Burkholderia xenovorans and uses thereof

Abstract

The present disclosure relates to a new Burkholderia arboris, a biocontrol agent and application thereof. The present disclosure provides a Burkholderia arboris, which is preserved in China General Microbiological Culture Collection Center on November 8, 2022, and the preservation number is CGMCC No. 26086. The Burkholderia arboris provided by the present disclosure has a more broad-spectrum bacteriostatic effect.

Description

Technical Field

[0001] This disclosure belongs to the field of biological control technology, specifically, it relates to Burkholderia forestica, biological control agents and their applications. Background Technology

[0002] Various crops are frequently threatened by bacterial and fungal infections during cultivation, with fungal diseases being the most common. Pathogenic fungi infect the roots, stems, and leaves of plants, leading to reduced yields and quality in crops and medicinal plants, causing significant losses. Root rot, rust rot, gray mold, and wilt, caused by pathogens of the genera *Fusarium*, *Ilyonectria*, *Botrytis*, and *Alternaria*, are all prevalent fungal diseases affecting medicinal plants, fruits, vegetables, and grain crops. In traditional producing areas, the incidence of root rot in ginseng and American ginseng is consistently around 30%, reaching 100% in severe cases. Strawberries grown in greenhouses are highly susceptible to gray mold outbreaks, often resulting in yield reductions of 20%, and in severe cases, over 50%, or even total crop failure.

[0003] Currently, plant disease control in production mainly relies on chemical methods, leading to widespread pesticide overuse. This not only causes environmental pollution and frequent pesticide residues but also results in increased pathogen resistance and reduced pesticide efficacy. With the development of green crop cultivation, the government has proposed requirements for reducing pesticide use and increasing efficiency, limiting pesticide residues, and protecting the environment. Biocontrol agents utilize beneficial microorganisms to effectively control crop diseases, offering advantages such as being green, safe, and highly effective. Biocontrol bacteria have achieved some commercial success in plant disease control. The most widely used Bacillus subtilis for agricultural use demonstrates excellent control efficacy against rice blast, powdery mildew and gray mold in cucumbers, strawberries, tomatoes, and other crops, sclerotinia rot in soybeans and rapeseed, and root rot in cereals. However, current biocontrol bacteria development faces challenges, including a limited number of strains and a narrow pathogen spectrum. Research on biocontrol bacteria targeting medicinal plant diseases is still in its early stages and falls far short of production needs. Therefore, screening for highly effective biocontrol bacteria, especially those targeting medicinal plants, is of great significance for the green control of medicinal plant diseases, reducing pesticide use, and ensuring the safety of Chinese medicinal materials. Summary of the Invention

[0004] The purpose of this disclosure is to provide a novel Burkholderia forestosa, a biological control agent, and its application for the prevention and control of plant diseases.

[0005] In one aspect, this disclosure provides a strain of Burkholderia arboris, which was deposited on November 8, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26086, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This Burkholderia arboris strain is named Burkholderia arboris MEC_B345.

[0006] Secondly, this disclosure provides a biological control agent, which includes Burkholderia forestosa provided in the first aspect.

[0007] Thirdly, this disclosure provides the application of Burkholderia foresticae provided in the first aspect or the biological control agent provided in the second aspect in the suppression of plant pathogenic fungi.

[0008] In some embodiments, the pathogenic fungi include one or more of the genera *Fusarium*, *Ilyonectria*, *Botrytis*, *Alternaria*, *Phytophthora*, *Valsa*, and *Drechslera*.

[0009] In some embodiments, the pathogenic fungus includes one or more of the following: Fusarium solani, Ilyonectria mors-panacis, Ilyonectria vredehoekensis, Botrytis cinerea, Alternaria solani, Fusarium oxysporum f.sp.cucumerinum, Phytophthora drechsleri, Alternariaraphani, Valsa mali, Fusarium oxysporum f.sp.niveum, and Drechslera Sofokiniana.

[0010] Fourthly, this disclosure provides the application of Burkholderia foresticae provided in the first aspect or the biological control agent provided in the second aspect in the prevention and control of plant diseases and the promotion of plant growth.

[0011] In some embodiments, the plant disease is caused by plant pathogenic fungi.

