Burkholderia cepacia ZJ15, microbial fertilizer and application of Burkholderia cepacia ZJ15 to prevention and treatment of diseases of mulberry trees and oak trees

Abstract

The invention discloses Burkholderia cepacia ZJ15, a microbial fertilizer and application of the Burkholderia cepacia ZJ15 and the microbial fertilizer in the aspect of preventing and treating diseases of mulberry trees and oak trees. The strain is preserved in Guangdong Microbial Culture Collection Center on June 05, 2025, and the preservation number is GDMCC No: 66469. The bacterial strain not only can promote germination of mulberry seeds, but also has the capabilities of dissolving inorganic phosphorus, dissolving organic phosphorus, producing auxin, producing siderophores and fixing nitrogen, can promote growth of mulberries, and has a relatively comprehensive growth promoting function; in addition, the bacterium has a relatively good antagonistic effect on mulberry and oak pathogenic bacteria such as colletotrichum truncatum, ailanthus guaiensis, enterobacter cloacae and the like; therefore, the bacterial manure can be developed into bacterial manure with the effects of preventing and treating diseases and promoting growth, and has good application prospects and application values in the aspects of preventing and treating diseases of mulberry trees and oak trees and promoting growth.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fertilizers. More specifically, it relates to a strain of Burkholderia cepacia ZJ15 and its application in preventing and treating mulberry and oak diseases, as well as in promoting mulberry seed germination and mulberry tree growth. Background Art

[0002] Mulberry (Morus alba L.), commonly known as the mulberry tree, is an important economic tree species in my country. Mulberry trees are widely distributed throughout China. Mulberry leaves are the primary food source for silkworms and a fundamental resource for sericulture. Mulberry leaf production directly determines the scale of sericulture. Oak leaves can also be used to feed tussah silkworms, and their starch-rich seeds are used for brewing wine and livestock feed. Their hard wood is used to manufacture vehicles, agricultural tools, flooring, and interior decoration. The bark of the cork oak is the raw material for cork production. Therefore, both mulberry and oak trees are important economic tree species in my country. Their application in sericulture is of great value to our sericulture industry.

[0003] Currently, the primary approach to increasing mulberry and oak leaf yields is to apply chemical fertilizers. However, excessive use of chemical fertilizers can cause soil compaction and degradation, and pollute the ecological environment. Furthermore, the presence of pests and diseases can severely impact leaf yield and quality. For example, Colletotrichum truncatum causes mulberry anthracnose, while Enterobacter cloacae causes mulberry wilt. Tubakiaseoraksanensis can cause obvious disease symptoms in oak leaves (First Report of Tubakiaseoraksanensis Parasitizing Quercus mongolica in Lesser Khingan Mountains, China, DOI: 10.1094 / PDIS-12-14-1284-PDN).

[0004] Currently, the research and industrialization of microbial fertilizers has entered a new stage of development. Using microbial fertilizers to replace traditional chemical fertilizers, combining growth promotion and biocontrol, has become a current research trend. Currently, the application of microbial fertilizers in the sericulture industry is a key path to breaking the bottleneck of traditional cultivation models and achieving sustainable green development. Screening for functional microorganisms is essential for the development of microbial fertilizers. Currently, among the microbial fertilizers registered with the "Quality Supervision, Inspection, and Testing Center for Microbial Fertilizers and Edible Fungi of the Ministry of Agriculture and Rural Affairs," only three are suitable for use on mulberry trees, and the only effective strains are Rhodopseudomonas palustris, Bacillus licheniformis, and Paenibacillus gelatinus. This is far from sufficient, as different strains have significant variations in their antibacterial spectrum and growth-promoting properties. Furthermore, strains are subject to the risk of degradation, leading to a decline in effectiveness. Continuously enriching the microbial library is of paramount importance.

[0005] Therefore, it is of great significance to develop beneficial microorganisms that can promote the growth of mulberry trees and prevent and control diseases, and enrich mulberry microbial fertilizers. Summary of the Invention

[0006] The present invention aims to explore beneficial microbial resources that can promote the growth of mulberry trees, prevent and control diseases of mulberry trees and oak trees, and enrich microbial fertilizers.

[0007] The present invention aims to provide a Burkholderia cepacia ZJ15 and its fermentation products and bacterial agents.

