Two efficient phosphorus-solubilizing burkholderia strains and application thereof

Abstract

The invention belongs to the technical field of microbiology and plant growth-promoting bacteria. More specifically, the invention relates to two efficient phosphorus-solubilizing burkholderia strains and application thereof. Two efficient phosphorus-solubilizing burkholderia strains, namely burkholderia metallica N19 and burkholderia cepacia N31, are separated from the citrus reticulata rhizosphere soil, and both the burkholderia metallica N19 and the burkholderia cepacia N31 can efficiently dissolve main insoluble aluminum phosphorus (Al-P) and calcium phosphorus (Ca-P) in the acid soil, so that the content of available phosphorus in the soil is remarkably increased. Pot experiments show that the N19 and N31 strains are compounded to irrigate roots, so that root development and overground part growth of Citrus chachiensis can be remarkably promoted, the phosphorus concentration of old leaves, new leaves and root systems is remarkably improved, and it is proved that the N19 and N31 strains enhance phosphorus absorption and transport capacity. The strain provided by the invention can adapt to a typical acidic, high-aluminum and organic-matter-rich rhizosphere micro-ecological environment, ensures long-acting expression of phosphate solubilizing and growth promoting functions under a complex soil background, and has a wide industrialization prospect and application value.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and plant growth-promoting bacteria technology. More specifically, it relates to two highly efficient phosphate-solubilizing Burkholderia strains and their applications. Background Technology

[0002] Phosphorus is a key macronutrient essential for plant growth and development, widely involved in core physiological processes such as energy metabolism, genetic information transmission, and membrane structure construction. However, in the acidic red soils widely distributed in my country, especially in the southern regions, large amounts of applied phosphate fertilizer are easily fixed by iron and aluminum ions in the soil, forming insoluble phosphorus pools mainly composed of aluminum-bound phosphorus (Al-P). This results in a long-term low content of available phosphorus in the soil, and the utilization rate of phosphate fertilizer in the current season is generally less than 20%.

[0003] Although phosphorus-solubilizing microorganisms can activate recalcitrant phosphorus through the secretion of organic acids, protonation, or enzymatic hydrolysis, and are considered an important strategy for green alternatives to chemical phosphate fertilizers, most reported phosphorus-solubilizing strains currently available focus on the efficient solubility of calcium phosphorus (Ca-P), with generally weaker solubility for Al-P, which dominates in acidic soils. Furthermore, most strains originate from non-target ecological niches, resulting in limited colonization in the rhizosphere of specialty economic crops such as tea taro and citrus, and their functional expression is easily affected by competition from indigenous microorganisms and fluctuations in rhizosphere physicochemical factors. In addition, a single phosphorus-solubilizing function is insufficient to systematically coordinate multiple growth-promoting needs such as root system architecture shaping, synergistic nutrient absorption, and enhanced stress resistance.

[0004] Therefore, it is of great significance to screen functional strains with multiple growth-promoting properties, such as broad-spectrum phosphorus solubilization (especially for difficult-to-solubilize phosphorus forms such as Al-P), plant hormone synthesis, and siderophore secretion, and to construct a microbial inoculant system with strong ecological adaptability and high functional synergy to improve the efficiency of plant phosphorus utilization. Summary of the Invention

[0005] The technical problem to be solved by this invention is that existing phosphorus-solubilizing microbial agents generally have the following defects and shortcomings: weak ability to dissolve aluminum-bound phosphorus (Al-P) in acidic soil, poor ecological adaptability, and difficulty in promoting growth through single functions. This invention provides a Burkholderia strain and its compound agent derived from the rhizosphere of tea branches and citrus, which has the ability to solubilize phosphorus, produce IAA and siderophores, and is stable in the field and environmentally friendly. This can be used to significantly improve the phosphorus absorption efficiency and overall growth performance of plants.

[0006] The first objective of this invention is to provide a strain of Burkholderia metalloids N19.

[0007] A second objective of this invention is to provide a microbial agent.

[0008] A third objective of this invention is to provide the application of the aforementioned Burkholderia metalloids or the aforementioned bacterial agents.