[0012] In some embodiments, the plant pathogenic fungi include one or more of the genera *Fusarium*, *Ilyonectria*, *Botrytis*, *Alternaria*, *Phytophthora*, *Valsa*, and *Drechslera*.

[0013] In some embodiments, the plant pathogenic fungus includes one or more of the following: Fusarium solani, Ilyonectria mors-panacis, Ilyonectria vredehoekensis, Botrytis cinerea, Alternaria solani, Fusarium oxysporum f.sp.cucumerinum, Phytophthora drechsleri, Alternariaraphani, Valsa mali, Fusarium oxysporum f.sp.niveum, and Drechslera Sofokiniana.

[0014] In some embodiments, the plant diseases are selected from ginseng root rot, ginseng rust rot, American ginseng root rot, Panax notoginseng root rot, Astragalus root rot, Atractylodes macrocephala root rot, Angelica sinensis root rot, Glycyrrhiza uralensis root rot, Salvia miltiorrhiza root rot, Ligusticum chuanxiong root rot, Ophiopogon japonicus root rot, Carthamus tinctorius root rot, Isatis indigotica root rot, Lycium barbarum root rot, Alfalfa root rot, Coptis chinensis root rot, Dendrobium officinale root rot, Zingiber officinale root rot, Sweet potato root rot, Aconitum carmichaelii root rot, Blackberry black rot, Strawberry gray mold, Tomato early blight, Cucumber wilt, Cucumber blight, Radish black spot, Apple rot, Watermelon wilt, and Wheat root rot.

[0015] This disclosure provides the application of either Burkholderia forestica, as provided in the first aspect, or the biological control agent provided in the second aspect, in controlling root rot in ginseng plants and promoting the growth of ginseng plants.

[0016] The Burkholderia forestosa disclosed herein has a broader spectrum of antibacterial activity.

[0017] The Burkholderia forestosa disclosed herein can effectively antagonize not only fungi of the Fusarium genus, but also fungi of the Agrostis genus, as well as fungi of other genera.

[0018] The Burkholderia forestosa disclosed herein can control plant diseases. Attached Figure Description

[0019] Figure 1The results show the inhibition of Fusarium solani, Ilyonectria mors-panacis, and Ilyonectria vredehoekensis by MEC_B345. Figure 1 In Figure A, MEC_B345 is shown to inhibit Fusarium solani, Ilyonectria mors-panacis, and Ilyonectria vredehoekensis; Figure B shows the inhibitory effect of MEC_B345 on Fusarium solani, Ilyonectria mors-panacis, and Ilyonectria vredehoekensis.

[0020] Figure 2 The image shows the colony growth morphology of MEC_B345. Figure 2 In the image, A shows a stereomicroscopic image of MEC_B345 colonies; B shows a scanning electron microscope image of MEC_B345; and C shows an optical microscope image of MEC_B345 after Gram staining.

[0021] Figure 3 The phylogenetic tree of Burkholderia forestii MEC_B345 is shown.

[0022] Figure 4 A diagram showing the biological characteristics of Burkholderia forestii MEC_B345 is displayed.

[0023] Figure 5 An image showing the inhibition of Burkholderia forestii MEC_B345 is displayed.

[0024] Figure 6 The inhibition rate of Burkholderia forestii MEC_B345 was shown.

[0025] Figure 7 The inhibition rate of volatile gases from Burkholderia forestica MEC_B345 was shown.

[0026] Figure 8 The inhibition rate of sterile filtrate of Burkholderia forestica MEC_B345 was shown.

[0027] Figure 9 This study demonstrated the preventive effect of Burkholderia forestica B345 against root rot in American ginseng. Figure 9In the table, A shows treatment with 10 μL 1×PBS; B shows treatment with 10 μL MEC_B345; C shows treatment with 10 μL Fusarium solani; D shows treatment with a mixed culture of 10 μL Fusarium solani and MEC_B345; E shows treatment with 10 μL Ilyonectria mors-panacis.; and F shows treatment with a mixed culture of 10 μL Ilyonectria mors-panacis. and MEC_B345. Detailed Implementation

[0028] The present disclosure will be further illustrated below through embodiments. It should be understood that the embodiments of the present disclosure are merely illustrative and not intended to limit the present disclosure. Simple modifications to the present disclosure based on the concept of the present disclosure are within the scope of protection claimed by the present disclosure.

