microbial agent or microbial fertilizer can be used for soil application or seed soaking.

[0021] The active ingredients of the above-mentioned microbial agent can also contain other biological components or non-biological components, and the other active ingredients of the above-mentioned microbial agent can be determined by those skilled in the art according to the inhibition effect on diseases.

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

[0023] The bacterial agent can be in various forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0024] If necessary, surfactants (such as Tween 20, Tween 80, etc.), adhesives, stabilizers (such as antioxidants), pH regulators, etc. can be added to the bacterial agent.

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

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

[0027] The active ingredient of the composition described above can be the Bacillus subtilis described above and / or the metabolite of the Bacillus subtilis described above,

[0028] The present application also provides the use of the Bacillus or the composition described above in the preparation of a product.

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

[0030] B1) antagonizing pathogenic bacteria,

[0031] B2) preventing and treating cucumber angular leaf spot,

[0032] B3) preventing and treating cucumber soft rot,

[0033] B4) preventing and treating cucumber gummy stem blight,

[0034] B5) preventing and treating melon fusarium wilt,

[0035] B6) preventing and treating black rot of broccoli,

[0036] B7) preventing and treating clubroot of broccoli,

[0037] B8) promoting the growth of plants.

[0038] In an embodiment of the present application, the promoting growth or biocontrol effect of the product on plants can be achieved by any of the following steps:

[0039] 1) spraying the above-mentioned Bacillus subtilis or the above-mentioned bacterial agent to the seedling of the cultivated plant.

[0040] 2) treating the seed of the cultivated plant with the above-mentioned Bacillus subtilis or the above-mentioned bacterial agent.

[0041] The present application also provides a method for treating and / or preventing the plant disease caused by the pathogenic bacteria, which comprises contacting the plant with the above-mentioned Bacillus or the above-mentioned composition to treat and / or prevent the plant disease caused by the pathogenic bacteria.

[0042] In the present application, the bacterial agent can be a pathogenic bacteria inhibitor or a disease inhibitor, and the pathogenic bacteria can be pathogenic fungi or pathogenic bacteria.

[0043] The pathogenic fungi can be Ascochyta citrullina, Rhizoctonia solani, Alternaria solani, Stemphylium solani, Phytophthora capsici Leonian, Fusarium oxysporum and Plasmodiophora brassicae;

[0044] The pathogenic bacteria can be Pseudomonas amygdali pv. lachrymans, Xanthomonas campestris pv. campestris and Pectobacterium brasiliense.

[0045] The disease can be: 1) cucumber angular spot; 2) cucumber soft rot; 3) melon wilt; 4) black rot of broccoli; 5) cucumber wilt; 6) broccoli clubroot.

[0046] In the above, the plant can be any one of the following:

[0047] C1) angiosperm,

[0048] C2) Magnoliidae or Dicotyledoneae or Monocotyledoneae,

[0049] C3) Cucurbitales or Brassicales or Poales plant,

[0050] C4) Cucurbitaceae or Brassicaceae or Poaceae plant;

[0051] C5) Cucumis or Brassica or Zea plant,

[0052] C6) cucumber or melon or broccoli or cabbage or maize.

[0053] The present application isolates and screens a bacillus subtilis ZF539 from healthy field soil. The strain ZF539 shows grayish or yellowish white colonies on LB plates, smooth and clean surface, and strong adhesion. Biolog determination and multi-gene phylogenetic analysis identify the strain as bacillus subtilis. The results of pot experiment show that the seed soaking and root irrigation treatment of strain ZF539 fermentation liquor have significant induced disease resistance effect on cucumber angular spot disease, and the control effects are 61.30% and 60.25% respectively; the root irrigation treatment of strain ZF539 fermentation liquor has significant induced disease resistance effect on melon fusarium wilt, and the control effect is 80.76%; the root irrigation treatment of strain ZF539 fermentation liquor has significant induced disease resistance effect on cucumber chlorotic yellow virus disease (CCYV), and the control effect is 59.49%.

[0054] In summary, strain ZF539 is a potential biocontrol application potential bacillus subtilis with induced plant disease resistance, which is the first systematic and comprehensive report of bacillus subtilis with obvious induced resistance effect on cucumber bacterial, fungal and viral diseases from seed treatment and root irrigation treatment, and has wide application prospect.

[0055] DEPOSIT DESCRIPTION

[0056] Strain name: bacillus subtilis

[0057] Latin name: Bacillus subtilis

[0058] Strain number: ZF539

[0059] Preservation agency: China General Microbiological Culture Collection Center

[0060] Abbreviation of preservation agency: CGMCC

[0061] Address: No.3, Beichen West Road, Beijing

[0062] Preservation date: May 30, 2025

[0063] Preservation center registration number: CGMCC No.34731. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1 Analysis of the antagonistic ability of ZF539 to different bacterial pathogenic bacteria.

[0065] Figure 2 Analysis of the antagonistic ability of ZF539 to different fungal pathogenic bacteria.

[0066] Figure 3 Figure 1. Colony photograph (A) and scanning electron microscope image (B) of strain ZF539 LB plate.

[0067] Figure 4 Figure 2. Phylogenetic tree of multi-genes of ZF539.

[0068] Figure 5 Figure 3. Laser confocal (A) and scanning electron microscope (B) images of strain ZF539 colonization on cucumber roots at 21 days after drenching.

[0069] Figure 6 Figure 4. Viable counts of strain ZF539 and 168 colonization on cucumber roots within 7 weeks after drenching.

[0070] Figure 7 Figure 5. Control effect of Bacillus subtilis ZF539 foliar spraying on cucumber angular leaf spot (pot). A: ZF539 foliar spraying; B: zhongshengjin foliar spraying; C: control.