[0009] The fourth objective of this invention is to provide a method for promoting plant growth.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: In the preliminary exploratory research of this invention, it was found that existing phosphorus-solubilizing strains generally have a significant mismatch between phosphorus form, soil pH, and host adaptation: On the one hand, most of the reported highly efficient phosphate-solubilizing bacteria have shown strong solubility for calcium phosphorus (Ca-P) and are suitable for neutral or calcareous soil environments. However, they have very weak or no response to aluminum phosphorus (Al-P), which is the dominant form of phosphorus in acidic soils, especially the main insoluble phosphorus form in the red soil of the main tea-branch citrus producing areas in southern my country.

[0011] On the other hand, the optimal growing soil for the traditional Chinese medicine Guangchenpi (dried tangerine peel) is a slightly acidic environment with a pH of 5.5-6.5. Under these conditions, Al 3+ The high activity of the strain makes it easy for phosphorus to be fixed as Al-P, leading to a deficiency of available phosphorus. If an unsuitable strain is used, even if it performs well in the laboratory Ca-P system, it is difficult to achieve a significant growth-promoting effect on tea branch citrus in a real acidic cultivation system.

[0012] Furthermore, the compatibility of the strain with the host's soil micro-ecosystem also has an impact. Previous exploratory experiments showed that while introducing exogenous phosphate-solubilizing strains from sources other than *Citrus aurantiacus* exhibited phosphate-solubilizing ability under laboratory conditions, it failed to significantly promote their growth in *Citrus aurantiacus* potted systems. This is presumably due to competitive exclusion from the indigenous microbial community, or intolerance to low pH / high Al content. 3+ Stress leads to short survival time and low population density, making it impossible to form a sustainable and effective functional community in the root microdomain. Therefore, this invention directionally isolates phosphorus-solubilizing and growth-promoting strains from rhizosphere soil samples of *Citrus aurantiacus*, ensuring that the obtained strains are naturally adapted to the rhizosphere microenvironment of *Citrus aurantiacus*, thereby significantly improving their colonization stability and effectiveness in field applications.

[0013] This invention provides a strain of Burkholderia metalloids (… Burkholderia metallica The strain N19 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 10, 2025, with accession number GDMCC NO.67261.

[0014] The present invention provides a microbial agent containing Burkholderia metalloid N19 or its fermentation broth.

[0015] Preferably, the above-mentioned microbial agent also contains Burkholderia cepacia (…). Burkholderia cepacia N31 or its fermentation broth, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 10, 2025, with accession number GDMCC NO.67262.

[0016] Optionally, in the above-mentioned bacterial agent, the mass ratio of Burkholderia metalloid N19 to Burkholderia cepacia N31 is (1~2):(1~2).

[0017] Optionally, in the above-mentioned bacterial agent, the mass ratio of Burkholderia metalloid N19 and Burkholderia cepacia N31 is 1:1.

[0018] Specifically, in the above-mentioned bacterial agents, the effective viable count of the fermentation broth of the *Burkholderia metalloids* N19 and *Burkholderia cepacia* N31 strains is not less than 1 × 10⁻⁶. 9 CFU / mL.

[0019] Specifically, the microbial agent is any one of plant growth-promoting microbial agents, plant root growth-promoting microbial agents, or plant phosphorus nutrient improvers.

[0020] This invention claims protection for the following applications: The application of Burkholderia metalloid N19 or the above-mentioned bacterial agent in promoting plant growth.

[0021] The application of Burkholderia metalloid N19 or the above-mentioned bacterial agent in the preparation of products that promote plant growth.

[0022] The application of Burkholderia metalloid N19 or the above-mentioned bacterial agent in the production of indoleacetic acid.

[0023] The application of Burkholderia metalloid N19 or the above-mentioned bacterial agent in the preparation of products for the production of indoleacetic acid.

[0024] Application of Burkholderia metalloid N19 or the above-mentioned bacterial agent in phosphorus solubilization.

[0025] The application of Burkholderia metalloid N19 or the above-mentioned bacterial agent in the preparation of products with phosphorus solubilizing ability. The application of Burkholderia metalloid N19 or the above-mentioned bacterial agent in promoting the absorption of phosphorus by plants.

[0026] The application of Burkholderia metalloid N19 or the above-mentioned bacterial agent in the preparation of products that promote the absorption of phosphorus by plants.

[0027] The application of Burkholderia metalloids N19 or the above-mentioned bacterial agents in the production of iron carriers.

