Burkholderia BI1 strain and application thereof

Abstract

The invention relates to the technical field of agricultural microorganisms, and discloses a Burkholderia metablica BI1 strain and application thereof, the Burkholderia metablica BI1 strain is classified and named as Burkholderia metablica BI1, and the Burkholderia metablica BI1 strain is preserved in the China Center for Type Culture Collection on March 27, 2025 with the preservation number of CCTCC No: M2025612. The strain has broad-spectrum antagonistic activity on various pathogenic bacteria causing ginger soil-borne diseases, the bacteriostasis rate reaches 50%-85%, and insoluble phosphorus and potassium minerals can be dissolved. The obtained microbial preparation can be prepared into a liquid or solid form, the viable count is high, and the storage stability is good. Field tests prove that the disease occurrence rate of the ginger can be remarkably reduced, plant growth is promoted, and the yield is increased by 20-25%. The invention provides an efficient biological control and biological fertilizer integrated solution for green and sustainable cultivation of ginger and other crops.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, specifically to a Burkholderia BI1 strain and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Ginger Zingiber officinale Ginger is an important economic crop in China; however, its production has long been severely hampered by soil-borne diseases. The main pathogens causing rhizome rot and wilt in ginger include various Fusarium species. Fusarium spp. ),like Fusarium fujikuroi , F. annulatum , F. foetens and oomycetes Pythium myriotylum and fungi Plectosphaerella cucumerina , Neocosmospora rubicola These pathogens can survive in the soil for a long time, spread rapidly, and often form complex infections, leading to a significant decline in ginger yield and quality, causing huge economic losses.

[0004] Currently, agricultural production still heavily relies on chemical fumigants (such as dazomet and chloropicrin) and chemical fungicides for the control of soil-borne diseases. However, the efficacy of chemical fumigants and fungicides is gradually decreasing, and they are causing damage to the ecological environment, resulting in damage to beneficial soil microbial communities, increased drug resistance, and chemical residue problems.

[0005] Therefore, there is an urgent need to develop sustainable resources of biocontrol and growth-promoting microorganisms. Burkholderia bacteria have shown potential as biocontrol agents in some crops. However, specific strains of Burkholderia optimized for the rhizosphere environment of ginger and field-validated to effectively inhibit various soil-borne pathogens of ginger are still rare in current technologies.

[0006] In summary, there is an urgent need in this field for a new strain of Burkholderia and its formulation that can simultaneously and efficiently inhibit multiple soil-borne pathogens of ginger, promote ginger growth, and is easy to industrialize and apply in the field. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention aims to solve the problems of reduced efficacy of chemical pesticides and fertilizers in controlling soil-borne diseases of ginger, ecological damage, and imbalance of soil microbial communities. Specifically, it provides a microbial strain and preparation that can simultaneously and efficiently inhibit multiple soil-borne pathogens of ginger, promote plant nutrient absorption and growth, and is suitable for both industrial production and field application.

[0008] The technical solution adopted in this invention is as follows: Firstly, a Burkholderia BI1 strain is provided, classified and named... Burkholderia metallica BI1, this strain was deposited at the China Center for Type Culture Collection (CCTCC) on March 27, 2025, with accession number CCTCC No: M2025612.

[0009] Secondly, a microbial mixture is provided, characterized in that it at least comprises the present invention. Burkholderia metallica BI1 strain, the microbial mixture may contain other biocontrol bacteria or bacteria that promote ginger growth and are compatible with the present invention. Burkholderia metallica Symbiotic microorganisms of strain BI1.

[0010] Optionally, the microbial mixture may further contain at least one microorganism selected from the following: Bacillus subtilis (Bacillus subtilis ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Pseudomonas fluorescens, etc.

[0011] Thirdly, a method comprising the above... Burkholderia metallica The microbial agent of strain BI1 or the microbial mixture of the present invention.

[0012] The preparation method of the microbial agent is as follows: [The following text appears to be a separate, unrelated section:] ...the... Burkholderia metallica BI1 can be fermented in solid or liquid fermentation media to prepare microbial agents of different formulations directly or through further processing (with carriers, other additives, etc.), or further mixed with other fertilizers to make compound fertilizers.