[0071] Figure 8 Figure 6. Control effect of Bacillus subtilis ZF539 foliar spraying on cucumber angular leaf spot (field). A: ZF539 foliar spraying; B: zhongshengjin foliar spraying; C: control.

[0072] Figure 9 Figure 7. Control effect of Bacillus subtilis ZF539 foliar spraying on cucumber soft rot (pot). A: ZF539 foliar spraying; B: zhongshengjin foliar spraying; C: control.

[0073] Figure 10 Figure 8. Control effect of Bacillus subtilis ZF539 foliar spraying on broccoli black rot (pot). A: ZF539 foliar spraying; B: chunlei mycin foliar spraying; C: control.

[0074] Figure 11 Figure 9. Control effect of Bacillus subtilis ZF539 foliar spraying on melon fusarium wilt (pot). A: ZF539 drenching; B: carbendazim drenching; C: control.

[0075] Figure 12 Figure 10. Control effect of Bacillus subtilis ZF539 drenching on broccoli clubroot (pot). A: ZF539 drenching; B: flonicamid drenching; C: control.

[0076] Figure 13 Figure 11. Control effect of Bacillus subtilis ZF539 foliar spraying on cucumber gummy stem blight (pot). A: ZF539 foliar spraying; B: kresoxim-methyl drenching; C: control.

[0077] Figure 14 Promoting effect of B. subtilis ZF539 root irrigation treatment on Chinese cabbage. A: ZF539 root irrigation treatment; B: IAA root irrigation treatment; left side is control, right side is treatment.

[0078] Figure 15 Promoting effect of B. subtilis ZF539 root irrigation treatment on corn. A: ZF539 root irrigation treatment; B: IAA root irrigation treatment; left side is control, right side is treatment. DETAILED DESCRIPTION

[0079] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the application.

[0080] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0081] In the quantitative experiments in the following examples, three repeated experiments are set, unless otherwise specified.

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

[0083] In the following examples, Ascochyta citrullina has been recorded in Zhao YJ, Li BQ, Shi YX, et al. Occurrence and control of melon wilt [J]. China Vegetables, 2008(02): 56-57+70.

[0084] Rhizoctonia solani in the following examples has been described in: Li, L., Chen, L. D., Huang, Y. S., Xie, X. W., Shi, Y. X., Chai, A. L., and Li, B. J. Establishment and application of real-time fluorescent quantitative PCR detection system for Rhizoctonia solani. Chinese Journal of Agricultural Biotechnology, 2021, 29(07): 1417-1425.

[0085] Alternaria solani in the following examples has been described in: Guo, R. T., Shi, Y. X., Zhao, Q., et al. Pathogen identification of Alternaria solani on lettuce. Plant Disease and Health, 2018, 48(03): 418-422.

[0086] Stemphylium solani in the following examples has been described in: Xie, X. W., Chen, L. D., Cao, J. Q., Han, D. J., Shi, Y. X., Chai, A. L., Li, L., and Li, B. J. Establishment and application of real-time fluorescent quantitative PCR detection method for Stemphylium solani. Plant Disease Report, 2021, 51(04): 618-625. DOI: 10.13926 / j.cnki.apps.000717.

[0087] Phytophthora capsici Leonian in the following examples has been described in: Jiang, H. C., Lv, G. H., Shi, Y. X., et al. Detection of Phytophthora capsici Leonian on pepper seeds by selective medium. Chinese Vegetables, 2011(06): 58-61.

[0088] Pseudomonas amygdali pv. lachrymans in the following examples has been described in: Yuan, B. J., Li, L., Zhang, H. J., Shi, Y. X., Chai, A. L., Xie, X. W., and Li, B. J. Screening of antagonistic bacteria against cucumber bacterial angular leaf spot and its control effect. Chinese Journal of Biological Control, 2022, 38(02): 421-427. DOI: 10.16409 / j.cnki.2095-039x.2021.06.016.

[0089] Pseudomonas syringae pv. tomato in the following examples has been described in: Chai, A. L., Patiguli, G. W. T., Shi, Y. X., Xie, X. W., Xi, S. M., and Li, B. J. Establishment and application of real-time fluorescent quantitative PCR detection method for Pseudomonas syringae pv. tomato. Acta Horticulturae Sinica, 2019, 46(01): 182-192.

[0090] The Plasmodiophora brassicae in the following examples has been described in: Chai A, Zhang SY, Li XJ, et al. Identification and control efficiency of biocontrol bacteria Bacillus paralicheniformis ZF480 against clubroot of Brassicaceae [J]. Plant Protection Science, 2022, 49(03): 938-945. DOI:10.13802 / j.cnki.zwbhxb.2022.2020208.

[0091] The above pathogenic bacteria are available to the public from the Chinese Academy of Agricultural Sciences, Institute of Vegetables and Flowers. This biological material is only used for repeating the experiments of the present application and cannot be used for other purposes.

[0092] The following examples use SPSS 11.5 statistical software to process the data, and the experimental results are expressed as mean ± standard deviation, using One-way ANOVA test, and different English letters represent differences.