[0008] Another object of the present invention is to provide the use of the Burkholderia cepacia ZJ15 and its fermentation products in promoting mulberry seed germination and promoting mulberry tree growth.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention isolated a strain of Burkholderia cepacia ZJ15 from the rhizosphere soil of mulberry trees. This strain has the ability to solubilize inorganic and organic phosphorus, produce arborescent amine (IAA), produce siderophores, and fix nitrogen. This strain can promote germination of mulberry seeds and growth. Furthermore, this strain has a strong antagonistic effect against mulberry and oak pathogens such as Colletotrichum truncatum, Tubakia seoraksanensis, and Enterobacter cloacae. Therefore, it can be developed into a microbial fertilizer that combines disease prevention and control with growth promotion.

[0011] The Burkholderia cepacia ZJ15 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 5, 2025, with the deposit number GDMCC No: 66469.

[0012] The present invention also provides fermentation products of Burkholderia cepacia ZJ15 and related products, including:

[0013] A fermentation broth of Burkholderia cepacia ZJ15.

[0014] As a specific embodiment, the above-mentioned Burkholderia cepacia ZJ15 or its bacterial agent is inoculated into a liquid culture medium for culture to obtain a fermentation broth containing indoleacetic acid and siderophore.

[0015] As an alternative embodiment, the culture medium is NB medium or LB medium.

[0016] The invention discloses a fermentation product obtained by removing the bacterial cells from the fermentation broth of Burkholderia cepacia ZJ15.

[0017] A bacterial agent containing Burkholderia cepacia ZJ15 and / or its fermentation liquid, and further containing a carrier.

[0018] A bacterial fertilizer containing Burkholderia cepacia ZJ15 and / or its fermentation liquid.

[0019] A bacterial fertilizer containing a fermentation product obtained by fermenting a substrate with Burkholderia cepacia ZJ15. The substrate is a fiber- and / or organic-rich substance, such as feces, crop straw, or oil meal. The oil meal is a byproduct of oil extraction from soybeans, peanuts, corn, sunflower seeds, sesame seeds, and the like.

[0020] The present invention also provides the use of the Burkholderia cepacia ZJ15 and related products in any one, any two, or any combination of the following aspects, or in the preparation of products having any one, any two, or any combination of the following functions:

[0021] (1) Promote germination of mulberry seeds,

[0022] (2) Promote the growth of mulberry trees,

[0023] (3) Phosphate dissolution,

[0024] (4) Nitrogen fixation,

[0025] (5) Produce growth hormone,

[0026] (6) Producing siderophores,

[0027] (7) Inhibit Colletotrichum truncatum,

[0028] (8) Inhibit Tubakia seoraksanensis,

[0029] (9) Inhibit Enterobacter cloacae

[0030] (10) Prevent and control plant diseases caused by Colletotrichum truncatum,

[0031] (11) Prevention and control of plant diseases caused by Tubakia seoraksanensis,

[0032] (12) Prevent and control plant diseases caused by Enterobacter cloacae.

[0033] The phosphorus dissolving includes dissolving inorganic phosphorus and dissolving organic phosphorus.

[0034] The auxin is indoleacetic acid.

[0035] The plants are mulberry trees and / or oak trees.

[0036] The present invention has the following beneficial effects:

[0037] The invention provides a Burkholderia cepacia ZJ15, which can not only promote the germination of mulberry seeds, but also has the ability to dissolve inorganic phosphorus, dissolve organic phosphorus, produce auxin (IAA), produce siderophores, and fix nitrogen, and can promote the growth of mulberry trees.

[0038] Moreover, Burkholderia cepacia ZJ15 has a good antagonistic effect on mulberry and oak pathogens such as Colletotrichum flatheadensis, Slovakian syringomyelia, and Enterobacter cloacae.

[0039] The Burkholderia cepacia ZJ15 of the present invention has a relatively comprehensive growth-promoting function and a good antagonistic effect on mulberry and oak pathogens. It can be developed into a microbial fertilizer with both disease prevention and control and growth-promoting functions. It has good application prospects and application value in preventing and controlling mulberry and oak diseases and promoting growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a diagram of the phosphate solubilization effect of strain ZJ15; a is the growth of strain ZJ15 on inorganic phosphorus culture medium, and b is the growth of strain ZJ15 on organic phosphorus culture medium.