[0028] The application of Burkholderia metalloid N19 or the above-mentioned bacterial agent in the preparation of products that produce siderophores.

[0029] This invention provides a method for promoting plant growth by treating plants with the aforementioned microbial agent.

[0030] As an alternative implementation, the treatment is root irrigation.

[0031] Specifically, the plant in question is the tea branch mandarin orange.

[0032] The present invention has the following beneficial effects: Two Burkholderia strains were isolated from the rhizosphere soil of Citrus reticulata Blanco cv. Chachi, namely Burkholderia metallica Burkholderia metallica N19 and Burkholderia cepacia Burkholderia cepacia N31. Both strains have the ability to dissolve aluminum-phosphorus (Al-P) and calcium-phosphorus (Ca-P), can convert insoluble phosphorus in the soil into soluble available phosphorus, increase soil nutrients, and also have the ability to produce IAA.

[0033] The pot experiment shows that the combined application of strain N19 and strain N31 by root irrigation can significantly improve the whole-plant biomass of Citrus reticulata Blanco cv. Chachi, and greatly increase the total root length and root surface area. At the same time, the phosphorus concentrations in the old leaves, new leaves and roots of the plants are all significantly increased, confirming that it can effectively enhance the absorption of phosphorus by plants. Further analysis of the phosphorus forms in the rhizosphere soil shows that the combined application of strain N19 and strain N31 by root irrigation can significantly reduce the proportion of insoluble phosphorus components in the soil.

[0034] In summary, the two Burkholderia strains provided by the present invention are isolated from the indigenous microbial community in the rhizosphere of Citrus reticulata Blanco cv. Chachi, are naturally adapted to the typical acidic, high-aluminum and rich-organic-matter rhizosphere microecological environment in the south, have excellent ecological compatibility and field colonization stability, can ensure the long-term expression of phosphorus-solubilizing and growth-promoting functions under complex soil backgrounds, and have broad industrialization prospects and application values. Brief Description of the Drawings

[0035] Figure 1 Colony morphologies of strain N19 and strain N31 on LB solid medium, PVK medium, Mengjinna organic phosphorus solid medium and King solid medium.

[0036] Figure 2 Phylogenetic dendrogram of 16S rDNA sequences of strain N19 and strain N31.

[0037] Figure 3 Determination results of Al-P dissolution ability and Ca-P dissolution ability of strain N19 and strain N31.

[0038] Figure 4 Effects of different treatments on the growth of Citrus reticulata Blanco cv. Chachi; Figure A is the growth phenotype diagram of Citrus reticulata Blanco cv. Chachi under different treatments; Figure B is the determination result of the whole-plant dry weight; Figure C is the determination result of the dry weight of new shoots; Figure D is the determination result of the dry weight of roots; Figure E is the determination result of the total root length; Figure F is the determination result of the root surface area; * represents 0.01 < P < 0.05, ** represents P < 0.01.

[0039] Figure 5Effects of different treatments on the phosphorus uptake of Citrus reticulata Blanco cv. Chachi; Figure A shows the determination results of phosphorus concentration in old leaves of Citrus reticulata Blanco cv. Chachi; Figure B shows the determination results of phosphorus concentration in new leaves of Citrus reticulata Blanco cv. Chachi; Figure C shows the determination results of phosphorus concentration in roots of Citrus reticulata Blanco cv. Chachi; * represents 0.01 < P < 0.05.

[0040] Figure 6 Effects of different treatments on soil phosphorus fractions; Figure A shows the determination results of total phosphorus; Figure B shows the determination results of water-soluble phosphorus; Figure C shows the determination results of phosphorus extracted by sodium bicarbonate; Figure D shows the determination results of phosphorus digested by sodium bicarbonate; Figure E shows the determination results of phosphorus extracted by sodium hydroxide; Figure F shows the determination results of phosphorus digested by sodium hydroxide; Figure G shows the determination results of phosphorus extracted by hydrochloric acid; Figure H shows the determination results of residual phosphorus; NS represents no significant difference, * represents 0.01 < P < 0.05, ** represents P < 0.01. Specific implementation mode

[0041] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0042] [[ID=​​​​​​​​​​​King liquid medium: 20.0 g peptone, 1.725 g dipotassium hydrogen phosphate, 15.0 mL glycerol, 1.5 g magnesium sulfate heptahydrate, 0.1 g tryptophan, bring the volume to 1 L with distilled water, and adjust the pH to 7.2. Sterilize at 121℃ for 15 min.