[0013] Optionally, the microbial inoculant includes an agriculturally acceptable carrier; the carrier can be a solid carrier or a liquid carrier. The solid or liquid carrier is a conventional carrier material, wherein the solid carrier can be selected from talc, peat, or vermiculite; the liquid carrier can be vegetable oil, mineral oil, or water.

[0014] Optionally, the formulation of the microbial agent is a wettable powder, a water dispersible agent, an aqueous suspension, a dispersible oil suspension, or other forms.

[0015] Optionally, the active ingredient of the microbial agent is... Burkholderia metallica BI1, this active ingredient can be used as Burkholderia metallica BI1 exists in the form of fermentation broth or fermentation supernatant.

[0016] Preferably, in the microbial agent, Burkholderia metallica The effective viable count of BI1 in liquid formulation is 10. 6 -10 9 CFU·mL -1 Or solid dosage form 10 6 -10 7 CFU·g -1 .

[0017] Fourthly, a method is provided that includes... Burkholderia metallica Fermentation broth of BI1.

[0018] Fifthly, a method is provided as described above. Burkholderia metallica The fermentation method of BI1, wherein the... Burkholderia metallica BI1 was inoculated into the fermentation medium for fermentation culture.

[0019] Optionally, the fermentation medium is LB medium.

[0020] Sixthly, the aforementioned Burkholderia metallica BI1, the use of the microbial mixture and / or the microbial agent in the preparation of compositions for any of the following purposes: (1) Prevention and control of soil-borne diseases in ginger; the soil-borne diseases in ginger are caused by one or more pathogens selected from the following: Pythium spp. ( Pythium myriotylum ), Fusarium oxysporum ( Fusarium fujikuroi ), Fusarium moniliforme ( Fusarium foetens Fusarium annulus ( ), Fusarium annulatum ), cucumber woven shell ( Plectosphaerella cucumerina ) or red clumps and red shells ( Neocosmospora rubicola ); (2) Promotes the growth of ginger plants; (3) Increase ginger yield; (4) Improve the physical and chemical properties of ginger cultivation soil.

[0021] In one or more embodiments of the present invention, the prevention in application (1) includes one or more of the following mechanisms: It directly inhibits the growth of pathogens by secreting antibacterial compounds; Indirectly inhibiting pathogens by competing for rhizosphere nutrients and colonization sites.

[0022] In one or more embodiments of the present invention, the improved ginger cultivation soil in (4) exhibits the following effects: Increase the content of available phosphorus and / or available potassium in the soil.

[0023] Seventhly, a method for preventing and controlling soil-borne diseases in ginger is provided, comprising: […]. ​ ​ BI1, the microbial mixture and / or the microbial agent are applied to the plant roots or the soil in which the plant grows.

[0024] Eighthly, a method for promoting ginger growth is provided, comprising: […]. ​ BI1, the microbial mixture and / or the microbial agent are applied to the roots of plants or the soil in which plants grow to enhance the absorption of nutrients by plants, increase plant biomass, or increase crop yield.

[0025] In one or more embodiments of the present invention, the promotion of plant growth includes dissolving insoluble phosphorus and / or insoluble potassium in the soil.

[0026] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) Broad-spectrum and highly efficient antagonistic activity: This strain exhibits strong antagonistic effects against a variety of soil-borne pathogenic fungi and oomycetes that cause serious damage to ginger. The in vitro inhibition rate can reach up to 85.79%, and it has the potential to "prevent multiple diseases with one strain".

[0027] (2) Multiple growth-promoting mechanisms: Biological control: It can produce a variety of antimicrobial metabolites (such as Macrophorin B, Pinolidoxin, etc.) and signaling molecules related to inducing systemic resistance, and secrete extracellular proteins related to microbial antagonism and plant immune induction.

[0028] Bio-fertilizers: can effectively dissolve insoluble inorganic phosphorus and potassium in the soil, converting ineffective nutrients into effective forms that plants can absorb.

[0029] (3) Significant field application effects: Field trials have shown that the application of this strain preparation can significantly increase the height and number of leaves of ginger plants, reduce the proportion of diseased leaves by about 40 percentage points, and increase crop yield by 20–25%.

