Burkholderia phenazinium MP4 and application thereof

Abstract

This invention discloses a strain of Burkholderia bancroftian MP4 that antagonizes the pathogen causing root rot of Gastrodia elata and its applications. The strain is classified and named as follows: Paraburkholderia bannensis The MP4 file was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 15, 2026, with accession number GDMCC No:68456; Paraburkholderia bannensis MP4 can inhibit the pathogen causing root rot in Gastrodia elata. Verticillium albo-atrum The growth of (Verticillium spp.) Paraburkholderia bannensis MP4 strains, selected from the native cultivation environment of Gastrodia elata, are more likely to survive in the rhizosphere environment of Gastrodia elata compared to exogenously introduced strains, exhibiting good environmental adaptability and being more susceptible to... Verticillium albo- atrum The inhibitory effect is significant and highly targeted, effectively hindering the proliferation of pathogens. Furthermore, this strain is a natural, native microorganism, free from chemical toxicity and pesticide residue risks, aligning with the development concept of green cultivation of Chinese medicinal herbs. This invention fills a gap in the antagonistic relationship between Gastrodia elata and other pathogens in its specific habitat. Verticillium albo-atrum The lack of high-quality microbial strains can serve as a foundation for ongoing research into microbial agents, their mechanisms of action, and their field applications.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology and biological control of plant diseases. More specifically, this invention relates to a strain of Burkholderia bancroftiana MP4 that antagonizes the pathogen of Gastrodia elata root rot and its application. Background Technology

[0002] Gastrodia elata is a precious traditional Chinese medicine, and its artificial cultivation is expanding year by year. According to identification... Verticillium albo- black (formerly Verticillium chrysogenum) is one of the core pathogens that cause various plant diseases such as root rot of Gastrodia elata. This pathogen can persist in the soil for a long time, and its damage is significantly aggravated with the extension of continuous cropping time, resulting in reduced yield and decreased quality of Gastrodia elata.

[0003] Currently, the mainstream control methods for root rot in Gastrodia elata still rely primarily on chemical pesticides. Long-term use easily leads to problems such as pathogen resistance, pesticide residues, and damage to the soil microecology, which does not meet the requirements for green cultivation of medicinal herbs. Existing biological control technologies utilize universal biocontrol bacteria... Verticillium albo-atrum It has extremely weak antibacterial effect and poor adaptability, and is specifically antagonistic to the specific habitat of Gastrodia elata. Verticillium albo-atrum No functional strains have been reported yet. Summary of the Invention

[0004] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0005] To achieve these objectives and other advantages of the present invention, a strain of Burkholderia bancroftiana MP4 antagonistic to the pathogen causing root rot of Gastrodia elata is provided, wherein Burkholderia bancroftiana MP4 is classified as... Paraburkholderia Bann MP4 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 15, 2026, with accession number GDMCC No: 68456; the described Burkholderia bancroftiana MP4 can inhibit the pathogen of root rot of Gastrodia elata. Verticillium wilt black and white The growth of.

[0006] Preferably, the 16S rDNA sequence of *Burkholderia bancroftian* MP4 is shown in SEQ ID NO.1.

[0007] Preferably, the *Burkholderia bancroftian* MP4 is... Verticillium albo-atrum The highest growth inhibition rate was 59.44%.

[0008] A method for culturing Burkholderia bancroftian MP4, which antagonizes the pathogen causing root rot of Gastrodia elata as described above, includes: taking Burkholderia bancroftian MP4 out of a -80℃ freezer, inoculating it into LB solid medium using the four-zone streak method to activate the strain, culturing it at a constant temperature of 28-30℃ for 25-36 h, then picking a single colony and inoculating it into LB medium, culturing it at a temperature of 28-30℃ for 20-36 h.

[0009] Preferably, the LB medium is either LB solid medium or liquid medium; when inoculated into LB liquid medium, the volume of LB liquid medium is 10-30 mL, and the shaking speed is set to 180-220 r / min; when inoculated into LB solid medium, the volume of LB solid medium is 10-30 mL; the LB solid medium comprises: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar, and 1000 mL distilled water; the LB liquid medium comprises: 10 g tryptone, 5 g yeast extract, 10 g NaCl, and 1000 mL distilled water.

[0010] Burkholderia banebrion MP4, as described above, antagonizes the pathogen causing root rot of Gastrodia elata. Verticillium albo-atrum Applications in [the field].

