Burkholderia cepacia S66, complex microbial inoculant and application of complex microbial inoculant

Abstract

The invention provides burkholderia cepacia S66, a complex microbial inoculant and application of the burkholderia cepacia S66, and belongs to the technical field of agricultural microorganisms. The preservation number of the Burkholderia cepacia S66 is GDMCC (China General Microbiological Culture Collection Center) No: 67740. The composite microbial agent is prepared from bacillus velezensis and burkholderia cepacia S66. The complex microbial inoculant can generate a synergistic effect through interaction, forms a stable biological membrane structure at rhizosphere, dissolves phosphorus which is difficult to utilize, enhances the absorption capacity of plants to iron, promotes plant growth, and is especially suitable for alkaline and / or iron-deficient soil environments. Results of the embodiment show that the complex microbial inoculant has efficient phosphate solubilizing and siderophore producing capacities, and can significantly promote the growth of arabidopsis thaliana and cucumbers in iron-deficient alkaline soil. The complex microbial inoculant can form a stable biological membrane through a metabolism complementation and siderophore sharing mechanism and promote the growth of plants in an iron-deficient environment, and has important theoretical value and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a strain of Burkholderia cepacia S66, a compound microbial agent, and its application. Background Technology

[0002] Iron is one of the essential micronutrients for plant growth, playing a crucial role in electron transport, chlorophyll synthesis, and enzyme activation. However, in alkaline soils, iron mainly exists in the form of insoluble iron oxides, with extremely low bioavailability, leading to widespread iron deficiency chlorosis and stunted growth in plants.

[0003] Traditional agriculture typically uses chemical fertilizers or regulators to alleviate iron deficiency in plants, but these methods suffer from drawbacks such as high cost, low absorption rates, and environmental pollution. In recent years, utilizing rhizosphere-promoting bacteria to improve plant growth conditions has become an important direction in green agriculture. However, single-ingredient microbial agents often suffer from poor stability and persistence, and are difficult to colonize long-term in complex rhizosphere environments.

[0004] Compound microbial agents can integrate the metabolic advantages of different strains to form a complementary symbiotic system in the rhizosphere, thereby enhancing colonization capacity and functional stability. Current research indicates that metabolic mutualism can enhance population stability, but systematic studies on mutually beneficial networks formed between different species through siderophore sharing are still lacking. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a strain of Burkholderia cepacia S66, which, when used in combination with Bacillus belye, forms a complementary symbiotic system, enhancing colonization capacity and functional stability. The present invention also provides a compound microbial agent that can form a stable biofilm through metabolic complementarity and siderophore sharing mechanisms, promoting plant growth in iron-deficient environments. This agent can synergistically enhance its effect on promoting plant growth in iron-deficient environments by forming a complementary symbiotic system in the rhizosphere.

[0006] The objective of this invention is achieved through the following technical solution: This invention provides a strain of Burkholderia cepacia (… Burkholderia contaminans The preservation number of Burkholderia cepacia S66 is GDMCC No: 67740.

[0007] This invention provides a compound microbial agent comprising: Bacillus belye and Burkholderia cepacia S66 as described in the above technical solution.

[0008] Preferably, the Bacillus belesii includes Bacillus belesii SQR9.

[0009] Preferably, the viability ratio of *Bacillus belyceae* and *Burkholderia cepacia* S66 in the compound microbial agent is 1:1; the OD of the compound microbial agent... 600 The value is 1.0 to 2.0.

[0010] This invention provides a method for preparing the compound microbial agent described in the above technical solution, comprising: A compound bacterial agent was obtained by mixing Bacillus bellis bacterial suspension and Burkholderia cepacia S66 bacterial suspension.

[0011] Preferably, the method for preparing the Bacillus bellis bacterial suspension includes: inoculating Bacillus bellis into a culture medium for culture to obtain a fermentation broth, separating the bacterial cells and resuspending them to obtain the Bacillus bellis bacterial suspension; The method for preparing Burkholderia cepacia S66 bacterial suspension includes: inoculating Burkholderia cepacia S66 into a culture medium for cultivation to obtain a fermentation broth, separating the bacterial cells and resuspending them to obtain Burkholderia cepacia S66 bacterial suspension.