[0041] Figure 2These are the quantitative analysis results of the amount of inorganic phosphorus dissolved by strain ZJ15; a is a standard curve of phosphorus drawn with the change of phosphorus content as the horizontal axis, and b is a phosphorus solubility curve drawn by plotting the change of the amount of phosphorus dissolved by strain ZJ15 inoculated into inorganic phosphorus culture medium over time.

[0042] Figure 3 Figure 2 is the quantitative analysis result of IAA production by strain ZJ15; a is the IAA standard curve drawn with the change in IAA concentration as the horizontal axis, and b is the bacterial IAA production curve drawn when the strain ZJ15 was inoculated into the fermentation medium and the concentration of IAA produced changed over time.

[0043] Figure 4 1 is a graph showing the effect of strain ZJ15's ability to produce siderophores and fix nitrogen; a is the growth of strain ZJ15 on CAS assay medium, and b is the growth of strain ZJ15 on Asu-Bai's medium for nitrogen-fixing bacteria.

[0044] Figure 5 This is the growth of strain ZJ15 on blood (agar) plate culture medium.

[0045] Figure 6 is the morphology of strain ZJ15; a is the growth of strain ZJ15 on NA medium, b and c are observation pictures of strain ZJ15 under a microscope.

[0046] Figure 7 This is the Gram staining result of strain ZJ15.

[0047] Figure 8 This is the phylogenetic tree of strain ZJ15 based on the 16S rRNA gene sequence.

[0048] Figure 9 This is the antagonistic effect of strain ZJ15 on pathogens. DETAILED DESCRIPTION

[0049] The present invention is further described below with reference to specific embodiments and accompanying drawings, but the embodiments do not limit the present invention in any form.

[0050] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0051] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0052] Main reagents: Gram staining kit from Beijing Solebow Technology Co., Ltd.; drug sensitivity paper from Hangzhou Microbiological Reagent Co., Ltd.; Salkowski colorimetric reagent from Feijing Biotechnology Co., Ltd.; Ezup column-type bacterial genomic DNA extraction kit, RNase A solution (10 mg / mL), lysozyme, lysozyme buffer, PCR reagents, etc. from Sangon Biotech (Shanghai) Co., Ltd.

[0053] The main culture medium is shown in Table 1:

[0054] Table 1 Main culture medium information of the experiment

[0055]

[0056]

[0057] Example 1 Isolation and purification of bacteria from the rhizosphere soil of healthy mulberry trees

[0058] Soil sample: Rhizosphere soil of healthy mulberry trees was collected on June 24, 2024 in Zijin County, Heyuan City, Guangdong Province (coordinates 114.84, 23.41).

[0059] Separation method: Take 10g of the collected soil sample and add it to 90ml of distilled water, shake it well, and put it into a shaker for 30 minutes to fully disperse the microorganisms. Take 1mL of liquid from the initial suspension and add it to a 9mL sterile water test tube. Mix it well to obtain 10 -2 Repeat this process to prepare 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Serial dilutions, select 10 -4 -10 -7 Pipette 100 μL of the dilution solution onto the center of the NA culture medium. Use a sterile applicator to gently spread the solution in the same direction, rotating the culture plate while spreading to ensure even distribution of the bacterial solution. Repeat the application three times for each dilution. Place the plate upside down in a 37°C constant temperature incubator and incubate for 48 hours. Select plates with 20-200 colonies and pick a single colony to transfer to a new culture medium. Purify the single colony to obtain a pure strain, and then store it in a -80°C freezer with glycerol.

[0060] Example 2 Detection of Growth-Promoting Function of Rhizosphere Soil Bacteria in Healthy Mulberry Trees and Screening of Strain ZJ15

[0061] The isolated strains were tested for their ability to dissolve inorganic phosphorus, dissolve organic phosphorus, produce auxin (IAA), produce siderophores, and fix nitrogen in order to screen plant growth-promoting bacteria.

[0062] 1. Experimental methods

[0063] (1) Phosphate solubility

[0064] Phosphorus solubilization ability test: The isolated and purified strain was inoculated into NB culture medium and cultured in a shaker at 28°C and 180 r / min until the OD600 of the bacterial solution was 1.0-1.2. Use a hole puncher to evenly punch 5 mm diameter holes on the inorganic phosphorus and organophosphorus bacterial culture medium plates, then pipette 50 uL of the bacterial solution onto the organophosphorus and inorganic phosphorus culture media to observe whether a transparent circle is produced.