[0011] Preferably, the *Burkholderia banebrionii* MP4 antagonizes the pathogen causing root rot of *Gastrodia elata* by inhibiting... Verticillium albo-atrum Growth.

[0012] Preferably, the Verticillium albo-atrum It is from the China General Microbiological Culture Collection Center, with accession number CGMCC No. 3.2869. Verticillium albo-atrum .

[0013] Application of Burkholderia bancroftian MP4, an antagonistic bacterium against root rot pathogens of Gastrodia elata as described above, in the preparation of biological agents for the prevention and control of diseases of Gastrodia elata.

[0014] Preferably, the disease is Gastrodia elata root rot, which is a disease that causes the tubers of Gastrodia elata to rot.

[0015] The present invention has at least the following beneficial effects: (1) The present invention provides a strain of Burkholderia banebrion MP4 that antagonizes the pathogen causing root rot of Gastrodia elata. Its physicochemical properties are as follows: the colonies are white, smooth, opaque, with neat, rounded edges and a slightly raised center; under microscopic observation, they are short, straight rods with blunt, rounded ends, arranged singly or in pairs, and turn pink after Gram staining, thus identifying it as a Gram-negative bacterium. It grows well at 28 ℃ in LB medium under aerobic conditions, and can reach the logarithmic growth phase in 20-36 h.

[0016] (2) The strains isolated and obtained in this invention Paraburkholderia bannensis MP4 strains, selected from the native cultivation environment of Gastrodia elata, are more likely to survive in the rhizosphere environment of Gastrodia elata compared to exogenously introduced strains, demonstrating good environmental adaptability. Plate confrontation experiments confirmed that... Paraburkholderia bannensis MP4 Verticillium albo-atrum It has a significant inhibitory effect and strong targeting, which can effectively inhibit the proliferation of pathogens; moreover, the strain is a natural native microorganism with no chemical toxicity and no risk of pesticide residues, which is in line with the development concept of green planting of Chinese medicinal materials.

[0017] (3) This invention fills the gap in the antagonistic relationship in the exclusive habitat of Gastrodia elata. Verticillium albo-atrum The lack of high-quality microbial strains can serve as a foundation for ongoing research into microbial agents, their mechanisms of action, and their field applications.

[0018] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0019] Figure 1 This is a colony morphology diagram of strain MP4 of the present invention; Figure 2 Gram staining image of strain MP4 of this invention; Figure 3 This is a phylogenetic diagram of strain MP4 based on its 16S rDNA sequence. Figure 4 This is Embodiment 2 of the present invention. Verticillium albo-atrum Colony morphology diagram; Figure 5 This is Embodiment 2 of the present invention. Verticillium albo-atrum Microscopic morphology of spores; Figure 6 This is Embodiment 2 of the present invention. Verticillium albo-atrum The pathogenicity test results are shown in the image. Figure 7 For example, strain MP4 of the present invention (Example 2) and Verticillium albo-atrum Image showing the antibacterial effect of a plate-on-plate confrontation; Figure 8 The strains in Example 2 and Comparative Example 1 of this invention and Verticillium albo-atrum Diagram showing the antibacterial effect of a plate-on-plate confrontation. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0021] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0022] In this invention, the formula for PDA solid culture medium plates is: 200 g potato, 20 g glucose, 15 g agar, and 1000 mL distilled water. The formula for LB solid medium is: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar, and 1000 mL distilled water. The formula for LB liquid culture medium is: 10 g tryptone, 5 g yeast extract, 10 g NaCl, and 1000 mL distilled water.

[0023] Example 1 A strain of Burkholderia bancroftian MP4, which antagonizes the pathogen causing root rot of Gastrodia elata, is classified as... Paraburkholderia bannensis MP4 (strain MP4) was registered and deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 15, 2026, with accession number GDMCC No:68456.