[0029] definition

[0030] Unless otherwise defined, all terms used herein (including technical and scientific terms) shall have the meanings commonly understood by those skilled in the art. It should be noted that the terms used herein shall be interpreted in a manner consistent with the context of this specification and shall not be interpreted in an idealized or overly rigid manner.

[0031] The term "about" as used herein is as understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is unfamiliar with the use of this term in the context in which it is used, "about" will mean a particular value plus or minus 10%.

[0032] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. In the following description, descriptions of well-known techniques are omitted to avoid unnecessarily obscuring the concepts of the disclosure. Such techniques are described in many publications, such as *Molecular Cloning: A Laboratory Manual (Fourth Edition)* (Cold Spring Harbor Laboratory Science Press).

[0033] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0034] Example

[0035] Example 1. Screening and Separation Method for MEC_B345

[0036] reagents

[0037] The formula for LB solid medium is as follows: 10.0g tryptone, 10.0g NaCl, 5.0g yeast extract, 15.0g agar powder, distilled water to a final volume of 1000mL, pH=7.0;

[0038] The formula for PDA solid culture medium is as follows: 200.0g potato, 20.0g glucose, 15.0g agar powder, distilled water to a final volume of 1000mL, pH=7.0 (purchased from Beijing Aoboxing Biotechnology).

[0039] Experimental methods

[0040] (1) Soil bacterial isolation: Rhizosphere soil from healthy American ginseng plants in Wendeng City, Weihai, Shandong Province was collected. 10g of soil sample was placed in a conical flask containing 90mL of sterile water. The flask was placed in a shaker and shaken for 30min (28℃, 180r / min). The soil suspension was then serially diluted, with 50μL of the diluted solution at a 10:10 ratio. -3 10 -4 10 -5 Soil suspension was prepared and evenly spread on LB solid medium, placed in a 25°C constant temperature incubator, and incubated in the dark for 2-3 days. The bacteria that grew were purified by obtaining single colonies using the streak method.

[0041] (2) Preliminary screening of antagonistic bacteria: The plate confrontation culture method was used for preliminary screening of antagonistic bacteria. Fusarium solani mycelium cake with a diameter of about 5 mm was placed in the center of a PDA plate, and the bacteria obtained in step (1) were spot-inoculated 2 cm away from the pathogenic fungus. The plate was placed in a constant temperature incubator at 25℃ and bacteria that inhibited the growth of fungi were selected.

[0042] (3) Verification of antagonistic bacterial function: The strains obtained in the initial screening in step (2) were re-screened using the confrontation culture method. The test strains were cultured overnight in LB liquid medium. The obtained bacterial cells were washed 1-2 times with 1×PBS buffer, and the bacterial concentration was adjusted to 1×10⁻⁶. 7 CFU / mL was kept on hand. Using *Fusarium solani*, a pathogenic bacterium of American ginseng, and *Ilyonectria mors-panacis* and *Ilyonectria vredehoekensis* as target fungi, 5 mm diameter fungal discs were placed in the center of PDA solid medium plates. 10 μL of antagonistic bacterial suspension was symmetrically inoculated 2 cm away from the discs. Each treatment was repeated three times, and the plates were incubated in the dark at 25°C to verify the antibacterial effect of the antagonistic bacteria.

[0043] MEC_B345's antibacterial effect against three pathogenic fungi of American ginseng is as follows: Figure 1As shown, MEC_B345 exhibited inhibition rates of 77.53%, 78.90%, and 75.04% against three pathogenic fungi of American ginseng: Fusarium solani, Ilyonectria mors-panacis, and Ilyonectria vredehoekensis, respectively. This indicates that MEC_B345 has a good inhibitory effect on the growth of pathogenic fungi of American ginseng, and the inhibitory effect is quite significant.