[0065] Quantitative detection of phosphate solubility: The amount of bacterial phosphate solubility was detected using the molybdenum antimony colorimetric method. The target bacteria were inoculated into NB medium and cultured to an OD600 of 1.0-1.2. Then, a 1% inoculum size was inoculated into an inorganic phosphate liquid culture medium. Samples were taken every 12 hours, and the fermentation broth was centrifuged at 8000 r / min for 10 minutes. The supernatant was collected and the soluble phosphate concentration was detected using the molybdenum antimony colorimetric method. Plotting the phosphorus standard curve: 0.4394 g of potassium dihydrogen phosphate (KH2PO4, analytical grade) dried at 50°C was weighed into a volumetric flask and the volume was adjusted to 1 L with water, resulting in a concentration of 100 mg / L phosphorus. Draw above-mentioned solution 10ml in 200ml volumetric flask, add water to scale, promptly obtain concentration and be 5mg / L phosphorus standard solution, accurately draw the 5mg / L phosphorus standard solution of 0,2,4,6,8,10,12ml respectively and add water to 30mL in volumetric flask, accurately add 5ml molybdenum antimony anti-developer, shake up, add water and be settled to 50mL, promptly obtain the phosphorus standard solution series that phosphorus (P) amount is respectively 0.0,0.2,0.4,0.6,0.8,1.0,1.2mg / L. Shake up, place 30min in room temperature.At wavelength 700nm place, measure the absorbance of each standard solution, take absorbance as ordinate, phosphorus concentration (mg / L) as abscissa, draw standard curve.Then calculate the dissolved phosphorus amount according to standard curve purpose bacterial strain.

[0066] (2) IAA production capacity

[0067] IAA production capacity test: The isolated and purified strain was inoculated into NB medium and cultured in a shaker at 28°C and 180 r / min until the OD600 of the bacterial liquid was 1.0-1.2. Then, a 1% inoculum size was inoculated into NB medium containing tryptophan (0.2 g / L) and cultured in a shaker at 28°C and 180 r / min for 7 days. The fermentation broth was centrifuged at 8000 r / min for 10 minutes, and the supernatant was mixed with an equal volume of Salkowski color developer and allowed to stand at room temperature in the dark for 30 minutes to observe the color change.

[0068] Quantitative detection of IAA production ability: The Salkowski colorimetric method was used to detect the amount of IAA produced by bacteria. The target bacteria were inoculated into NB medium containing L-tryptophan (0.2 g / L) and cultured. The resulting fermentation broth was centrifuged and the supernatant was mixed with an equal volume of Salkowski color developer. The color was developed in the dark for 30 minutes. NB liquid culture medium without inoculation was mixed with an equal volume of Salkowski color developer as a blank control, and the absorbance value of OD530 was measured. Samples were taken every 24 hours. To prepare an IAA standard curve: Accurately weigh 20 mg of IAA standard and add it to 100 mL of NB medium containing L-tryptophan (0.2 g / L) to fully dissolve it, thereby obtaining a 200 mg / L IAA standard solution. Appropriate amounts of this 200 mg / L IAA standard solution were then diluted with NB medium containing L-tryptophan (0.2 g / L) to prepare 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, and 120 mg / L IAA standard solutions. The resulting standard solutions were mixed with an equal volume of Salkowski colorimetric reagent. A blank control was prepared by mixing NB medium containing L-tryptophan (0.2 g / L) with an equal volume of Salkowski colorimetric reagent. The mixture was incubated at room temperature in the dark for 30 min, and the OD530 absorbance was measured to prepare an IAA standard curve. The IAA production of the strain was calculated based on the IAA standard curve.

[0069] (3) Siderophore production capacity

[0070] Siderophore production capacity test: The isolated and purified strain was inoculated into NB medium and cultured in a shaker at 28°C and 180 rpm until the OD600 of the bacterial solution reached 1.0-1.2. Then, 8 μL of the bacterial solution was transferred to CAS detection medium and observed for the formation of a light yellow transparent halo.