[0047] Salkowski reagent: 12 g FeCl3•6H2O + 500 mL distilled water + 7.9 mL concentrated H2SO4 (add slowly, cool in an ice bath).

[0048] Stock solution of antimony molybdate sulfate: Measure 126 mL of concentrated sulfuric acid and slowly add it to 100 mL of water, stirring constantly, and cool. Separately, weigh 10 g of finely ground ammonium molybdate and dissolve it in 300 mL of water at approximately 60°C, then cool. Slowly pour the sulfuric acid solution into the ammonium molybdate solution. Add 100 mL of 0.5% potassium antimony phenolate solution, cool, dilute with water to 1000 mL, shake well, and store in a brown reagent bottle.

[0049] The preparation method of the molybdenum-antimony anti-stock solution is as follows: Add solution A to solution B, then add 0.5 g of potassium antimony tartrate, cool, dilute with pure water to 1 L, shake well, and store at 4 ℃ away from light for later use.

[0050] Solution A: Slowly add 153 mL of concentrated sulfuric acid to 400 mL of pure water while stirring with a stirring rod, and then cool.

[0051] Solution B: Weigh 10 g of ammonium molybdate into 300 mL of pure water.

[0052] For molybdenum-antimony anti-indicator, add 1.5g of ascorbic acid to every 100ml of molybdenum-antimony anti-indicator stock solution. Prepare fresh and use immediately, and store away from light.

[0053] Ascorbic acid: Seller: Dingguo Biotechnology, Product No.: DH030-1, Brand: FLUKA.

[0054] Ca-P medium (Monkina inorganic phosphorus liquid medium with calcium phosphate as the sole phosphorus source): 10.0 g glucose, 0.5 g ammonium sulfate, 0.3 g sodium chloride, 0.3 g potassium chloride, 0.3 g magnesium sulfate, 0.03 g ferrous sulfate, 0.03 g manganese sulfate, 5.0 g calcium phosphate, 4 g agar. Dilute to 1 L with distilled water and adjust pH to 7.0. Sterilize at 121℃ for 15 min.

[0055] Al-P medium (Monginna inorganic phosphorus liquid medium with aluminum phosphate as the sole phosphorus source): glucose 10.0 g, ammonium sulfate 0.5 g, sodium chloride 0.3 g, potassium chloride 0.3 g, magnesium sulfate 0.3 g, ferrous sulfate 0.03 g, manganese sulfate 0.03 g, aluminum phosphate 5.0 g, agar 4 g, diluted to 1 L with distilled water, and the pH adjusted to 7.0. Sterilize at 121℃ for 15 min.

[0056] SSM liquid culture medium: per 1L: 4 g succinic acid, 1 g ammonium sulfate, 0.2 g magnesium sulfate, 4.075 g potassium hydroxide. Simultaneously, dissolve 60 g of dipotassium hydrogen phosphate and 30 g of potassium dihydrogen phosphate in 200 ml of water, add 20 ml of this solution per liter, sterilize separately, and then mix. Preparation method of CAS detection solution: Solution A: 60.5 mg Chromium Azure S (CAS) + 50 mL deionized water; Solution B: 13.5 mg FeCl3·6H2O + 50 mL water; CAS test solution: Mix 7.5 ml of solution A and 1.5 ml of solution B, add 25 mL of 0.0219 g of hexadecyltrimethylammonium bromide (HDTMA, CTAB), add 4.3079 g of piperazine and 30 mL of water, and bring the volume to 100 mL (adjust the pH to 5.6 with concentrated hydrochloric acid).

[0057] Pretreatment before use: Before using the CAS test solution, add 43.9 mg of 5-sulfosalicylic acid to every 50 mL of CAS test solution.

[0058] Example 1: Screening, isolation and identification of Burkholderia I. Screening and Isolation of Strains Both strains N19 and N31 were isolated from rhizosphere soil samples of potted *Citrus aurantiacus*.