[0030] (4) Environmental friendliness and safety: This formulation complies with the Chinese microbial fertilizer standard (NY / T 798-2015), is environmentally safe, can reduce the input of chemical pesticides and fertilizers, and is conducive to sustainable agricultural development.

[0031] (5) Feasibility of industrial production: ​ BI1 strain is easy to ferment and culture. It can be industrially produced through liquid or solid-state fermentation and can be prepared into stable bio-fertilizers or bio-pesticides. Attached Figure Description

[0032] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0033] ​ . ​ Isolation and identification of strain BI1; a) Morphology of pure colonies on LB agar; b) Observation of bacterial cell morphology under an optical microscope (scale bar = 50 µm); c) Phylogenetic tree based on 16S rRNA gene sequence, showing the phylogenetic relationship of strain BI1. ​ and ​ The systematic classification status among genera.

[0034] ​ . ​ Phosphate- and potassium-solubilizing activities of strain BI1; a: Uninoculated NBRIP medium (negative control); b: ​ A distinct clear zone formed on NBRIP medium, indicating its ability to dissolve inorganic phosphorus; c: uninoculated modified Aleksandrov medium; d: ​ ​ The pale yellow color around the colonies of BI1 on modified Aleksandrov medium indicates that it achieves potassium dissolution through medium acidification. Detailed Implementation

[0035] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0037] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0038] Example 1 ​Isolation and Identification of BI1 Rhizosphere soil samples collected from ginger plants (from Laiwu District, Jinan City, Shandong Province) were serially diluted and inoculated onto LB agar plates and cultured at 30℃. Purified colonies were obtained through multiple isolations, and their morphological and molecular characteristics were identified. [Strain details omitted] ​ BI1 colonies are light brown and raised, and the bacteria are Gram-negative, motile short rod-shaped bacteria (see...). ​ a, 1b).

[0039] The sequence was identified by 16S rRNA gene sequencing. It was amplified using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO:2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO:3) and confirmed by Sanger sequencing. Its sequence is similar to that of the type strain. ​ The similarity of LMG 24068ᵀ exceeds 99.5% (see...) ​ c, sequence number SEQ ID NO:1), identified as ​ .

[0040] Example 2 ​ In vitro antagonistic activity of BI1 Evaluation was conducted using the double-culture plate method. ​ The antagonistic activity of BI1 against major soil-borne pathogens of ginger was tested, including the following: ​ , ​ , ​ ​ , ​ , ​ and ​ ​ wait.

[0041] On 9 cm diameter PDA plates, pathogenic bacterial discs and BI1 strain streaks were simultaneously inoculated, with a 2 cm gap between them; the control group was inoculated only with pathogenic bacterial discs. The colony growth area (mm²) of the pathogen was measured at 6, 12, 18, and 24 days post-incubation (dpi). 2 The antagonism rate is calculated using the following formula: , Where C is the control colony area and T is the double-culture colony area.

[0042] The results show that Burkholderia metallica BI1 exhibits broad-spectrum antagonistic activity, producing 50–85% inhibition against a variety of pathogens, depending on the pathogen species and culture time. The maximum inhibition rate occurs at [specific point in time]. Neocosmospora rubicola(85.79%) Fusarium annulatum (73.66%) and Plectosphaerella cucumerina (74.10%). The above results confirm that this strain has the potential to serve as a biocontrol agent for ginger root rot complex.

[0043] Table 1 Burkholderia metallica In vitro antagonistic activity of BI1 against different fungal pathogens , , , This table lists the average colony growth area (mean ± standard deviation) of each pathogen on control plates, the area of ​​the inhibition zone in the two-culture experiment, and the percentage of antagonism relative to the control (n = 5).

[0044] Example 3 Burkholderia metallica Metabolite analysis of BI1 Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to... Burkholderia metallica Secondary metabolites of BI1 were analyzed, and the structures of the compounds were confirmed by MS / MS to identify the antifungal, signaling, and chelate iron compounds produced during fermentation. After inoculation in LB broth, supernatants were collected at 6, 18, 24, 48, and 72 hours (culture conditions: 30℃, 200 rpm), filtered through a 0.22 μm filter, and detected by ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS / MS).