[0093] Example 1, isolation and screening of high-efficiency biocontrol strains

[0094] 228 bacterial strains were isolated from healthy rhizosphere soil of cucumber fusarium wilt in Shouguang, Shandong. The isolation method is as follows:

[0095] 1. Isolation and preservation of strains

[0096] Take 10 g of field rhizosphere soil and dissolve it in 90 mL of sterile water, and treat it with a constant temperature shaker at 28°C for 30 min to mix it thoroughly. Dilute the mixed soil sample by 10 -1 to 10 -5 gradient, and take 100 μL of the suspension with dilution concentrations of 10 -3 , 10 -4 , 10 -5 and spread them on NA plates (10.0 g / L proteose peptone, 3.0 g / L beef powder, 5.0 g / L sodium chloride, 15.0 g / L ribose powder, solvent water, pH 7.2±0.2), 3 plates for each concentration, and incubate at 28°C for 24 h, then pick different morphological single colonies on sterile NA plates and streak them for purification until single colonies with the same morphology are obtained. After single colony purification, store them in glycerol tubes for future use.

[0097] 2. Plate screening of high-efficiency antagonistic strains

[0098] The 228 bacterial strains isolated from the rhizosphere soil of cucumber were subjected to antagonistic ability analysis against 3 pathogenic bacteria and 7 pathogenic fungi using filter paper piece culture method.

[0099] The plant pathogenic bacteria are specifically Pseudomonas amygdali pv. lachrymans, Xanthomonas campestris pv. campestris and Pectobacterium brasiliense.

[0100] The specific method for analyzing antagonistic ability is as follows:

[0101] Analysis of antagonistic ability of bacteria: the fermentation broth of the above different pathogenic bacteria cultured for 48 hours was diluted to 10 8 CFU / mL, 100 μL was coated on the NA plate and dried. Filter paper was pasted in the middle of the plate, 5 μL of the tested bacterial solution was added to the filter paper, and the plate was placed in a 28°C incubator for 48 hours. The diameter of the inhibition zone was observed. Each treatment was repeated 3 times. Inhibition rate (%) = inhibition diameter / control colony diameter x 100.

[0102] Analysis of antagonistic ability of fungi: a fungal cake with a diameter of 8 mm was inoculated in the center of the PDA plate in the experimental group, and after 24 hours of culture at 28°C, 5 μL of the culture solution of the tested strain was added to the opposite 4 points at a distance of 3 cm from the center of the plate by cross intersection method. The cake inoculated with only fungi was used as a control group (CK), and the culture was carried out at 28°C for 5 days. Each treatment was repeated 3 times, and each repetition was 3 plates.

[0103] From 228 bacterial strains isolated from the rhizosphere soil of cucumber, 1 strain of high-efficiency biocontrol bacteria ZF539 with antagonistic effect on 3 kinds of pathogenic bacteria and 7 kinds of pathogenic fungi was screened. The inhibition spectrum results are shown in Figure 1 and Figure 2 .

[0104] Example 2, classification and identification of Bacillus subtilis ZF539

[0105] 1. Morphological observation of strain ZF539

[0106] The strain ZF539 with the highest control effect screened in Example 1 above was cultured on LB plate for 36 hours, and the colony morphology was observed. The results are shown in Table 1. Figure 3 As shown in Table 1, the colony morphology was opaque, milky white or light beige, regular, with neat edges and occasionally slightly wavy, about 2-5 mm in diameter. The colony surface was dry, often rough and granular or with obvious wrinkled texture, and the central area was slightly raised. Scanning electron microscopy showed that the strain ZF539 was rod-shaped, about 2-3 mm long and 1-1.5 mm wide, with flagella all over the body. Figure 3

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

[0108] According to the methods of "Berger's Bacterial Identification Manual" and "Common Bacterial System Identification Manual", the strain ZF539 was subjected to physiological and biochemical tests such as Gram staining, growth temperature test, salt tolerance test, motility test, contact enzyme test, V-P test, starch hydrolysis test, gelatin liquefaction test, citrate utilization test, nitrate reduction test, etc.

[0109] The test results are shown in Table 1. The results show that the strain ZF539 is a gram-positive bacterium, with an optimal pH of 5-6 and an optimal NaCl content of 1%. It has motility. The contact enzyme, V-P and gelatin liquefaction reactions are positive, and the citrate utilization, starch hydrolysis and nitrate reduction reactions are negative.

[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 test

[0114] A single colony of strain ZF539 was inoculated into an LB culture medium test tube slope and cultured at 28°C for 24 hours. The BIOLOG GENIII kit (operated according to the kit instructions) was used by the China Agricultural Microbial Culture Collection Center to determine the utilization of unique carbon sources by strain ZF539.

[0115] The results are shown in Table 2: the strain ZF539 is a gram-positive bacterium with certain motility. The optimal growth environment is pH 5-6 and NaCl content of 1%-8%. The Biolog results show that the strain ZF517 can utilize glucan, D-maltose, sucrose, D-cellubiose and D-trehalose, etc.

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

[0117]

[0118] Note: "+" means positive, "-" means negative, and "w" means weakly positive.

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

[0120] After extracting the genomic DNA of strain ZF539 using a bacterial genomic DNA extraction kit (DP302, Tiangen Biochemical Technology (Beijing) Co., Ltd.), the sequences of the bacterial 16S rDNA gene, gyrA, atpD, and rpoA genes were amplified by PCR. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 45 s, 35 cycles; and extension at 72°C for 10 min. The PCR amplification products were sent to Bomeide Biological Company for sequence determination. After alignment using MEGA 6.0, a phylogenetic tree was constructed using the maximum likelihood method to determine the taxonomic status of strain ZF539 and analyze its genetic relationship. The multi-gene phylogenetic tree of ZF539 is shown in Figure 1. Figure 4 .