[0012] Preferably, the culture medium includes TSB medium; the culture temperature is 25~37℃; the culture time is ≥24h; and the culture rotation speed is 170~180rpm.

[0013] This invention provides the application of Burkholderia cepacia S66 described in the above technical solution, the compound microbial agent described in the above technical solution, and the compound microbial agent prepared by the preparation method described in the above technical solution in improving soil, promoting plant growth, and enhancing plant stress resistance, or in any two or more of these applications.

[0014] Preferably, the plant includes Arabidopsis thaliana and / or cucumber.

[0015] Preferably, the improvement of soil includes improving soil physicochemical properties; the improvement of soil physicochemical properties includes realizing the bioavailability conversion of iron and / or increasing the available phosphorus content in the soil.

[0016] This invention provides a method for improving soil and / or promoting plant growth, comprising: During plant growth, the above-described Burkholderia cepacia S66, the above-described compound microbial agent, or the compound microbial agent prepared by the above-described preparation method are applied.

[0017] The beneficial effects of this invention are: This invention provides a strain of Burkholderia cepacia (… Burkholderia contaminansBurkholderia cepacia S66, with accession number GDMCC No: 67740, possesses phosphate solubilization and siderophore production capabilities. It can utilize the metabolites secreted by Bacillus belyssus, forming a complementary symbiotic system with Bacillus belyssus, thus enhancing its colonization ability and functional stability.

[0018] This invention provides a compound microbial agent comprising *Bacillus belyssae* and *Burkholderia cepacia* S66. In this compound microbial agent, *Burkholderia cepacia* S66 and *Bacillus belyssae* can form a symbiotic system in the rhizosphere through metabolic advantages, based on siderophore sharing and metabolic complementarity. The compound microbial agent can produce a synergistic effect through interaction, forming a stable biofilm structure in the rhizosphere, dissolving poorly utilized phosphorus, enhancing the plant's iron absorption capacity, and promoting plant growth, especially suitable for alkaline or iron-deficient soil environments. The results of the embodiments of this invention show that the compound microbial agent has highly efficient phosphorus solubilization and siderophore production capabilities, and can significantly promote the growth of *Arabidopsis thaliana* and cucumber in iron-deficient alkaline soils. In summary, the compound microbial agent provided by this invention forms a stable biofilm and promotes plant growth in iron-deficient environments through metabolic complementarity and siderophore sharing mechanisms, possessing significant theoretical value and application prospects.

[0019] Biological Preservation Instructions Burkholderia cepacia S66, Latin scientific name: Burkholderia contaminans It was deposited on January 26, 2026 at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67740. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0021] Figure 1 Phenotypic observation of biofilm formed by compound microbial agents; Figure 2 The graph shows the fresh weight test results of the biofilm formed by the compound microbial agent. Figure 3 A diagram illustrating the metabolic symbiotic relationship between Bacillus belyssus SQR9 and Burkholderia cepacia S66. Figure 4 The graph shows the test results of the phosphorus solubilizing (organic and inorganic phosphorus) ability of the compound microbial agent; Figure 5 The graph shows the test results of the iron-producing capacity of the compound bacterial agent. Figure 6Figure 1 shows the effect of compound microbial agents on the growth of potted Arabidopsis thaliana. Figure 7 The graph shows the effect of compound microbial agents on the growth of potted cucumbers. Figure 8 A graph showing the comparison of the phosphorus-solubilizing capacity of various bacterial cultures; Figure 9 The graph shows the results of the siderophore production capacity determination for each bacterial culture. Detailed Implementation

[0022] This invention provides a strain of Burkholderia cepacia (… Burkholderia contaminans The preservation number of Burkholderia cepacia S66 is GDMCC No: 67740.