[0071] (4) Nitrogen fixation ability

[0072] Nitrogen fixation ability test: The isolated and purified strain was inoculated into Ashby solid culture medium for nitrogen-fixing bacteria, and the growth of the strain was observed.

[0073] 2. Experimental results

[0074] By testing the ability of the isolated and purified strains to solubilize inorganic phosphorus, solubilize organic phosphorus, produce auxin (IAA), produce siderophores, and fix nitrogen, a plant growth-promoting bacterium with the ability to solubilize inorganic phosphorus, solubilize organic phosphorus, produce auxin (IAA), produce siderophores, and fix nitrogen was screened out, and the plant growth-promoting bacterium was named strain ZJ15.

[0075] The data related to the growth-promoting function of strain ZJ15 are as follows:

[0076] (1) Phosphate solubility

[0077] The inorganic phosphorus solubility ability of strain ZJ15 is as follows Figure 1 As shown in Figure a, the ability to dissolve organic phosphorus is as follows Figure 1 As shown in Figure b, strain ZJ15 can produce obvious transparent zones on both inorganic phosphorus and organic phosphorus culture media, indicating that strain ZJ15 can dissolve inorganic phosphorus and organic phosphorus.

[0078] The amount of dissolved inorganic phosphorus in strain ZJ15 was quantitatively detected, and the experimental results are shown in Table 2 and Figure 2 As shown, its maximum dissolved inorganic phosphorus content can reach 101.82±0.41 mg / L.

[0079] Table 2 Dissolved inorganic phosphorus content of strain ZJ15 at different fermentation times

[0080]

[0081] (2) IAA production capacity

[0082] The results of the strain ZJ15's ability to produce IAA are shown in Tables 3 and Figure 3 As shown, its maximum IAA production can reach 29.36±0.70 mg / L.

[0083] Table 3 IAA production of strain ZJ15 at different fermentation times

[0084]

[0085] (3) Siderophore production capacity

[0086] The results of the siderophore production test of strain ZJ15 are as follows Figure 4 As shown in Figure a, strain ZJ15 can grow on the CAS detection medium and produce an obvious orange-yellow halo around the colony, indicating that strain ZJ15 has a good ability to produce siderophores.

[0087] (4) Nitrogen fixation ability

[0088] The nitrogen fixation ability of strain ZJ15 was tested as follows Figure 4 As shown in Figure b, the results show that strain ZJ15 can grow well on Ashby solid culture medium for nitrogen-fixing bacteria, indicating that strain ZJ15 has good nitrogen-fixing ability.

[0089] Example 3 Safety Testing of Strain ZJ15 and Testing of Its Ability to Promote Mulberry Seed Germination

[0090] 1. Experimental methods

[0091] (1) The strain ZJ15 was inoculated onto a blood (agar) plate culture medium, and then the plate was inverted and placed in a 37°C constant temperature incubator for 48 hours to observe the hemolytic characteristics of the strain ZJ15.

[0092] (2) Preparation of soaking solution: strain ZJ15 was inoculated into NB medium and cultured in a shaking incubator at 28°C and 180 r / min until the OD600 of the culture solution was 1.0-1.2. The fermentation solution was then placed in a 50 ml sterile centrifuge tube and centrifuged at 8000 r / min for 5 min. The supernatant was discarded and 30 ml of distilled water was poured into the tube to wash the strain ZJ15 to remove the culture medium components. The washing was repeated three times. The washed strain ZJ15 was then diluted with distilled water to adjust the OD600 to 0.5±0.02.

[0093] Place a 90mm sterile filter paper in a 90mm sterile Petri dish, then add 10ml of the diluted bacterial solution. Randomly place 30 plump mulberry seeds (Guisangyou 12) in the Petri dish, and use 10ml of distilled water as a control group. Five replicates were set for both the experimental and control groups.

[0094] 2. Experimental results

[0095] (1) Safety test of strain ZJ15 Figure 5 As shown, strain ZJ15 exhibited γ-hemolysis (no hemolysis, no hemolysis around the colonies) in blood (agar) plate culture medium, indicating that strain ZJ15 had good safety.

[0096] (2) The results of strain ZJ15 promoting mulberry seed germination are shown in Table 4. The germination rate of seeds infiltrated with strain ZJ15 can reach 92.77%, while the germination rate of the control group is only 78.77%, indicating that strain ZJ15 has a good effect in promoting seed germination.