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

[0122]

[0123]

[0124] Strain ZF539 has a 16S rRNA nucleotide sequence of SEQ ID NO: 1 (1510 bp) in the sequence listing, a gyrA gene nucleotide sequence of SEQ ID NO: 2 (1025 bp) in the sequence listing, an atpD gene nucleotide sequence of SEQ ID NO: 3 (1053 bp) in the sequence listing, and a rpoD gene nucleotide sequence of SEQ ID NO: 4 (898 bp) in the sequence listing. Based on the 16S rDNA gene, gyrA, atpD, and rpoD genes, the multi-gene phylogenetic tree of strain ZF539 was constructed to identify it 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, which was preserved in China General Microbiological Culture Collection Center (CGMCC, Beijing, China) on May 30, 2025, and the preservation number is CGMCC No. 34731. Hereinafter, it is referred to as Bacillus subtilis ZF539 or strain ZF539.

[0128] Example 3, Analysis of the colonization ability of ZF539 in cucumber root system

[0129] 1. Strain preparation

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

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

[0132] 2. Plant material

[0133] Cucumber seedlings (Zhongnong No. 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 (OD 600 ≈0.8). After centrifugation, it was resuspended in sterile PBS to a final concentration of 1×10 8 CFU / mL to obtain ZF539 strain fermentation broth.

[0136] Inoculation method: root irrigation was used, 10 mL per cucumber seedling, and the seedlings were cultured after inoculation.

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

[0138] 4. Detection method

[0139] 1 g of sterilized root system was ground and suspended in 1 mL of PBS, then gradient diluted and plated on LB plates containing tetracycline (50 μg / mL), and incubated at 37°C for 24-48 hours. The number of colonies (CFU / g of fresh root weight) was counted and the colonization density was calculated.

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

[0141] The plant sample at the highest detection point was taken for scanning electron microscope detection.

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

[0143] Gold spraying and imaging: After the sample was sprayed with a gold film, the root surface and internal bacterial attachment morphology were observed by field emission scanning electron microscope (such as Hitachi SU8010) (accelerating voltage 5kV, secondary electron mode).

[0144] Under the scanning electron microscope, it was observed that strain ZF539 colonized the surface of cucumber roots in large quantities Figure 5 ). The highest colonization amount was about 8x10 6 CFU / g( Figure 6 ) at 14-21 days after inoculation, and the lowest colonization amount still maintained above 10 4 CFU / g at 60 days after inoculation. The colonization effect was significantly higher than that of standard strain 168 (ATCC 23857), indicating that strain ZF539 was a strain with high colonization ability and had the development potential for field biocontrol application.

[0145] Example 4, Determination of the control effect of strain ZF539 foliar spraying treatment on cucumber angular leaf spot (pot experiment)

[0146] Cucumber seeds of Zhongnong "6" were planted in 72-hole seedling pots in a greenhouse and cultivated until the cucumber seedlings grew to 1 true leaf.

[0147] The stored test cucumber angular leaf spot pathogen Pseudomonas syringae pv. Lachrymans Pal-nm002 was taken out from the -80°C refrigerator. After thawing in an ice bath, it was activated on LB plates at 28°C and transferred to LB liquid medium for 200r·min -1 . The content of Pseudomonas syringae pv. Lachrymans Pal-nm002 in the bacterial suspension was 10 6 cfu / mL, ready for use.

[0148] The cultured Pseudomonas syringae pv. Lachrymans Pal-nm002 bacterial suspension with a content of 10 6 cfu / mL was inoculated with a micro-sprayer to 1 true leaf stage cucumber with 10 mL bacterial suspension. After inoculation, it was placed in a humidity cabinet to maintain a temperature of about 20°C and a relative humidity of 90% RH.

[0149] The following treatments were performed 12 hours after inoculation of the pathogen:

[0150] C. cucumerina + ZF539 treatment group: 12 cucumber plants were taken and sprayed with 120 ml of 100-fold dilution of ZF539 strain fermentation broth (concentration of 1 x 10 6 CFU / mL, same below) 12 hours after inoculation of the pathogen. The 100-fold dilution of ZF539 strain fermentation broth was prepared by diluting the ZF539 strain fermentation broth with sterile water to 100-fold, obtaining the 100-fold dilution of ZF539 strain fermentation broth (same below).

[0151] C. cucumerina + zhongshengjin treatment group: 12 cucumber plants were taken and sprayed with 120 ml of 1000-fold wettable powder of 3% zhongshengjin. The 1000-fold wettable powder of 3% zhongshengjin was prepared by diluting the commercially purchased 3% zhongshengjin with sterile water to 1000-fold, obtaining the 1000-fold wettable powder of 3% zhongshengjin (same below).

[0152] C. cucumerina treatment (control group) ZF539: 12 cucumber plants were taken and sprayed with 120 ml of sterile water 12 hours after inoculation of the pathogen.

[0153] After inoculation, they were placed in a humidity cabinet, and the temperature was maintained at 26°C. The relative humidity was 90% RH, and the indoor healthy control was observed for disease, and the disease index was recorded. The experiment was repeated 3 times.

[0154] The experimental results were repeated 3 times, and the disease grading standard was as follows:

[0155] 0: no disease spot;

[0156] 1: disease spot accounted for less than 5% of leaf area;

[0157] 3: disease spot accounted for more than 5% to 25% of leaf area;

[0158] 5: disease spot accounted for more than 25% to 50% of leaf area;

[0159] 7: disease spot accounted for more than 50% to 75% of leaf area;

[0160] 9: disease spot accounted for more than 75% of 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 of each grade x representative value of relative grade) / (total number of leaves x representative value of highest grade)] x 100

[0163] Efficiency (%) = ((disease index of control group - disease index of treatment group) / disease index of control group) x 100

[0164] Results show (Table 4 and Figure 7 ): the cucumber angular leaf spot disease prevention effect of ZF539 bacterial agent treatment is as high as 76.19%, higher than that of the control agent, myceliophthora (67.92%).