[0044] Example 2. Identification and biological function of MEC_B345

[0045] reagents

[0046] The formula for Monkina organic phosphorus medium is as follows: 10.0g glucose, 0.5g (NH4)2SO4, 0.3g NaCl, 0.3g KCl, 0.03g FeSO4·7H2O, 0.03g MnSO4·4H2O, 0.2g egg yolk lecithin, 5g CaCO3, 0.4g yeast extract, 20g agar, and distilled water to a final volume of 1000mL, pH=7.0;

[0047] The formula for inorganic phosphorus medium is as follows: 10.0g glucose, 0.5g (NH4)2SO4, 0.2g NaCl, 0.2g KCl, 5.0g Ca(PO4)2, 0.03g MgSO4·7H2O, 0.03g MnSO4, 0.003g FeSO4, 0.5g yeast extract, 20.0g agar, and distilled water to a final volume of 1000mL. The pH is 6.8–7.0.

[0048] The formulation of the protease detection medium is as follows: 1.0g soluble starch, 5.0g peptone, 5.0g NaCl, 5.0g beef extract, 15.0g agar, and distilled water to a final volume of 1000mL.

[0049] The formula for Coolaber's modified CAS agar / ferrophilic assay medium is as follows: 100.0g glucose, 20.0g peptone, 0.5g MgSO4·7H2O, 0.5g CaCl2, 20.0g agar powder, CAS assay solution (sterilized), and distilled water to a final volume of 1000mL.

[0050] CAS test solution (sterilized): 0.6g CAS, 0.0027g FeCl3·6H2O, 0.073g HDTMA (hexadecyltrimethylammonium bromide).

[0051] Experimental methods

[0052] (1) Morphological characteristics of MEC_B345

[0053] 1) Stereomicroscopic characterization

[0054] MEC_B345 was inoculated into LB liquid medium and cultured overnight at 28°C and 180 rpm. The resulting bacterial suspension was centrifuged at 5000 rpm for 10 min. The centrifuged bacterial cells were washed 1-2 times with 1×PBS buffer, and the bacterial concentration was adjusted to approximately 1×10⁻⁶. 7 CFU / mL, add 10 μL of bacterial suspension to LB agar plates, repeat 3 times, and incubate in the dark at 25°C for 48-72 h. Photograph bacterial colonies using a stereomicroscope (Leica S8APO).

[0055] The morphology of this bacterium on LB agar plates is as follows: Figure 2 As shown in Figure A: The colony is slightly concave in the middle, generally shaped like a platform, with smooth and neat edges, glossy, viscous, and yellowish-brown, opaque, round, with a relatively smooth surface.

[0056] 2) Scanning electron microscopy characterization

[0057] The bacterial suspension cultured for 24-48 hours was centrifuged, and the bacterial cells were washed twice with 1×PBS and fixed with 2.5% glutaraldehyde solution (for electron microscopy, Solarbio). The samples fixed at 4℃ for 24 hours were then photographed using a scanning electron microscope (SU8100 biological scanning electron microscope + Oxford UltimMax65 energy dispersive spectroscopy, Hitachi MC1000 gold-sprayed microscope).

[0058] Characterizing single cell morphology using scanning electron microscopy, such as... Figure 2 As shown in B: rod-shaped, with a relatively rough surface, a diameter of about 1 to 2 μm, and abundant secretions between cells.

[0059] 3) Gram staining characterization

[0060] After the bacteria are smeared and fixed, they are first stained with crystal violet solution, mordanted with iodine solution, and counterstained with safranin. The slides are then placed under an optical microscope for observation.

[0061] like Figure 2 As shown in Figure C, MEC_B345 stains red with Gram stain, indicating that this bacterium is Gram-negative.

[0062] (2) Molecular identification of MEC_B345

[0063] Using bacterial suspension as DNA template, the bacterial 16S rRNA gene (as shown in SEQ ID No:1) was amplified by PCR using 2×Taq PCR StarMix with Loading Dye (Beijing Kangrun Chengye Biotechnology Co., Ltd.). Universal primers for the 16S rRNA gene (such as 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′ shown in SEQ ID No:2; and 1492R: 5′-ACGGTTACCTTACCTTGTTACGACTT-3′ shown in SEQ ID No:3) were synthesized by Beijing Liuhe Huada Genomics Co., Ltd. The PCR reaction system consisted of 40 μL: 2.0 μL DNA template, 0.8 μL each of forward and reverse primers (10 mM), 20 mL 2×Taq PCR StarMix, and ddH2O to a final volume of 40 μL. PCR reaction conditions: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, 32 cycles; final extension at 72℃ for 10 min. The PCR products were sent to BGI Genomics Co., Ltd. for sequencing.