[0059] 1. Methods for screening and isolating strains Soil samples were resuspended in sterile water, serially diluted, and spread onto Monkina organophosphate solid medium for incubation. Strains with strong phosphorus-solubilizing abilities were initially screened based on the size of the clear zone around the colonies. Furthermore, multiple isolated strains were inoculated onto PVK medium, Monkina organophosphate solid medium, and King solid medium, respectively, to test each strain's ability to solubilize inorganic phosphorus, organic phosphorus, and produce IAA.

[0060] 2. Separation Results Based on a comprehensive evaluation of growth-promoting functions (phosphate solubilization and IAA production capacity), two strains with outstanding functions were finally screened and, after purification and culture, named strain N19 and strain N31, respectively. The results of the determination of inorganic phosphorus solubilization, organic phosphorus solubilization, and IAA production capacity of strains N19 and N31 are as follows: Figure 1 As shown.

[0061] II. Colony characteristics and morphological identification of strains N19 and N31 Strains N19 and N31 were cultured and observed on LB solid medium. The results are as follows: Figure 1 As shown. The colony characteristics of strain N19 are: round colonies with a raised center, pale yellow color, smooth and neat edges, and moist cell structure. The colony characteristics of strain N31 are: round colonies with a raised center, pale yellow color, smooth and neat edges, and moist cell structure.

[0062] III. Sequence determination and analysis of strains N19 and N31 The 16S rDNA genes of strains N19 and N31 were amplified by PCR using universal primers, and the amplified products were purified and then sequenced.

[0063] The 16S rDNA gene sequence of strain N19 is shown in SEQ ID NO.1. SEQ ID NO.1:

[0064] The 16S rDNA gene sequence of strain N31 is shown in SEQ ID NO.2. SEQ ID NO.2:

[0065] The 16S rDNA sequence of the amplified product was BLAST aligned, and a phylogenetic tree was constructed. The results are as follows: Figure 2 As shown, the results indicate that N19 shares more than 98% similarity with several different strains of Burkholderia metalloids, and N31 shares more than 98% similarity with several different strains of Burkholderia cepacia. Based on the morphological, cultural characteristics and physiological and biochemical analysis results of the strains, strain N19 was identified as Burkholderia metalloids and N31 as Burkholderia cepacia.

[0066] Burkholderia metalloids strain N19 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 10, 2025, with accession number GDMCC No. 67261.

[0067] Burkholderia cepacia strain N31 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 10, 2025, with accession number GDMCC No. 67262.

[0068] Example 2: Determination of the ability of N19 and N31 strains to decompose inorganic phosphorus, produce siderophores, and produce IAA. I. Quantitative Detection of Ca-P Solving Ability Take three Erlenmeyer flasks containing 50 ml of sterile Ca-P medium and label them 1, 2, and 3. Inoculate the bacterial suspension (N19 strain or N31 strain) into the numbered medium at a 1% inoculum volume. Incubate at 30°C with shaking at a speed of 180 r / min. After 3 days, remove the Erlenmeyer flasks. Centrifuge the inoculated Ca-P medium and the uninoculated medium at 10000 rpm for 10 min. Take the supernatant and mix it with the molybdenum antimony indicator at a ratio of 1:4 (v / v). After standing at room temperature in the dark for 30 min, measure the absorbance at a wavelength of 700 nm.

[0069] The method for calculating the Ca-P solution is as follows: The standard curve formula for available phosphorus concentration in Ca-P medium is Y = 1.089X + 0.0152, where X is the OD value after inoculation. 700 Compared with unvaccinated OD 700 The difference. Multiply the Y value by 200 to obtain the available phosphorus concentration (μg / mL) in the Ca-P medium.

[0070] II. Quantitative Detection of Al-P Degradation Capacity Take three Erlenmeyer flasks containing 50 ml of sterile Al-P medium and label them 1, 2, and 3. Inoculate the bacterial suspension (N19 or N31 strain) at a 1% inoculum volume into the numbered mediums and incubate at 30°C with shaking at a speed of 180 r / min. After 3 days, remove the Erlenmeyer flasks. Centrifuge the inoculated Al-P medium and the uninoculated medium at 10000 rpm for 10 min. Take the supernatant and mix it with the molybdenum antimony indicator at a ratio of 1:4 (v / v). After standing at room temperature in the dark for 30 min, measure the absorbance at a wavelength of 700 nm.