[0024] The process of isolating, purifying, and identifying strain MP4 from the habitat of Gastrodia elata in this invention: Figure 1 This is a colony morphology diagram of strain MP4 of this invention. Rhizosphere soil and surface soil from *Gastrodia elata* tubers were collected from the *Gastrodia elata* planting area in Wangcang County, Guangyuan City. 5 g of soil sample was placed in 45 mL of sterile physiological saline and incubated with shaking at 200 r / min and 28 ℃ for 30 min. After standing for 10 min, 1 mL of the supernatant was added to 9 mL of sterile physiological saline, and the mixture was added at a ratio of 10... -1 ~10 -8 After serial dilution, the culture medium was spread onto PDA solid agar plates and LB solid agar plates, with each dilution repeated three times. The plates were then incubated at 28 °C. Daily observations were performed, and colonies of different morphologies were isolated by streaking. This streaking was repeated until a single colony exhibited a uniform growth pattern. A single colony was then picked and incubated in 20 mL of LB liquid agar until OD (Organic Degrees). 600 The concentration was 0.7, and the strain was then preserved at -80°C using the glycerol tube preservation method. The MP4 strain colonies were white, smooth, opaque, with neat, rounded edges and a slightly raised center (as shown in the image). Figure 1 ).

[0025] Figure 2 This is a Gram staining image of strain MP4 from this invention. A single colony of the purified strain MP4 was picked and... Figure 2The procedure involves Gram staining to observe the staining and cell morphology. The bacteria appear pink, short and straight, with blunt, rounded ends, and are arranged singly or in pairs; they are Gram-negative bacteria (e.g., ...). Figure 2 ).

[0026] Figure 3 This is a phylogenetic diagram of strain MP4 based on its 16S rDNA sequence. The 16S rDNA sequence of strain MP4 was amplified and sequenced, and then subjected to BLAST alignment and NJ cluster analysis (e.g.,...). Figure 3 The strain was identified as belonging to the genus Burkholderia, and its classification was named accordingly. Paraburkholderia bannensis (Burkholderia bancroftiana).

[0027] Example 2 strain MP4 and Verticillium albo-atrum Plate confrontation antibacterial test: Figure 4 This is Embodiment 2 of the present invention. Verticillium albo-atrum Colony morphology diagram. Verticillium wilt black and white Purchased from the China General Microbiological Culture Collection Center, accession number CGMCC No. 3.2869. Verticillium albo-atrum The bacteria were inoculated in the center of a PDA solid medium plate and incubated at 28 °C. Initially, the mycelium was white with neat edges. As the incubation time increased, the inner mycelium turned grayish-black, while the outer mycelium remained white (e.g., ...). Figure 4 ).

[0028] Figure 5 This is Embodiment 2 of the present invention. Verticillium albo-atrum Microscopic morphology of spores. Place one drop of lactophenol-cotton blue staining solution on a glass slide. Use an inoculation loop to pick up a small amount of edge hyphae, gently spread them in the staining solution, and stain for 2 minutes. Cover with a coverslip, absorb excess staining solution, and then observe under a microscope. Figure 5 As shown, the conidiophores are whorled, branching in two whorls. Conidia are elliptical, single, and septate, produced continuously at the apex of flask-shaped conidiophores, aggregating to form easily dispersed spore balls. On PDA solid agar plates, dark brown dormant mycelium is produced, forming tuberculate hyphal knots and thick-walled spore chains (such as...). Figure 5 ).

[0029] Figure 6 For the present invention Verticillium albo-atrum The pathogenicity test results are shown in the image. First, a suspension of pathogenic conidia was prepared. Verticillium albo-atrum The bacteria were inoculated onto PDA solid medium plates using the four-zone streak method. After 6 days of incubation, a mycelial cake was taken from the edge of a single colony using a 5 mm diameter sterile punch. Then, a mycelial cake was inoculated into the center of a new PDA solid medium plate using a sterile inoculation needle, with the mycelial side of the cake facing down and in close contact with the new PDA solid medium plate. The culture was continued until... Verticillium albo-atrum Spread the culture medium evenly, add an appropriate amount of sterile water, then gently wash off the conidia with a sterile medical brush and transfer them to a sterile test tube containing sterile water. Repeat this process at least three times to remove the pathogens. Verticillium albo-atrum Conidial suspension diluted to 10 7 / mL. Secondly, the selection and processing of the tested Gastrodia elata samples: Four fresh, healthy Gastrodia elata tubers of similar size were selected and collected from the Gastrodia elata planting area in Wangcang County, Guangyuan City (i.e., the isolation source of strain MP4). The samples were rinsed with tap water to remove surface soil and impurities, then rinsed with sterile water, and after being dried with absorbent paper, transferred to a clean bench and immersed in 75% ethanol solution for 2 minutes. They were then rinsed again with sterile water and air-dried in a sterile environment. Next, the pathogen infection experiment was conducted. Micro-wounds were created on the surface of the Gastrodia elata using a sterile syringe, and then a conidial suspension was sprayed onto the wounds using a small spray bottle as the experimental group. A blank control group was prepared by spraying sterile water. Mycelial cakes from the edge of a single colony on a PDA solid medium plate were inoculated onto the surface of the Gastrodia elata using a 1.1 cm diameter sterile punch. An equal volume of uncontaminated blank PDA solid medium plates was used as a negative control. Each experimental group was treated three times, and then incubated in a 28℃ constant temperature incubator. The disease progression of *Gastrodia elata* was observed for 7 consecutive days. The infection results on day 7 are as follows: Figure 6 As shown, the first row is the blank control group, and the last three rows are the experimental group. It can be seen that... Verticillium albo-atrum It can cause the tubers of Gastrodia elata to rot.