[0064] The sequencing results were assembled, and the resulting sequences were uploaded to the NCBI website for BLAST alignment. Closely related strains were identified, and the 16S rRNA sequences of the corresponding strains were downloaded. A phylogenetic tree was constructed using MEGA 7.0 with the Neighbor-Joining algorithm selected and a repetition count of 1000. The phylogenetic tree of the 16S rRNA sequence of MEC_B345 was then analyzed to determine its phylogenetic relationships. Figure 3 As shown. The strain MEC_B345 was ultimately identified as Burkholderia arboris.

[0065] Burkholderia arboris MEC_B345 was deposited on November 8, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26086, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This Burkholderia arboris strain is named Burkholderia arboris MEC_B345.

[0066] (3) Biological functions of MEC_B345

[0067] 1) Phosphorus dissolving capacity test

[0068] 10 μL of bacterial suspension was spot-inoculated onto Monkina organic and inorganic phosphorus agar plates, with one inoculation point in the center of each petri dish, and three replicates per bacterium. The plates were incubated at 25°C for 14 days, and the presence or absence of phosphate-soluble zones was observed. The diameter of the transparent zone (D / mm) and the colony diameter (d / mm) were measured.

[0069] MEC_B345's ability to dissolve organic and inorganic phosphorus, such as Figure 4 As shown in Table 1, MEC_B345 formed transparent zones on both inorganic and organic phosphorus media, indicating that MEC_B345 has phosphorus-solubilizing ability.

[0070] 2) Protease production capacity test

[0071] The clear zone method was used to determine the presence of clear zones. The activated strain was inoculated into the protease detection medium and incubated at 25°C for 2 days. The appearance of clear zones was observed, and the diameter of the clear zones (D / mm) and the diameter of the colonies (d / mm) were measured.

[0072] MEC_B345's ability to produce proteases, such as Figure 4 As shown in Table 1, MEC_B345 forms a clear zone on the protease detection medium, indicating that MEC_B345 can produce proteases to hydrolyze the proteins in the medium.

[0073] 3) Detection of ferrophilic production capacity

[0074] Spot 10 μL of bacterial suspension onto the heptaphilin detection medium, one inoculation point per center of each petri dish, with three replicates. Incubate at 25°C for 5 days. The presence of an orange-yellow halo indicates the ability to produce heptaphilin. Measure the diameter of the orange-yellow halo (D / mm) and the colony diameter (d / mm). See details below. Figure 4 See Table 1.

[0075] The assay was performed using Coolaber modified CAS agar / ferrophilic detection medium: Weigh 141g of basal medium and add distilled water to bring the volume to 800mL; sterilize at 121℃ for 30min; cool to 50-60℃, slowly add 100mL of 10× buffer solution and 100mL of 10× CAS detection solution preheated to 60℃ to the medium, mix well without producing bubbles, and pour into a petri dish.

[0076] MEC_B345's ability to produce heptatrophic substances, such as Figure 4 As shown in Table 1, MEC_B345 can form an orange-yellow halo on CAS agar / ferrophilic assay medium, indicating that MEC_B345 has the ability to produce ferrophilic elements, which can convert iron elements in the soil that are difficult for plants to absorb into iron elements that are easy for plants to absorb.

[0077] Table 1. Biological characteristics of MEC_B345

[0078]

[0079] 4) Ammonia production capacity testing

[0080] MEC_B345 strain was inoculated at 0.1% in 0.5% peptone liquid medium in triplicate, and cultured on a shaker at 28°C and 180 rpm. Uninoculated 0.5% peptone medium served as a control (CK). After 5 days of culture, 100 μL of the bacterial culture was transferred to a white ceramic plate, and 3–5 drops of Nessler's reagent (Tianjin Aopsheng Chemical Co., Ltd.) were added to each well.