[0165] Table 4 Bacillus subtilis for pot cucumber angular leaf spot disease prevention effect

[0166] Treatment Disease index Relative control / % C. cucumerina + ZF539 16.4±1.11a 76.19 C. cucumerina + Zhongshengjin 22.1±2.13b 67.92 C. cucumerina 68.9±3.21c -

[0167] Example 5, strain ZF539 leaf spray treatment of cucumber angular leaf spot disease prevention effect determination (field test)

[0168] Cucumber seeds of Zhongnong No. 6 were planted in 72-hole seedling pots in a greenhouse, and when the cucumber seedlings grew to 1 true leaf, they were planted in the experimental shed of the Chinese Academy of Agricultural Sciences on April 10, 2024, and drip irrigation was carried out every 2 days. This experiment is divided into three zones, each zone is 3m x 3m in size, with a planting interval of 30cm, and there are 16 cucumber plants in each zone.

[0169] After 21 days of cucumber seedling transplanting and planting, the preserved Pseudomonas syringae pv. Lachrymans Pal-nm002 of the test cucumber angular leaf spot pathogen was taken out from the -80°C refrigerator. After thawing in an ice bath, it was activated on LB plates at 28°C and transferred to LB liquid medium for 200r·min -1 The content of Pseudomonas syringae pv. Lachrymans Pal-nm002 in the bacterial suspension was 10 6 cfu / mL, and 120ml of bacterial suspension was sprayed on the leaves with a micro-sprayer. After 12 hours of inoculation of the pathogen, the following treatments were carried out:

[0170] Cucumber angular leaf spot fungus + ZF539 treatment group: 16 cucumber plants were taken, and 320ml of 100-fold diluted ZF539 strain fermentation liquid was sprayed on the leaves.

[0171] Cucumber angular leaf spot fungus + myceliophthora treatment group: 16 cucumber plants were taken, and 320ml of 1000-fold wettable powder of 3% myceliophthora was sprayed on the leaves.

[0172] Cucumber angular leaf spot fungus treatment (control group): 16 cucumber plants were taken, and 320ml of sterile water was sprayed on the leaves.

[0173] After the cucumber plants were diseased, the relative humidity was 90% RH, and the health of the control in the observation room was observed, and the disease index was recorded.

[0174] The ZF539 disease grading standard and data processing method are the same as in Example 4, and the test results are repeated 3 times.

[0175] Results show (Table 5 and Figure 8):In field test, the control effect of ZF539 on cucumber angular leaf spot was up to 70.69%, higher than that of the control fungicide (66.32%).

[0176] Table 5 Control effect of Bacillus subtilis on cucumber angular leaf spot in field

[0177] Treatment Disease index Relative control / % C. cucumerina + ZF539 11.4±2.23a 70.69 C. cucumerina + Zhongshengjin 13.1±3.35b 66.32 C. cucumerina 38.9±3.98c -

[0178] Example 6, Determination of the control effect of ZF539 foliar spray treatment on cucumber soft rot (pot experiment)

[0179] Cucumber seeds of Zhongnong No. 6 were planted in 72-hole seedling pots in a greenhouse and cultivated until the cucumber seedlings grew to 1 true leaf.

[0180] The preserved cucumber soft rot pathogen Exzerohilum brasiliense SX309 was taken out from the -80℃ refrigerator, thawed in an ice bath, activated on LB plates at 28℃, and transferred to LB liquid medium for 200 r·min -1 The content of Exzerohilum brasiliense SX309 in the bacterial suspension was about 10 6 cfu / mL, 10 ml of Exzerohilum brasiliense SX309 bacterial suspension was inoculated on 1 true leaf stage cucumber using a micro-sprayer. After inoculation, the plants were placed in a humidity cabinet, and the temperature was maintained at about 20℃, and the relative humidity was 90% RH.

[0181] The following treatments were performed 12 hours after inoculation of the pathogen:

[0182] Cucumber soft rot fungus + ZF539 treatment group: 12 cucumber plants were inoculated with 120 ml of 100-fold diluted ZF539 strain fermentation broth.

[0183] Cucumber soft rot fungus + Zhongshengmycin treatment group: 12 cucumber plants were inoculated with 120 ml of 1000-fold wettable powder of 3% Zhongshengmycin.

[0184] Cucumber soft rot fungus treatment (control group): 12 cucumber plants were inoculated with 120 ml of sterile water.

[0185] After inoculation, they were placed in a humidity cabinet, and the temperature was maintained at 26℃, and the relative humidity was 90% RH. The health of the control plants in the room was observed, and the disease index was recorded.

[0186] After 12 hours of inoculation of pathogenic bacteria, 100 times diluted ZF539 strain fermentation liquor was sprayed on leaves, and the plants were placed in a humidity cabinet at a temperature of about 26°C, and relative humidity of 90% RH. The occurrence of disease on healthy control plants was observed, and the disease index was recorded. 3% Zhongshengjin 1000 times wettable powder was used as a control agent, and sterile water was used for seed treatment as a negative control. The disease grading standard and data processing method were the same as in Example 4, and the test was repeated 3 times.

[0187] The results (Table 4 and Figure 9 ) show that the control effect of ZF539 bacterial agent on cucumber soft rot was as high as 64.53%, which was higher than that of the control agent Zhongshengjin (57.65%).