[0030] The above Verticillium albo-atrum The bacteria were inoculated onto PDA solid medium plates using the four-zone streak method. After 6 days of cultivation, a mycelial patch from the edge of a single colony was punched using a 5 mm diameter sterile punch. Then, a mycelial patch was inoculated into the center of a new PDA solid medium plate using a sterile inoculation needle, with the mycelial side of the patch facing down and in close contact with the new PDA solid medium plate. Two μL of MP4 culture solution was then inoculated at four equidistant points 1.5 cm from the mycelial patch using a pipette. The specific cultivation method for MP4 culture solution was as follows: The MP4 strain, preserved in glycerol at -80℃ as described in Example 1, was taken out and inoculated onto LB solid medium using the four-zone streak method for strain activation. It was incubated at 28℃ for 30 h. Then, a single colony was picked up using sterile forceps and inoculated into 20 mL of LB liquid medium. The culture was incubated at 28℃ and 200 r / min for 20 h, yielding a bacterial concentration of 7 × 10⁻⁶. 8 MP4 bacterial suspension at CFU / mL. Inoculated alone. Verticillium albo-atrum As a blank control group, the experimental group was repeated in triplicate and incubated at 28 ℃. The pathogens were measured. Verticillium albo-atrum The colony diameters are shown in Table 1. The inhibition rate of the bacterial strains to be screened against the pathogens was calculated. The calculation formula is as follows: Inhibition rate (%) = (Coronary diameter of blank control group - Colony diameter of experimental group) / Colony diameter of blank control group × 100% Table 1

[0031] Figure 7 For example, strain MP4 of the present invention (Example 2) and Verticillium albo-atrum Plate contrast diagram showing antibacterial effect. The first column of plates represents the blank control group, and the following three columns represent the experimental group. From Figure 7 As can be seen from the blank control group Verticillium albo-atrum The mycelia grew normally and spread evenly across the culture medium. The experimental group strain MP4 grew normally, but the inoculated MP4 plates showed [a certain pattern / effect]. Verticillium albo-atrum The growth of mycelium on the outer edge was significantly inhibited, and the inhibitory effect remained strong even after 14 days of culture. The antibacterial effect of the initially screened strain MP4 was highly consistent with the antibacterial effects of the three replicates in the secondary screening. Even under conditions where the mycelium did not completely cover the entire PDA solid medium plate, strain MP4 showed strong inhibition against [the virus / mycelium]. Verticillium albo-atrum The mycelial growth inhibition rate of strain MP4 reached a maximum of 59.44% after 14 days of culture. Therefore, strain MP4 has a significant inhibitory effect on mycelial growth. Verticillium albo-atrum Its antibacterial effect is quite prominent.

[0032] Comparative Example 1 Chryseobacterium sp. (Aureobacillus) 1Y-6 was purchased from the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 822601. The above... Verticillium albo-atrum The bacteria were inoculated onto PDA solid medium plates using the four-zone streak method. After 6 days of cultivation, a mycelial disc was taken from the edge of a single colony using a 5 mm diameter sterile punch. Then, a mycelial disc was inoculated into the center of a new PDA solid medium plate using a sterile inoculation needle, with the mycelial side of the disc facing down and in close contact with the new plate. Two μL of cultured bacteria was then inoculated at four equidistant points 1.5 cm from the disc using a pipette. Chryseobacterium sp. 1Y-6 bacterial culture. Chryseobacterium The specific culture method for sp. 1Y-6 bacterial suspension is as follows: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Chryseobacterium After being removed from the -80℃ freezer, sp. 1Y-6 was activated by streak plating on LB solid medium using the four-zone method and incubated at 28℃ for 30 h. Then, single colonies were picked using sterile forceps and inoculated into 20 mL of LB liquid medium and incubated at 28℃ and 200 r / min for 14 h, yielding a bacterial concentration of 7 × 10⁻⁶. 8 CFU / mL Chryseobacterium sp. 1Y-6 bacterial culture. Inoculated alone. Verticillium albo-atrum The experimental group served as a blank control group, and the experimental group was repeated in 3 groups and incubated in a constant temperature incubator at 28℃.