[0023] In this invention, *Burkholderia cepacia* S66 was screened from the rhizosphere soil of healthy cucumbers from sites affected by Fusarium wilt. When cultured on TSB plates, *Burkholderia cepacia* S66 forms small, pale yellow colonies that are Gram-negative and uniformly stained. The nucleotide sequence of the 16S rDNA of *Burkholderia cepacia* S66 is shown in SEQ ID NO. 1.

[0024] The *Burkholderia cepacia* S66 provided by this invention possesses phosphate solubilization and iron-producing capabilities. *Burkholderia cepacia* S66, used alone, significantly promotes the growth of *Arabidopsis thaliana* and cucumber in iron-deficient saline-alkali soils. In this invention, *Burkholderia cepacia* S66 can utilize the metabolites secreted by *Bacillus belyssiensis* SQR9, and can be used in combination with *Bacillus belyssiensis* to form a complementary symbiotic system, enhancing colonization ability and functional stability.

[0025] This invention provides a compound microbial agent comprising: Bacillus belye and Burkholderia cepacia S66 as described in the above technical solution.

[0026] As an optional embodiment of the present invention, the *Bacillus belyssiensis* includes *Bacillus belyssiensis* SQR9. In this invention, the viability ratio of *Bacillus belyssiensis* and *Burkholderia cepacia* S66 in the compound microbial agent can be 1:1; the OD of the compound microbial agent... 600 The value can be 1.0 to 2.0.

[0027] This invention provides a method for preparing the composite microbial agent described in the above-mentioned technical solution, comprising: mixing *Bacillus bellis* bacterial suspension and *Burkholderia cepacia* S66 bacterial suspension to obtain the composite microbial agent. As an optional embodiment of this invention, the method for preparing the *Bacillus bellis* bacterial suspension comprises: inoculating *Bacillus bellis* into a culture medium for cultivation to obtain a fermentation broth, separating the bacterial cells and resuspending them to obtain *Bacillus bellis* bacterial suspension. This invention does not specifically limit the culture medium, as long as it allows *Bacillus bellis* to grow normally. In this invention, the culture medium can be TSB medium; the cultivation temperature can be 30℃; the cultivation time can be ≥24h, 24~48h, or 24h; the cultivation rotation speed can be 170~180rpm, or 170, 175, or 180rpm. This invention does not specifically limit the method for separating the bacterial cells; conventional separation methods in the art can be used. In this invention, the method for separating bacterial cells can be centrifugation; the centrifugation speed can be 5000-6000 rpm or 5500 rpm; the centrifugation time can be 2-5 min or 3-4 min. Preferably, this invention uses a sodium chloride solution to resuspend the bacterial cells; the mass fraction of sodium chloride in the sodium chloride solution can be 0.9%. As an optional embodiment of this invention, the OD of the *Bacillus belyssioides* bacterial suspension... 600 The value can be 1.0 to 2.0.

[0028] As an optional embodiment of the present invention, the method for preparing the Burkholderia cepacia S66 bacterial suspension includes: inoculating Burkholderia cepacia S66 into a culture medium for cultivation to obtain a fermentation broth; separating the bacterial cells and resuspending them to obtain the Burkholderia cepacia S66 bacterial suspension. The present invention does not specifically limit the culture medium, as long as it allows Burkholderia cepacia S66 to grow normally. In the present invention, the culture medium can be TSB medium; the cultivation temperature can be 25~37℃ or 30℃; the cultivation time can be ≥24h, 24~48h, or 24h; the cultivation rotation speed can be 170~180 rpm, or 170, 175, or 180 rpm. The present invention does not specifically limit the method for separating the bacterial cells; conventional separation methods in the art can be used. In the present invention, the method for separating the bacterial cells can be centrifugation; the centrifugation speed can be 5000~6000 rpm or 5500 rpm; the centrifugation time can be 2~5 min or 3~4 min. The present invention preferably uses a sodium chloride solution to resuspend the bacterial cells; the mass fraction of sodium chloride in the sodium chloride solution can be 0.9%. As an optional embodiment of the present invention, the OD of the Burkholderia cepacia S66 bacterial suspension... 600 The value can be 1.0 to 2.0.