[0188] Table 6 Control effect of Bacillus subtilis on pot-cultured cucumber soft rot

[0189] Treatment Disease index Relative control / % P. aphanidrum + ZF539 29.4±2.02a 64.53 P. aphanidrum + kaili 35.1±1.43b 57.65 P. aphanidrum 82.9±4.01c -

[0190] Example 7, Determination of the control effect of ZF539 foliar spray treatment on black rot of broccoli (pot culture test)

[0191] Broccoli seeds of Zhongqing No. 16 were planted in 72-hole seedling pots in a greenhouse, and the preserved Xanthomonas campestris pathogenic to black rot of broccoli was taken out from a -80°C refrigerator when the broccoli seedlings grew to 2 true leaves. The bacteria were thawed in an ice bath, activated on LB plates at 28°C, and transferred to LB liquid medium for 200 r·min -1 The content of Xanthomonas campestris in the bacterial suspension was about 10 6 cfu / mL, and 10 ml of the bacterial suspension was sprayed on the 2-true-leaf broccoli using a micro-sprayer. After inoculation, the plants were placed in a humidity cabinet at a temperature of about 20°C and a relative humidity of 90% RH.

[0192] The following treatments were performed 12 hours after inoculation of pathogenic bacteria:

[0193] Xanthomonas campestris + ZF539 treatment group: 12 broccoli plants were inoculated with 120 ml of 100 times diluted ZF539 strain fermentation liquor by foliar spray.

[0194] Xanthomonas campestris + kasugamycin treatment group: 12 broccoli plants were inoculated with 120 ml of 2% kasugamycin wettable powder by foliar spray.

[0195] Xanthomonas campestris treatment (control group): 12 broccoli plants were inoculated with 120 ml of sterile water by foliar spray.

[0196] After inoculation, the plants were placed in a humidity cabinet, and the temperature was maintained at 26°C. The relative humidity was 90% RH. The health of the control plants was observed, and the disease index was recorded.

[0197] The disease grading standard and data processing method were the same as in Example 4. The test was repeated 3 times.

[0198] The results (Table 7 and Figure 10 ) showed that the control effect of ZF539 on black rot of broccoli was 54.69%, which was higher than that of the control agent kasugamycin (44.62%).

[0199] Table 7 Control effect of Bacillus subtilis on black rot of potted broccoli

[0200] Treatment Disease index Relative control / % X. campestris + ZF539 24.44±2.31a 54.69 X. campestris + kaili 29.87±1.86b 44.62 X. campestris 53.94±4.32c -

[0201] Example 8, Determination of the control effect of ZF539 root irrigation treatment on Fusarium wilt of melon

[0202] The biocontrol strain ZF539 preserved in a tube at -80°C was activated on an LB plate by streaking. After 1 day of culture at 28°C, a single colony was picked and placed in a shaking tube. After 18 hours of culture at 28°C and 180 rpm, the bacterial solution was diluted to OD 600 about 0.8.

[0203] The cultured P. aculeatum colony was picked and placed in PD medium, and cultured at 28°C on a shaker for 7 days. The shaking liquid was filtered with two layers of gauze to obtain a spore suspension. The P. aculeatum spore suspension was diluted to a spore concentration of 1 x 10 6 spores / mL using a hemocytometer.

[0204] The pathogenic soil was prepared according to 4 L of substrate per 1 L of spore suspension, and the melon was planted in a 72-hole seedling tray and induced in a greenhouse.

[0205] On the 1st, 5th, and 10th days after sowing, the following treatments were performed:

[0206] Melon + ZF539 treatment group: 24 melons were taken, and each melon was sprayed with 20 mL of 100-fold diluted ZF539 strain fermentation liquid.

[0207] Melon + kasugamycin treatment group: 24 melons were taken, and each melon was root-irrigated with 20 mL of 500-fold diluted 60% carbendazim wettable powder. The 500-fold diluted 60% carbendazim wettable powder was prepared by diluting commercially purchased 60% carbendazim with sterile water to 500 times to obtain 500-fold diluted 60% carbendazim wettable powder.

[0208] Fusarium wilt of melon (control group): 24 melons were taken and irrigated with 20 ml of sterile water.

[0209] After inoculation, they were placed in a humidity cabinet, maintaining a temperature of 26°C, and the cucumber plants were allowed to develop disease, with a relative humidity of 90% RH, and the indoor healthy controls were observed for disease development, and the disease index was recorded.

[0210] The disease grading standard for ZF539 is as follows, and the test results were repeated 3 times:

[0211] 0: no disease;

[0212] 1: cotyledon slight wilting;

[0213] 2: cotyledon severe wilting;

[0214] 3: true leaf wilting;

[0215] 4: whole plant mild wilting;

[0216] 5: whole plant dead or no seedling emergence.

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

[0218] Disease index = [number of strains at each level * representative value of that level] * 100 / [total number of strains surveyed * highest representative value].

[0219] The results show (Table 8 and Figure 11 ): the control effect of ZF539 on melon fusarium wilt is as high as 64.87%, higher than that of the control fungicide carbendazim (54.38%).

[0220] Table 8, control effect of Bacillus subtilis ZF539 on melon fusarium wilt

[0221] Treatment Disease index Relative control / % F. oxysporum + ZF539 24.34±1.89a 64.87 F. oxysporum + carbendazim 31.61±3.29b 54.38 F. oxysporum 69.29±5.21c -

[0222] Example 9, determination of the control effect of ZF539 root irrigation treatment on brassica oleracea clubroot

[0223] The refrigerated Chinese cabbage root tumor was taken out, thawed at room temperature, then placed in a cell wall breaking machine, and a suitable amount of sterile water was added to break it. The root tumor suspension was filtered through 4 layers of gauze into a new clean container to obtain the bacterial suspension of brassica oleracea clubroot pathogen Plasmodiophora brassicae, and the prepared bacterial suspension was mixed with the substrate to make the concentration of P. brassicae dormant spores in the soil reach 10 8 spores / L substrate.