[0097] Table 4 Statistical results of germination rate in treatment group and control group

[0098]

[0099] Example 4 Drug resistance detection of strain ZJ15

[0100] 1. Experimental methods

[0101] The disk diffusion method was used to test the drug susceptibility of strain ZJ15 to 30 antibiotics (penicillin G, oxacillin, ampicillin, carbenicillin, piperacillin, cephalexin, cefazolin, cefradine, cefuroxime, ceftazidime, ceftriaxone, cefoperazone, amikacin, gentamicin, kanamycin, neomycin, tetracycline, doxycycline, minocycline, erythromycin, midecamycin, norfloxacin, ofloxacin, ciprofloxacin, vancomycin, polymyxin B, co-trimoxazole, furazolidone, chloramphenicol, and clindamycin).

[0102] Strain ZJ15 was inoculated into NB medium and cultured in a shaker at 28°C and 180 rpm until the OD600 reached 0.1 ± 0.02. Antibiotic-sensitive paper discs were then placed on a plate evenly coated with 200 μL of bacterial culture. The cells were incubated at 28°C for 24 hours. Each treatment was repeated three times, and the inhibition zones of strain ZJ15 for different antibiotics were calculated.

[0103] 2. Experimental results

[0104] The results of the drug resistance experiment of strain ZJ15 are shown in Table 5. The results show that strain ZJ15 is sensitive to 15 of the 30 antibiotics tested (piperacillin, cefuroxime, ceftazidime, ceftriaxone, cefoperazone, amikacin, kanamycin, neomycin, doxycycline, minocycline, norfloxacin, ofloxacin, ciprofloxacin, co-trimoxazole, and chloramphenicol).

[0105] Table 5 Detection of antibiotic resistance of strain ZJ15

[0106]

[0107]

[0108] Note: Value = mean ± standard error, - means no inhibition zone was produced.

[0109] Example 5 Identification of strain ZJ15

[0110] 1. Experimental methods

[0111] (1) Inoculate strain ZJ15 onto NA medium and place in a 37°C incubator. Observe the growth and colony morphology of the strain and perform Gram staining. Then, use an optical microscope to observe the colony morphology and Gram staining results of strain ZJ15. Morphological observation and Gram staining should be performed according to the Manual of Identification of Common Bacteria and the Bergey Manual of Bacteria Identification.

[0112] (2) The 16S rRNA gene of strain ZJ15 was sequenced and the obtained 16S rRNA gene was aligned using NCBI-BLAST. A phylogenetic tree was constructed using the neighbor joining (NJ) method using Mega11 software.

[0113] 2. Experimental results

[0114] (1) The growth of strain ZJ15 on NA medium is as follows Figure 6 As shown in Figure a, the observation results under the microscope are as follows Figure 6 As shown in Figures b and c, strain ZJ15 grew well on NA medium. The colonies were light yellow and opaque, with smooth and neat edges. Under a microscope, the surface of a single colony was smooth, moist, and shiny. The Gram staining results of strain ZJ15 are shown in Figures b and c. Figure 7 .

[0115] (2) The results of the NCBI-BLAST comparison of the strain 16S rRNA gene sequences are shown in Table 6. The phylogenetic tree of strain ZJ15 based on the 16S rRNA gene is shown in Table 6. Figure 8 As shown, strain ZJ15 is in the same phylogenetic branch as Burkholderia cepacia.

[0116] Table 6 Partial results of NCBI-BLAST comparison of 16S rRNA gene of strain ZJ

[0117]

[0118] In summary, combined with the results of morphological and molecular identification, strain ZJ15 was determined to be Burkholderia cepacia, and was deposited in Guangdong Provincial Microbiological Culture Collection (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou) on June 5, 2025, with the deposit number GDMCC No: 66469.

[0119] Example 6 Antagonistic effect of strain ZJ15 on pathogens

[0120] 1. Experimental strains

[0121] Biocontrol bacteria: strain ZJ15.

[0122] Pathogens: Colletotrichum truncatum, Tubakiaseoraksanensis, and Enterobacter cloacae; all strains were isolated and preserved in our laboratory.

[0123] Colletotrichum truncatum and Enterobacter cloacae were isolated from mulberry trees, and Tubakia seoraksanensis was isolated from oak trees; all were kept in the laboratory of the Asia-Pacific Sericulture Training Center of South China Agricultural University.