[0029] After obtaining the *Bacillus belyssioides* bacterial suspension and the *Burkholderia cepacia* S66 bacterial suspension, the present invention mixes the two to obtain a composite bacterial agent. The present invention does not specifically limit the mixing method; any conventional mixing method in the art can be used. As an optional embodiment of the present invention, the mixing can be an equal-volume mixture.

[0030] This invention provides the application of Burkholderia cepacia S66 described in the above technical solution, the compound microbial agent described in the above technical solution, and the compound microbial agent prepared by the preparation method described in the above technical solution in improving soil, promoting plant growth, and enhancing plant stress resistance, or in any two or more of these applications.

[0031] The compound microbial agent provided by this invention has the ability to solubilize phosphorus and produce iron carriers, and can be applied to improve soil by solubilizing phosphorus and / or providing iron carriers. The compound microbial agent can improve the physicochemical properties of soil; this improvement includes activating insoluble iron in the soil to achieve the bioavailability transformation of iron; and increasing the available phosphorus content in the soil.

[0032] The compound microbial agent provided by this invention has the effect of promoting plant growth. As an optional embodiment of this invention, the plant includes Arabidopsis thaliana and / or cucumber. Results from the examples show that the compound microbial agent provided by this invention can significantly promote the growth of Arabidopsis thaliana and cucumber in iron-deficient alkaline soil, increase the fresh weight of Arabidopsis thaliana, and increase the plant height of cucumber.

[0033] The compound microbial agent provided by this invention has the effect of improving plant stress resistance. As an optional embodiment of this invention, the plant includes Arabidopsis thaliana and / or cucumber. The compound microbial agent provided by this invention can significantly improve the alkali resistance and / or iron deficiency resistance of plants. The results of the embodiments of this invention show that the compound microbial agent can significantly improve the growth status of cucumber and Arabidopsis thaliana in iron-deficient alkaline soil.

[0034] This invention provides a method for improving soil and / or promoting plant growth, comprising: During plant growth, the *Burkholderia cepacia* S66 described in the above-described technical solution, the compound microbial agent described in the above-described technical solution, or the compound microbial agent prepared by the preparation method described in the above-described technical solution are applied. This invention does not specifically limit the application method; any conventional application method in the art can be used.

[0035] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0036] Bacillus belesiensis ( Bacillus velezensisSQR9 was deposited on February 27, 2012, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 5808 and address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It is described in published patent CN 120866069 A.

[0037] TSB culture medium preparation method: TSB powder 30 g / L, autoclave at 115℃ for 30 min.

[0038] Pancreatic Soy Peptone Soy Broth (TSB), Haibo Biotechnology, product number: HB 4114, composition: 17.0 g / L tryptone, 3.0 g / L soy peptone, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, pH value 7.5.

[0039] Organophosphate bacteria culture medium (Haibo Biotechnology HB8673): Glucose 10 g / L, ammonium sulfate 0.5 g / L, yeast extract 0.5 g / L, sodium chloride 0.3 g / L, potassium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, manganese sulfate 0.03 g / L, lecithin 0.2 g / L, calcium carbonate 1.0 g / L, agar 15.0 g / L. pH 7.0~7.5.

[0040] Inorganic phosphorus bacteria culture medium (Haibo Biotechnology HB8549-2): glucose 10 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, potassium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, manganese sulfate 0.03 g / L, calcium phosphate 5.0 g / L, agar 15.0 g / L. pH 7.0~7.5.

[0041] The sterile fermentation broth involved in the following scheme is obtained by filtering and sterilizing the culture supernatant.

[0042] In the following technical solutions, OD 600 A compound bacterial agent solution with a value of 1.0 refers to the OD value of each bacterium in the mixed system. 600 The OD is 1.0. For example, if equal volumes of *Bacillus belye* SQR9 and *Burkholderia cepacia* S66 are mixed, the OD will be 1.0. 600 The compound bacterial agent solution is 1.0, and the OD of Bacillus vesiculosus SQR9 in the compound bacterial agent solution is... 600 The OD value of Burkholderia cepacia S66 was 1.0. 600 It is 1.0.