[0224] Three treatments were set up, with 15 seedlings per treatment, and three replicates, as follows:

[0225] Broccoli root rot + ZF539 treatment group: before sowing, spray 100 times dilution of ZF539 strain fermentation liquor on the surface of the substrate, each broccoli plant is irrigated with 10 mL of ZF539 strain fermentation liquor, and 15 days after sowing, each plant is irrigated with 50 mL of 100 times dilution of ZF539 strain fermentation liquor.

[0226] Broccoli root rot + 500g / L fluazinam suspension treatment group: before sowing, spray 500g / L fluazinam suspension on the surface of the substrate, each broccoli plant is irrigated with 10 mL of ZF539 strain fermentation liquor, and 15 days after sowing, each plant is irrigated with 50 mL of 500g / L fluazinam suspension.

[0227] Broccoli root rot treatment group (control): before sowing, spray sterile water on the surface of the substrate, each broccoli plant is irrigated with 10 mL of ZF539 strain fermentation liquor, and 15 days after sowing, each plant is irrigated with 50 mL of sterile water.

[0228] After sowing, the seedlings are placed in a greenhouse at about 20°C, and water is sprayed to keep the soil humidity at 100% for the first 14 days; after 14 days, water is sprayed as needed, and pests and diseases are prevented and treated. About 30 days after sowing, the water control is evenly diseased, the disease condition is investigated, and the disease index is calculated.

[0229] Broccoli root rot investigation and grading standards:

[0230] 0, no root swelling;

[0231] 1, the swollen roots are attached only to the lateral roots, accounting for 1% to 25% of the total root system;

[0232] 2, the swollen roots are attached to the main roots, and the swollen roots account for more than 25% to 50% of the lateral roots;

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

[0234] 4, the swollen roots are attached to the main roots, and the swollen roots account for more than 75% of the root system.

[0235] Disease incidence (%) = number of diseased plants in treatment / total number of plants in treatment * 100

[0236] Disease index = [number of plants at each level * representative value at that level] * 100 / [total number of plants investigated * highest level representative value]

[0237] Control efficiency (%) = (control disease index - treatment disease index) / control disease index * 100

[0238] The results show (Table 9 and Figure 12 ): the control efficiency of broccoli root rot treated with ZF539 fungicide is as high as 61.49%, which is slightly lower than that of the control drug fluazinam (70.46%).

[0239] Table 9, Control effect of Bacillus subtilis ZF539 on root rot of broccoli

[0240] Treatment Disease index Relative control / % P. aphanidrum + ZF539 28.34±2.19b 61.49 P. aphanidrum + 500g / L flonicamid suspension 21.74±1.31a 70.46 P. aphanidrum 73.61±6.09c -

[0241] Example 10, Determination of the control effect of leaf spraying treatment of strain ZF539 on cucumber gummy stem blight (pot experiment)

[0242] Cucumber seeds of Zhongnong No. 6 were planted in 72-hole seedling pots and cultured in a greenhouse until the cucumber seedlings grew to 1 true leaf.

[0243] The cucumber gummy stem blight pathogen Colletotrichum higginsianum was transferred to PDA plate medium and cultured for 4 days, and then a sterile puncher with a diameter of 0.8 cm was used to punch along the edge of the colony. Fresh cucumbers without wounds were selected, washed with tap water, wiped with 75% alcohol, and then dried. A sterile puncher with a diameter of 0.8 cm was used to lightly punch the epidermis on the fruit surface, and the cultured mycelium blocks of different strains were placed on it and directly placed in natural scattered light at 18-22°C (generally suitable for indoor shady places) and light intensity <1000 lx for 4 days.

[0244] The bacterial suspension was uniformly sprayed on the cucumber seedlings with a spray bottle, and cultured in a closed greenhouse at 22-27°C for 24-48h. The disease incidence of the seedlings was observed regularly, and the disease rate, disease index and control effect were calculated.

[0245] 4. Inoculation treatment,

[0246] After 12 hours of inoculation of the pathogen, 10 mL of 100-fold diluted ZF539 strain fermentation liquid was sprayed on the inoculated part of each treatment, and then placed in a humidity cabinet at a temperature of about 26°C. When the cucumber plants were diseased, the relative humidity was 90% RH, and the indoor healthy control was observed for disease, and the disease index was recorded. 50% kresoxim-methyl water dispersible granules were used as the pesticide control, and sterile water was used for seed treatment as the negative control. The disease grading standard is as follows, and the test results were repeated 3 times.

[0247] 5. Effect investigation

[0248] Disease grading standard:

[0249] Leaf disease grading standard: the growth status of the plants was observed every day, the disease incidence was recorded, and the disease level was determined.

[0250] Leaf disease severity grading standard:

[0251] 0 level: healthy seedlings;

[0252] 1 level: lesion area accounts for less than 5% of cotyledon area;

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

[0254] Grade 5: Lesions occupying more than 10-25% of cotyledon area;

[0255] Grade 7: Lesions occupying more than 25-50% of cotyledon area;

[0256] Grade 9: Lesions occupying more than 51% of cotyledon area.

[0257] Disease index = [∑(number of leaves at each grade x representative value of relative grade number) / (total number of leaves x representative value of highest grade number)] x 100

[0258] Control effect = [(average disease index of control - average disease index of treatment) / average disease index of control] x 100%

[0259] The results show (Table 10 and Figure 13 ): the control effect of ZF539 on cucumber gummy stem blight is as high as 53.96%, higher than that of the control agent kresoxim-methyl (45.12%).