[0124] 2. Experimental methods

[0125] The strain ZJ15 was inoculated into NB medium and cultured in a shaking incubator at 28°C and 180 rpm until the OD600 of the bacterial solution reached 1.0±0.2.

[0126] The plate confrontation method was used to test the antibacterial effect of biocontrol bacteria on pathogenic fungi. 8 μL of biocontrol bacteria ZJ15 was dropped onto the PDA culture medium, and then the pathogen cake was inoculated onto the PDA culture medium.

[0127] The agar diffusion method was used to test the antibacterial effect of biocontrol bacteria on pathogenic bacteria. The pathogenic bacteria solution was evenly spread on the NA culture medium, and then 8 μL of biocontrol bacteria ZJ15 solution was dropped in the center of the culture medium.

[0128] 3. Experimental results

[0129] like Figure 9 The results show that strain ZJ15 has a good antagonistic effect on several pathogens. Figure a shows the antagonistic effect of ZJ15 against Colletotrichum truncatum, Figure b shows the antagonistic effect of strain ZJ15 against Tubakia seoraksanensis, and Figure c shows the antagonistic effect of strain ZJ15 against Enterobacter cloacae.

[0130] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A strain of Burkholderia cepacia ZJ15, characterized in that: The Burkholderia cepacia ZJ15 was deposited in the Guangdong Provincial Microbial Culture Collection Center on June 5, 2025, with the deposit number GDMCC No: 66469.

2. A Burkholderia cepacia fermentation broth, characterized in that: The fermentation liquid of Burkholderia cepacia ZJ15 according to claim 1.

3. A bacterial agent, characterized in that Contains the Burkholderia cepacia ZJ15 according to claim 1 and / or its fermentation liquid.

4. A microbial fertilizer, characterized in that: The bacterial fertilizer contains the Burkholderia cepacia ZJ15 according to claim 1 and / or its fermentation liquid.

5. A microbial fertilizer, characterized in that: The bacterial fertilizer contains fermentation products, which are obtained by fermenting a substrate with the Burkholderia cepacia ZJ15 according to claim 1, and the substrate is a substance rich in fiber and / or organic matter.

6. A Burkholderia cepacia fermentation product, characterized in that: The product is obtained by removing the bacterial cells from the fermentation liquid of Burkholderia cepacia ZJ15 as described in claim 1.

7. Use of the Burkholderia cepacia ZJ15 of claim 1, the fermentation liquid of claim 2, the bacterial agent of claim 3, the bacterial fertilizer of claim 4 or 5, or the fermentation product of claim 6 in any one, any two, or any combination of the following aspects: (1) Promote germination of mulberry seeds, (2) Promote the growth of mulberry trees, (3) Phosphate dissolution, (4) Nitrogen fixation, (5) Produce growth hormone, (6) Producing siderophores, (7) Inhibit Colletotrichum truncatum, (8) Inhibit Tubakia seoraksanensis, (9) Inhibit Enterobacter cloacae (10) Prevent and control plant diseases caused by Colletotrichum truncatum, (11) Prevention and control of plant diseases caused by Tubakia seoraksanensis, (12) Prevent and control plant diseases caused by Enterobacter cloacae.

8. Use of the Burkholderia cepacia ZJ15 of claim 1, the fermentation liquid of claim 2, the bacterial agent of claim 3, the bacterial fertilizer of claim 4 or 5, or the fermentation product of claim 6 in the preparation of a product having any one, any two, or any combination of the following functions: (1) Promote germination of mulberry seeds, (2) Promote the growth of mulberry trees, (3) Phosphate dissolution, (4) Nitrogen fixation, (5) Produce growth hormone, (6) Producing siderophores, (7) Inhibit Colletotrichum truncatum, (8) Inhibit Tubakia seoraksanensis, (9) Inhibit Enterobacter cloacae (10) Prevent and control plant diseases caused by Colletotrichum truncatum, (11) Prevention and control of plant diseases caused by Tubakia seoraksanensis, (12) Prevent and control plant diseases caused by Enterobacter cloacae.

9. The use according to claim 7 or 8, characterized in that: The plant is a mulberry tree.

10. The use according to claim 7 or 8, characterized in that: The plant is oak.

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