[0081] The ammonia production capacity test results of MEC_B345 are as follows: Figure 4 As shown, no yellow or reddish-brown precipitate appeared in the uninoculated culture medium after Nessler's reagent was added, while the culture medium inoculated with this strain showed a yellow or reddish-brown precipitate, indicating that the strain has the ability to produce ammonia.

[0082] comprehensive Figure 4 According to the results in Table 1, MEC_B345 has the ability to dissolve organic and inorganic phosphorus, hydrolyze proteins, produce ferrophosphate and ammonia, thus having the potential to promote plant growth.

[0083] Example 3. Validation of the broad-spectrum antibacterial function of MEC_B345

[0084] The confrontation culture method was used to observe whether MEC_B345 has an antagonistic effect on pathogenic fungi.

[0085] Centrifuge the bacterial suspension after overnight incubation to obtain bacterial cells, wash the cells twice with 1×PBS buffer, and adjust the bacterial concentration to 1×10⁻⁶. 7 CFU / mL was kept on hand. The following fungi were used as target bacteria: *Fusarium solani* and *Rhizoctonia solani* (preserved by Professor Gao Weiwei's research group at the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences), *Botrytis cinerea* (strawberry gray mold), *Early blight* (tomato early blight), *Fusarium wilt* (cucumber wilt), *Phytophthora blight* (cucumber blight), *Black spot* (radish black spot), *Fungi rot* (apple rot), *Fusarium wilt* (watermelon wilt), and *Plant Pathology Laboratory* (College of Horticulture and Landscape Architecture, Tianjin Agricultural University). Pre-cultured 5mm target bacteria were placed in the center of PDA medium. 10L of antagonistic bacterial suspension was symmetrically inoculated 2cm away from the bacterial cake. Each treatment was repeated three times. The cultures were incubated in the dark at 25℃. Photos were taken when the strains in the control group were about to cover a 90mm culture dish. For detailed antibacterial results, see [link to relevant documentation]. Figure 5 , Figure 6 And Table 2.

[0086] Statistical antibacterial rate: The formula for calculating the antibacterial rate is as follows:

[0087]

[0088] Table 2 shows the antibacterial rate of MEC_B345.

[0089]

[0090] Note: Plant diseases caused by pathogenic fungi. Fusarium solani: root rot of ginseng, American ginseng, Panax notoginseng, Astragalus membranaceus, Atractylodes macrocephala, Angelica sinensis, Glycyrrhiza uralensis, Salvia miltiorrhiza, Ligusticum chuanxiong, Ophiopogon japonicus, Carthamus tinctorius, Isatis indigotica, Lycium barbarum, Alfalfa, Coptis chinensis, Dendrobium officinale, Zingiber officinale, Dioscorea opposita, Aconitum carmichaelii; Ilyonectria mors-panacis: rust rot of ginseng, American ginseng, Panax notoginseng; Ilyonectria vredehoekensis: rust rot of American ginseng, blackberry black rot; Botrytis cinerea: gray mold of strawberry; Alternaria solani: early blight of tomato; Fusarium oxysporum f.sp.Cucumerinum: wilt of cucumber; Phytophthora drechsleri: cucumber blight; Alternaria raphani: radish black spot; Valsa mali: fruit rot; Fusarium oxysporum f.sp. Niveum: watermelon wilt; Drechslera Sofokiniana: wheat root rot.

[0091] Depend on Figure 5 , Figure 6 As shown in Table 2, MEC_B345 not only exhibits excellent antagonistic effects against the main pathogenic fungi of ginseng root rot, such as Fusarium solani, Ilyonectria morspanacis, and Ilyonectria vredehoekensis, but also against pathogens of other crops, such as Botrytis cinerea, Alternaria solani, Fusarium oxysporum f.sp.cucumerinum, Phytophthora drechsleri, Alternaria raphani, Valsa mali, Fusarium oxysporum f.sp.niveum, and Drechslera Sofokiniana.

[0092] Example 4. Verification of the antibacterial function of secondary metabolites of MEC_B345 against the pathogen causing root rot in American ginseng.