[0071] The method for calculating the Al-P solution capability is as follows: The standard curve formula for effective phosphorus concentration is Y = 1.089X + 0.0152, where X is the OD after inoculation. 700 Compared with unvaccinated OD 700 The difference. Multiply Y by 40 to obtain the effective phosphorus concentration (μg / mL) in Al-P medium.

[0072] III. Quantitative Detection of Iron Production Capacity Take three Erlenmeyer flasks containing 30 ml of sterile SSM liquid culture medium and label them 1, 2, and 3. Inoculate the bacterial suspension (N19 strain or N31 strain) into the numbered SSM medium at a 1% inoculum volume, and incubate at 30°C with shaking at a speed of 180 r / min. After 3 days, remove the Erlenmeyer flasks. Centrifuge the inoculated SSM culture medium and the uninoculated SSM culture medium at 10000 rpm for 10 min. Take the supernatant and mix it with an equal volume of CAS detection solution. After standing at room temperature in the dark for 30 min, measure the absorbance at a wavelength of 630 nm.

[0073] The calculation method for iron production capacity is as follows: Ferrocarrier percentage =

[0074] The standard curve formula for calculating ferritin production is Y = 32.99X + 0.2746, where X is the measured percentage of ferritin.

[0075] IV. Quantitative Testing of IAA Production Capacity Take three Erlenmeyer flasks containing 50 ml of sterile King liquid culture medium and label them 1, 2, and 3. Inoculate the bacterial culture (N19 strain or N31 strain) into the numbered King culture medium at a 1% inoculum volume, and incubate at 30°C with shaking at a speed of 180 r / min. After 3 days, remove the Erlenmeyer flasks. Centrifuge the inoculated King culture medium and the uninoculated King culture medium at 10000 rpm for 10 min. Take the supernatant and mix it with an equal volume of Salkowski reagent. After standing at room temperature in the dark for 30 min, measure the absorbance at a wavelength of 530 nm.

[0076] The method for calculating IAA production capacity is as follows: The standard curve formula for calculating IAA is Y = 0.0167X, where Y is the OD after vaccination. 530 Compared with unvaccinated OD 530 The difference, X, is the measured IAA concentration (i.e. IAA production capacity).

[0077] V. Analysis of Growth-Promoting Characteristics Colony morphology of strains N19 and N31 on King solid medium is shown in the figure below. Figure 1 As shown, the results indicate that both strains N19 and N31 can turn King solid medium pink after the Salkowski reaction, demonstrating significant IAA synthesis capabilities.

[0078] The results of the determination of the siderophore production capacity, phosphorus solubilization capacity and IAA production capacity of strains N19 and N31 are shown in Table 1. The results show that both strains can dissolve Ca-P and Al-P precipitates ( Figure 3 The results demonstrate that strains N19 and N31 possess an extremely strong ability to dissolve different insoluble phosphorus forms, indicating their excellent phosphorus activation potential in soils with different phosphorus forms. Strains N19 and N31 also exhibit the ability to produce siderophores and IAA, demonstrating significant potential for growth promotion.

[0079] Table 1. Characteristics of iron carrier, IAA production, and phosphorus solubilization capacity.

[0080] Example 3: Effects of Burkholderia metalloids N19 and Burkholderia cepacia N31 on the growth of Citrus aurantiaca var. chamomile. I. Cultivation of Chazhigan (a type of mandarin orange) Healthy seedlings of Xinhui Chazhigan oranges with uniform growth were transplanted into greenhouse pots. Each pot contained 1.7 kg of acidic soil and planted one seedling. Base fertilizer was applied uniformly, with the following treatment: superphosphate: 0.6 mg / kg soil; urea: 0.2 g / kg soil; potassium chloride: 0.2 g / kg soil.

[0081] II. Vaccination Procedure One week after transplanting and stabilizing, the seedlings of *Citrus medica* were inoculated. *Burkholderia metalloids* N19 and *Burkholderia cepacia* N31 were inoculated separately into liquid LB medium and cultured at 28°C and 180 r / min until OD... 600 The concentration was 0.8. After centrifugation at 5000 rpm for 5 min, the culture was resuspended in an equal volume of sterile water to prepare the bacterial agent (10). 9 (CFU / mL).