[0033] Figure 8 The strains in Example 2 and Comparative Example 1 of this invention and Verticillium albo-atrum Diagram showing the antibacterial effect of a plate-on-plate confrontation. Figure 8 The images shown in the middle are the results after 14 days of cultivation. It can be seen that, compared to Example 1, after 14 days of cultivation... Chryseobacterium sp. 1-6 showed normal growth and was inoculated. Chryseobacterium On the flat surface of sp. 1Y-6 Verticillium albo-atrum The growth of mycelium on the outer edge was somewhat inhibited, but Verticillium albo-atrum The colony diameter of the bacteria was significantly larger than that of the plate inoculated with strain MP4, indicating that strain MP4 is related to... Chryseobacterium Compared to sp. 1Y-6, it showed a significant inhibitory effect.

[0034] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A strain of Burkholderia bancroftian MP4 that antagonizes the pathogen causing root rot of Gastrodia elata, characterized in that, The classification name of *Burkholderia banebrionis* MP4 is... Paraburkholderia bannensis MP4 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 15, 2026, with accession number GDMCC No: 68456; the described Burkholderia bancroftiana MP4 can inhibit the pathogen of root rot of Gastrodia elata. Verticillium albo-atrum The growth of.

2. The Burkholderia banebrion MP4 antagonistic to the pathogen causing root rot of Gastrodia elata as described in claim 1, characterized in that, The 16S rDNA sequence of *Burkholderia bancroftian* MP4 is shown in SEQ ID NO.

1.

3. The Burkholderia banebrion MP4 antagonistic to the pathogen causing root rot of Gastrodia elata as described in claim 1, characterized in that, The MP4 of *Burkholderia banebrionis* Verticillium albo-atrum The highest growth inhibition rate was 59.44%.

4. A method for culturing Burkholderia bancroftian MP4, which antagonizes the pathogen causing root rot of Gastrodia elata, as described in any one of claims 1-3, characterized in that, include: After removing Burkholderia banebrion MP4 from the -80℃ freezer, the strain was activated by inoculating it onto LB solid medium using the four-zone streak method and incubated at 28-30℃ for 25-36 h. Then, single colonies were picked and inoculated onto LB medium and incubated at 28-30℃ for 20-36 h.

5. The method for culturing Burkholderia baneformis MP4, which antagonizes the pathogen causing root rot of Gastrodia elata, as described in claim 4, is characterized in that... The LB medium is either LB solid medium or liquid medium; when inoculating with LB liquid medium, the volume of LB liquid medium is 10~30 mL, and the shaking speed is set to 180~220 r / min. When inoculating onto LB solid medium, the volume of LB solid medium should be 10-30 mL. LB solid medium consists of: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar, and 1000 mL distilled water. LB liquid medium consists of: 10 g tryptone, 5 g yeast extract, 10 g NaCl, and 1000 mL distilled water.

6. A Burkholderia banebrion MP4 antagonistic to the pathogen causing root rot of Gastrodia elata as described in any one of claims 1-3, in inhibiting... Verticillium albo-atrum Applications in [the field].

7. The application as described in claim 6, characterized in that, The *Burkholderia bane* MP4 antagonizes the pathogen causing root rot of *Gastrodia elata* by inhibiting [the pathogen's activity] in vitro or in soil. Verticillium albo-atrum Growth.

8. The application as described in claim 6, characterized in that, The Verticillium albo-atrum It is from the China General Microbiological Culture Collection Center, with accession number CGMCC No. 3.2869. Verticillium albo-atrum .

9. The use of Burkholderia bancroftian MP4, which antagonizes the pathogen causing root rot of Gastrodia elata as described in any one of claims 1-3, in the preparation of a biological agent for the prevention and control of diseases of Gastrodia elata.

10. The application as described in claim 9, characterized in that, The disease mentioned is root rot of Gastrodia elata, which is a disease that causes the tubers of Gastrodia elata to rot.

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