[0093] (1) Inhibition of the growth of the pathogen causing root rot of American ginseng by the volatile gas of MEC_B345

[0094] Centrifuge the bacterial suspension after overnight incubation to obtain bacterial cells, wash the cells twice with 1×PBS buffer, and adjust the bacterial concentration to 1×10⁻⁶. 8 CFU / mL, 1×10 6 The target fungus was *Fusarium solani*, a pathogenic bacterium of American ginseng, at a concentration of CFU / mL. The experiment employed a double-plate inverted method. The upper basal medium was PDA medium, with 5mm fungal discs placed in the center. The lower basal medium was LB medium, with 50μL of different concentrations of fungal suspension evenly spread on the LB medium. The two plates were inverted, sealed with sealing film, and each treatment was repeated three times. The plates were incubated in the dark at 25°C. After 7 days, the colony diameter was measured using the cross-hatching method. The inhibition rate was calculated as in Example 3.

[0095] Table 3. Antibacterial rate of volatile gases from MEC_B345 against F. solani.

[0096]

[0097] like Figure 7 As shown in Table 3, the volatile gases from MEC_B345 have a certain inhibitory effect on F. solani.

[0098] (2) Effects of MEC_B345 sterile filtrate on the growth of pathogens causing root rot in American ginseng

[0099] Centrifuge the bacterial suspension after overnight incubation to obtain bacterial cells, wash the cells twice with 1×PBS buffer, and take 0.1 mL of 1×10⁻⁶ PBS buffer. 8 CFU / mL bacterial suspension was inoculated into 100mL LB liquid medium (250mL Erlenmeyer flask) and cultured at 28℃ with shaking at 180r / min for 48h. The resulting sterile filtrate was then filtered for later use. *Fusarium solani*, a pathogenic bacterium of American ginseng, was used as the target fungus, and PDA medium was used as the basal medium. A 5mm diameter fungal cake was placed in the center of a petri dish, and four 8mm diameter wells (Axygen) were punched 2cm away from the center. 100μL of MEC_B345 sterile filtrate was injected into each well. Each treatment was repeated three times, and cultured in the dark at 25℃. After 4 days, the diameter of the fungal colonies was measured using the cross-sectional method. The inhibition rate was calculated as in Example 3.

[0100] The inhibitory effect of MEC_B345 sterile filtrate on the growth of F. solani is as follows: Figure 8 As shown, the inhibition rate was 19.90%, indicating that the metabolites of MEC_B345 can inhibit the growth of F. solani.

[0101] Example 5. Verification of the disease prevention effect of MEC_B345 on root rot of American ginseng.

[0102] reagents

[0103] CMC medium: 15.0g CMC, 1.0g NH4NO3, 1.0g KH2PO4, 0.5g MgSO4·7H2O, 1.0g yeast extract, distilled water to a final volume of 1000mL.

[0104] Experimental methods

[0105] (1) Preparation of bacterial suspension:

[0106] Preparation of MEC_B345 suspension: The strain was inoculated into LB medium and cultured overnight at 28°C and 180 rpm. The cells were then obtained by centrifugation (5000 rpm, 10 min). The cells were washed twice with 1×PBS solution, and the bacterial concentration was adjusted to 1×10⁻⁶. 7 CFU / mL.

[0107] Preparation of pathogenic fungal spore suspension: The fungal discs were inoculated into CMC medium. *Fusarium solani* was cultured at 25°C with shaking at 175 rpm for 3 days, and *Ilyonectria morspanacis* was cultured at 20°C with shaking at 175 rpm for 5–6 days. The suspension was centrifuged (8000 rpm, 10 min), the supernatant was discarded, and the cells were washed twice with 1×PBS solution. The spore concentration was adjusted to 1×10⁻⁶. 7 CFU / mL.

[0108] (2) Experimental procedure: Healthy American ginseng roots were washed with clean water and then disinfected. They were rinsed with 75% alcohol for 2 min, 0.8% sodium hypochlorite solution for 10 min, and sterile water for 7-8 times. After disinfection, the ginseng roots were placed in a 15 mm diameter petri dish (the petri dish was lined with 3 layers of sterile filter paper and 10 mL of sterile water was added). Three 2 mm deep wounds were made on each ginseng root with a sterile syringe needle. There were 6 treatments in the experiment. Treatment A was a blank control: 10 μL of PBS solution was added. Treatment B: 10 μL of MEC_B345 bacterial solution. Treatment C: 10 μL of Fusarium solani bacterial solution. Treatment D: 10 μL of Ilyonectria mors-panacis bacterial solution. Treatment E: 10 μL of equal volume mixed MEC_B345 and Fusarium solani bacterial solution. Treatment F: 10 μL of equal volume mixed MEC_B345 and Ilyonectria mors-panacis bacterial solution. Each treatment was repeated 3 times. Incubate at 25℃ in a constant temperature incubator in the dark for 5 days, and observe continuously.