[0045] Detected metabolites included antifungal compounds (such as 4-methylbenzoic acid, macrophorin B, pulcherosine, and pinolidoxin), signaling molecules (such as Val-Val-Phe, N-jasmonoyltyrosine, and capryloylglycine), and chelate iron compounds (such as aerobactin). Results from different time points reflect the dynamic metabolic activity of BI1 during growth (see Table 2). Time points refer to... Burkholderia metallica BI1 The sampling period during which the metabolite was detected during liquid fermentation culture is used to indicate its production stage in the fermentation process.

[0046] Table 2 Burkholderia metallica Metabolites detected in fermentation broth at different culture time points using BI1

[0047] Note: pos = positive ion mode, neg = negative ion mode.

[0048] The various metabolites identified by LC-MS / MS, such as Macrophorin B and Pinolidoxin, are all compounds with known antifungal activity reported in the literature; Val-Val-Phe and N-jasmonoyltyrosine are known plant signaling molecules or inducers. LC-MS / MS combined with proteomics analysis indicates that… Burkholderia metallica BI1 can synthesize a complex group of metabolites. These metabolites, along with the secreted type VI secretion system proteins and antioxidant enzymes (see Table 3), together explain the broad-spectrum antagonistic effect of this strain.

[0049] Table 3 Burkholderia metallica BI1 is an extracellular protein involved in plant protection.

[0050] This table lists from Burkholderia metallica BI1 Extracellular proteins identified in the study have potential microbial antagonistic and plant immune-inducing functions. These proteins are involved in pathogen inhibition, oxidative stress tolerance, and plant systemic resistance responses.

[0051] Example 4: Mineral Dissolving Capacity Evaluation using the standard plate method Burkholderia metallica Mineral solubility of BI1. The phosphorus solubility and potassium solubility of the strain were determined by NBRIP medium (Nautiyal, 1999) and modified Aleksandrov medium (Etesami et al., 2017), respectively.

[0052] After incubation at 30°C for 5–7 days Burkholderia metallica BI1 The formation of a distinct clear zone on NBRIP medium indicates the release of soluble phosphorus. A pale yellow area around the colony was observed on modified Aleksandrov medium, due to acidification caused by bacterial metabolism. No such changes were observed in the uninoculated control group (see...). Figure 2 ).

[0053] Example 5: Formulation and Storage Stability Fermentation was carried out in LB liquid medium at 30 °C and 200 rpm for 72 hours, and the resulting fermentation broth had a colony forming unit (CFU) count exceeding 10. 8 CFU·mL -1The fermentation broth was mixed with sterile talc powder at a ratio of 1:2 (w / w) to prepare a solution containing 10... 8 CFU·g -1 A stabilized powder. This formulation maintains a viable bacterial count above 10⁻⁶ after being stored at 25°C for 6 months. 7 CFU·g -1 .

[0054] Example 6 Field Evaluation Field trials were conducted in Shandong Province, with two treatment concentrations: 10... 6 and 10 8 CFU·mL -1 Each ginger plant was irrigated with 10 mL of bacterial suspension one month after transplanting and again one month later. Results measured on December 12, 2024, showed that 10 8 CFU·mL -1 The average plant height in the treatment group was 31.5 ± 3.5 cm, and the average number of leaves was 7.2 ± 2.4, while in the control group they were 26.1 ± 4.3 cm and 5.6 ± 2.5 leaves, respectively. The proportion of leaves damaged by disease decreased from approximately 50% in the control group to BI1-10. 8 Approximately 11% of the treatment group was affected. Field estimates showed a yield increase of approximately 20–25% compared to the control (see Table 4).

[0055] Table 4. At different concentrations Burkholderia metallica Field performance of ginger plants treated twice with BI1

[0056] Data are presented as mean ± standard deviation (n = 5). The measurement date was December 12, 2024, and the harvest date was December 20, 2024.

[0057] Example 7 Safety and Standards Compliance Evaluation According to the Chinese agricultural industry standard NY / T 798–2015 "Technical Requirements for Microbial Fertilizers", the following applies to Example 5. Burkholderia metallica The BI1 formulation was tested.