[0260] Table 10 Control effect of Bacillus subtilis on pot-cultured cucumber gummy stem blight

[0261] Treatment Disease index Relative control / % P. aphanidrum + ZF539 22.24±1.21a 53.96 P. aphanidrum + kresoxim-methyl 26.51±2.67b 45.12 P. aphanidrum 48.31±3.45c -

[0262] Example 10 Evaluation of the growth-promoting effect of strain ZF539 on Chinese cabbage by root irrigation treatment

[0263] The seeds of Chinese vegetable fast-growing green cabbage were planted in 72-hole seedling pots and cultured in a greenhouse. When the Chinese cabbage grew to two leaves and one heart, ZF539 treatment was started. Each treatment was set up with 3 biological replicates, and each replicate had 20-30 seedlings. The treatments were as follows:

[0264] ZF539 treatment: 10 mL of 100-fold fermented diluent of ZF539 was irrigated to each Chinese cabbage; secondary irrigation treatment was performed on the 5th day after the first irrigation, and 10 mL of 100-fold fermented diluent of ZF539 was irrigated to each Chinese cabbage.

[0265] IAA treatment: 10 mL of 10 ng / ml IAA solution was irrigated to each Chinese cabbage; secondary irrigation treatment was performed on the 5th day after the first irrigation, and 10 mL of 10 ng / ml IAA solution was irrigated to each Chinese cabbage.

[0266] CK treatment: 10 mL of sterile water was irrigated to each Chinese cabbage; secondary irrigation treatment was performed on the 5th day after the first irrigation, and 10 mL of sterile water was irrigated to each Chinese cabbage.

[0267] The growth of Chinese cabbage was observed every day, and the plant height and fresh weight of Chinese cabbage were determined on the 7th day after the secondary treatment, and photographs were taken for record.

[0268] The results show that the strain ZF539 has a significant growth-promoting effect on Chinese cabbage (Table 11 and Figure 14 ), which can significantly increase the plant height and fresh weight of Chinese cabbage, and the growth-promoting rates are 29.54% and 24.41%, respectively, which are higher than the growth-promoting effect of IAA control agent.

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

[0270]

[0271]

[0272] Example 11 Evaluation of the growth-promoting effect of strain ZF539 on corn by root irrigation treatment

[0273] The corn seeds of Nongke 368 were planted in 36-hole seedling pots and cultured in a greenhouse. After 3 weeks of sowing, the corn seedlings of Jingke Nuo 2000 grew to two-leaf-one-heart stage, and the root irrigation treatment was started. Three biological replicates were set for each treatment, and each replicate had 18 seedlings. The specific treatments were as follows:

[0274] ZF539 treatment: 10 mL of 100-fold fermented diluent of ZF539 was irrigated to each corn; the second irrigation treatment was performed on the 5th day after the first irrigation, and 10 mL of 100-fold fermented diluent of ZF539 was irrigated to each corn.

[0275] IAA treatment: 10 mL of 10 ng / ml IAA solution was irrigated to each corn; the second irrigation treatment was performed on the 5th day after the first irrigation, and 10 mL of 10 ng / ml IAA solution was irrigated to each corn.

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

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

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

[0279] Table 12 Growth-promoting effect of strain ZF539 on corn

[0280] Treatment 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 application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the application.

Claims

1. A Bacillus characterized in that, The Bacillus is Bacillus subtilis ZF 539, which is deposited with the China General Microbiological Culture Collection Center and has a preservation number of CGMCC No. 34731.

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

3. The composition of claim 2, wherein, The composition is a culture, which is a substance obtained by culturing the Bacillus in a microbial culture medium.

4. The composition of claim 2, wherein, The composition is a microbial inoculant.

5. The composition according to any one of claims 2-4, characterized in that, The composition has at least one of the following properties: A1) antagonizing pathogenic bacteria, A2) controlling cucumber angular leaf spot, A3) controlling cucumber soft rot, A4) controlling cucumber gummy stem blight, A5) controlling melon fusarium wilt, A6) controlling broccoli black rot, A7) controlling broccoli clubroot, A8) promoting plant growth.

6. Process for the preparation of a composition, characterized in that, The composition is the composition of any one of claims 2-5, and the method comprises the step of using the Bacillus of claim 1 as a component of the composition.

7. Use of the Bacillus of claim 1 or the composition of claims 2-5 in the manufacture of a product.

8. The product of claim 7, wherein, The product has at least one of the following properties: B1) antagonizing pathogenic bacteria, B2) controlling cucumber angular leaf spot, B3) controlling cucumber soft rot, B4) controlling cucumber gummy stem blight, B5) controlling melon fusarium wilt, B6) controlling broccoli black rot, B7) controlling broccoli clubroot, B8) promoting plant growth.

9. A method for the treatment and / or prevention of plant diseases caused by pathogenic fungi, characterized in that, The method comprises contacting a plant with the Bacillus of claim 1 or the composition of claims 2-5 to treat and / or prevent a plant disease caused by pathogenic bacteria.

10. The composition of claim 5 or the product of claim 8 or the method of claim 9, wherein, The plant is any one of the following: C1) an angiosperm, C2) a Magnoliidae or Dicotyledoneae or Monocotyledoneae plant, C3) a Cucurbitaceae or Brassicaceae or Poaceae plant, C4) a Cucumis or Brassica or Zea plant, C5) a Cucumis or Brassica or Zea plant, C6) a Cucumis or Cucumis or Brassica or Brassica or Zea.

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