[0082] The prepared Burkholderia metalloid N19 and Burkholderia cepacia N31 inoculants were mixed in equal volumes to obtain a Burkholderia-treated compound inoculant.

[0083] Burkholderia treatment group (N19+N31): 60mL of compound bacterial agent was applied to the roots of each plant. Control group: roots were irrigated with an equal volume of sterile water.

[0084] Sampling was performed 90 days after inoculation. The following indicators were measured in different treatment groups after sampling: 1. Plant growth indicators (total dry weight / new shoot dry weight / root dry weight, root length, root surface area); 2. Phosphorus content in various tissues (old leaves, new leaves, roots); 3. Phosphorus components in rhizosphere soil.

[0085] III. Experimental Results Ninety days after planting the seedlings of Chazhigan in flowerpots, it was found that the plants in the treatment group inoculated with Burkholderia spp. compound bacterial agent grew significantly better than the uninoculated control group.

[0086] The effects of different treatments on the growth of tea branch tangerines are as follows: Figure 4 As shown, the results indicated that, compared with the control group, the Burkholderia spp. compound inoculant treatment significantly increased the whole plant dry weight, new shoot dry weight, and root dry weight of Chazhigan citrus (P<0.05), and also significantly increased the total root length and root surface area. These results suggest that the compound inoculant can effectively promote root development (including lateral root formation) and shoot growth in Chazhigan citrus, thereby enhancing the overall biomass accumulation of the plant.

[0087] The effects of different treatments on phosphorus absorption in tea branches are as follows: Figure 5 As shown, the results indicate that Burkholderia compound inoculant treatment has a significant regulatory effect on phosphorus absorption in plants, and can significantly increase the phosphorus concentration in old leaves, new leaves and roots. This suggests that Burkholderia compound inoculant can improve the absorption of phosphorus by plants.

[0088] The effects of different treatments on soil phosphorus composition are as follows: Figure 6 As shown, the results indicate that, compared with the control group, significant changes occurred in different phosphorus components in the rhizosphere soil of *Citrus medica* after inoculation, with a significant reduction in the concentration of insoluble phosphorus in the soil. This suggests that the *Burkholderia* compound inoculant treatment converts insoluble phosphorus in the soil into a form available to plants, thereby alleviating soil phosphorus fixation problems and improving phosphorus availability.

[0089] 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 strain of Burkholderia metalloids ( Burkholderia metallica N19, characterized in that, The bacteria were deposited in the Guangdong Microbial Culture Collection Center on November 10, 2025, and the deposit number is GDMCC NO.67261.

2. An inoculant characterized in that, The metal Burkholderia N19 or fermentation liquor thereof according to claim 1.

3. The bacterial agent of claim 2, wherein Also contains Burkholderia cenocepacia (Bcav Burkholderia cepacia ) N31 or its fermentation liquor, which has been preserved in Guangdong Microbial Culture Collection Center on November 10, 2025, with the preservation number of GDMCC NO. 67262.

4. The bacterial agent of claim 3, wherein the bacterial agent is a Bacillus strain. The mass ratio of the metal Burkholderia N19 and the onion Burkholderia N31 is (1~2):(1~2).

5. The metal Burkholderia N19 according to claim 1 or the microbial agent according to claims 2~4 is applied to promote plant growth, or is applied to prepare a product for promoting plant growth.

6. The metal Burkholderia N19 according to claim 1 or the microbial agent according to claims 2~4 is applied to produce indole acetic acid, or is applied to prepare a product for producing indole acetic acid.

7. The metal Burkholderia N19 according to claim 1 or the microbial agent according to claims 2~4 is applied to decompose phosphorus, or is applied to prepare a product with the ability to decompose phosphorus.

8. The metal Burkholderia N19 according to claim 1 or the microbial agent according to claims 2~4 is applied to promote the absorption of phosphorus elements by plants, or is applied to prepare a product for promoting the absorption of phosphorus elements by plants.

9. The metal Burkholderia N19 according to claim 1 or the microbial agent according to claims 2~4 is applied to produce siderophores, or is applied to prepare a product for producing siderophores.

10. A method of promoting plant growth, characterized by, The plant is treated with the microbial agent according to any one of claims 2~4.

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