[0109] The experimental results on day 5 after vaccination are as follows Figure 9 As shown, inoculation with MEC_B345 can alleviate root rot in American ginseng caused by inoculated pathogenic fungi. The blank control and American ginseng inoculated solely with MEC_B345 did not develop the disease (see...). Figure 9 The disease was more severe in the control group inoculated only with pathogenic fungi (A and 9B), while the control group inoculated only with pathogenic fungi showed more severe disease. The mycelial growth in the F. solani inoculated group was vigorous, and the cross-section of the American ginseng showed severe rot (see [link to treatment]). Figure 9 C), while the treatment group simultaneously inoculated with MEC_B345 and F. solani showed no obvious mycelial growth ( Figure 9 D); the root surface of the I. mors-panacis inoculated group showed signs of rot. Figure 9 E), the treatment group simultaneously inoculated with a mixed bacterial solution of MEC_B345 and I. mors-panacis showed that, when viewed on the cross-section of the American ginseng, the color of the rotten parts was lighter and the area of ​​rotten parts was significantly reduced. Figure 9 F).

[0110] MEC_B345 can significantly reduce root rot in American ginseng caused by F. solani and I. mors-panacis, thus preventing and treating root rot in American ginseng.

[0111] The preferred embodiments of this disclosure have been described in detail above. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0112] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0113] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. Burkholderia forestosa ( Burkholderia arboris ), characterized in that, It was deposited on November 8, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 26086.

2. A biological control agent, characterized in that, The biological control agent includes Burkholderia forestica as described in claim 1.

3. The application of Burkholderia forestosa as described in claim 1 or the biological control agent as described in claim 2 in inhibiting plant pathogenic fungi; wherein the pathogenic fungi are selected from... Fusarium solani, Ilyonectria mors- panacis, Ilyonectria vredehoekensis, Botrytis cinerea, Alternaria solani, Fusarium oxysporum f.sp. cucumerinum ,Phytophthora drechsleri, Alternaria raphani, Valsa mali, Fusarium oxysporum f. sp. niveum and Drechslera Sofokiniana One or more of them.

4. The application of Burkholderia forestosa as described in claim 1 or the biological control agent as described in claim 2 in the control of plant diseases; The plant disease is caused by plant pathogenic fungi; the plant pathogenic fungi are selected from... Fusarium solani Ilyonectria mors-panacis , Ilyonectria vredehoekensis , Botrytis cinerea , Alternaria solani , Fusarium oxysporum f.sp. cucumerinum、 Phytophthora drechsleri , Alternaria raphani, Valsa mali , Fusarium oxysporum f. sp. niveum and Drechslera Sofokiniana One or more of them.

5. The application according to claim 4, characterized in that, The plant diseases mentioned are selected from ginseng root rot, ginseng rust rot, American ginseng root rot, Panax notoginseng root rot, Astragalus root rot, Atractylodes macrocephala root rot, Angelica sinensis root rot, Glycyrrhiza uralensis root rot, Salvia miltiorrhiza root rot, Ligusticum chuanxiong root rot, Ophiopogon japonicus root rot, Carthamus tinctorius root rot, Isatis indigotica root rot, Lycium barbarum root rot, Alfalfa root rot, Coptis chinensis root rot, Dendrobium officinale root rot, Zingiber officinale root rot, Sweet potato root rot, Aconitum carmichaelii root rot, Blackberry black rot, Strawberry gray mold, Tomato early blight, Cucumber wilt, Cucumber blight, Radish black spot, Apple rot, Watermelon wilt, and Wheat root rot.

6. The application of Burkholderia foresticae as described in claim 1 or the biological control agent as described in claim 2 in the control of root rot in ginseng plants.

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