[0058] Test results showed that the number of live bacteria was higher than 10. 8 CFU·g -1 The pH value was 7.1, the water content was 8.7%, and no pathogenic bacteria (Escherichia coli, Salmonella, Staphylococcus aureus) were detected. After storage at 25 °C for 6 months, the product maintained a viable count ≥ 9 × 10⁻⁶.7 CFU·g -1 Furthermore, it showed no adverse effects on ginger seedlings.

[0059] The above data proves that the product has good environmental safety and meets the requirements of NY / T 798–2015 standard, and can be safely promoted and used in the field as a certified microbial fertilizer.

[0060] In conclusion, Burkholderia metallica The BI1 strain can be industrially produced through liquid or solid-state fermentation, and can be formulated into stable bio-fertilizers or bio-pesticides. This strain exhibits broad-spectrum control effects, good storage stability, and environmental safety, making it suitable for large-scale application in ginger and various vegetable crops, and contributing to the development of green and organic agriculture in my country. This bio-formulation meets China's microbial fertilizer registration standards, and the viable cell count remains at 10 after six months of storage. 7 CFU·g -1 The above methods can reduce the incidence of root and stem rot by 65–75% and increase field yield by 20–25% compared to the untreated control.

[0061] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Burkholderia BI1 strain, characterized in that, Category naming Burkholderia metallica BI1, this strain was deposited at the China Center for Type Culture Collection (CCTCC) on March 27, 2025, with accession number CCTCC No: M2025612.

2. A mixture of microorganisms, characterized in that, At least comprising the contents of claim 1 Burkholderia metallica BI1 strain.

3. A device comprising the features described in claim 1 Burkholderia metallica The microbial agent of strain BI1 or the microbial mixture of claim 2.

4. The microbial agent as described in claim 3, characterized in that, It includes an agriculturally acceptable carrier; the carrier is a solid carrier or a liquid carrier; the solid carrier is selected from at least one of talc, peat, and vermiculite; the liquid carrier is selected from vegetable oil, mineral oil, or water; the formulation of the microbial agent is a wettable powder, water-dispersible granule, water suspension, or dispersible oil suspension.

5. A device comprising the features described in claim 1 Burkholderia metallica Fermentation broth of BI1.

6. A claim 1 Burkholderia metallica The fermentation method of BI1 is characterized by, The Burkholderia metallica BI1 was inoculated into the fermentation medium for fermentation culture.

7. The claim 1 Burkholderia metallica BI1, the use of the microbial mixture of claim 2 and / or the microbial agent of claim 3 or 4 in the preparation of compositions for any of the following uses: (1) Prevention and control of soil-borne diseases in ginger; the soil-borne diseases in ginger are caused by one or more pathogens selected from the following: Pythium spp. ( Pythium myriotylum ), Fusarium oxysporum ( Fusarium fujikuroi ), Fusarium moniliforme ( Fusarium foetens Fusarium annulus ( ), Fusarium annulatum ), cucumber woven shell ( Plectosphaerella cucumerina ) or red clumps and red shells ( Neocosmospora rubicola ); (2) Promotes the growth of ginger plants; (3) Increase ginger production.

8. The application as described in claim 7, characterized in that, The prevention and control in application (1) includes one or more of the following mechanisms: It directly inhibits the growth of pathogens by secreting antibacterial compounds; Indirectly inhibiting pathogens by competing for rhizosphere nutrients and colonization sites; The improved ginger cultivation soil in application (4) exhibits the following effects: Increase the content of available phosphorus and / or available potassium in the soil.

9. A method for preventing soil-borne diseases in ginger, comprising using the method described in claim 1 Burkholderia metallica BI1, the microbial mixture of claim 2 and / or the microbial agent of claim 3 or 4 are applied to the roots of plants or to the soil in which plants grow.

10. A method for promoting ginger growth, characterized in that, Including the one described in claim 1 Burkholderia metallica BI1, the microbial mixture of claim 2 and / or the microbial agent of claim 3 or 4 are applied to the roots of plants or the soil in which plants grow, to enhance the absorption of nutrients by plants, increase plant biomass or increase crop yield.

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