[0043] Example 1 Isolation and identification of disease-resistant strains Bacillus belyssus SQR9, with the strain preservation number CGMCC No. 5808, was isolated in the laboratory in a previous stage.

[0044] Burkholderia cepacia S66 was obtained by screening healthy cucumber rhizosphere soil from wilt disease-affected areas.

[0045] The specific screening method is as follows: Cucumber roots are removed from the soil, and rhizosphere soil is collected and shaken at 30℃ and 170 r / min for 20 minutes to form a soil suspension. 10 μL of each suspension is then extracted. -6 10 -7 10 -8 0.1 mL of soil suspension diluted by a certain factor was spread on TSB plates, with 3 replicates for each concentration. After incubation at 30°C for 3 days, colonies were selected from the plates and streaked for purification.

[0046] A bacterial strain named S66 was obtained. When strain S66 was cultured on TSB plates, it formed small, pale yellow colonies that were Gram-negative and showed uniform staining.

[0047] Based on the 16S rDNA sequence of strain S66, strain S66 was identified as Burkholderia cepacia (…). Burkholderia contaminans It is preserved at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCCNo: 67740.

[0048] The nucleotide sequence of the 16S rDNA of strain S66 is shown in SEQ ID NO.1, specifically:

[0049] Example 2 1. Obtaining the fermentation broth of compound microbial agents: Bacillus belye SQR9 and Burkholderia cepacia S66, preserved in glycerol tubes at -80℃, were streaked onto TSB solid agar plates and incubated upside down at 30℃ for 2 days. Single colonies were then selected and cultured overnight at 30℃ with shaking at 170 rpm. The fermentation broth was centrifuged at 6000 rpm for 2 minutes to collect the cells. The culture medium was removed, and the cells were resuspended in 0.9% sodium chloride solution and the OD was adjusted. 600 The concentration was 2.0, and bacterial suspensions of *Bacillus belyssiensis* SQR9 and *Burkholderia cepacia* S66 were obtained. Equal volumes of *Bacillus belyssiensis* SQR9 and *Burkholderia cepacia* S66 bacterial suspensions were mixed to obtain the OD value. 600 This is a 1.0g compound bacterial inoculum solution. Prepared for subsequent experiments.

[0050] 2. Quantitative Cultivation of Biofilms Phenotypic observation: 24-well plates (2 mL TSB medium per well) were used to inoculate bacterial suspension at a rate of 1% (v / v), and the plates were incubated at 30°C for 24 hours before taking pictures.

[0051] Specifically, the *Bacillus belyssioides* SQR9 culture from step 1 was inoculated into a 24-well plate at a 1% (v / v) inoculation rate and incubated at 30°C for 24 hours; this was designated as group Bv. Burkholderia cepacia S66 bacterial suspension from step 1 was inoculated into a 24-well plate at an inoculation rate of 1% (v / v) and incubated at 30°C for 24 hours; this was designated as group Bc.

[0052] The compound bacterial agent solution from step 1 was inoculated into a 24-well plate at an inoculation rate of 1% (v / v) and incubated at 30°C for 24 hours; this was designated as the BcBv group.

[0053] After static incubation, take photos of each item.

[0054] Fresh weight measurement: Using a 6-well plate (10 mL TSB medium per well), add a 40 μm filter membrane to the well, inoculate the bacterial solution at a rate of 1% (v / v), incubate at 30℃ for 24 h, remove the filter membrane and remove the moisture with absorbent paper, and weigh the fresh weight of the biofilm.

[0055] Specifically, a 6-well plate (10 mL TSB medium per well) was used. A 40 µm filter membrane was added to each well, and the Bacillus berberis SQR9 bacterial suspension from step 1 was inoculated at a rate of 1% (v / v). The plate was then incubated at 30°C for 24 hours and designated as group Bv.

[0056] Using a 6-well plate (10 mL TSB medium per well), a 40 µm filter membrane was added to each well, and Burkholderia cepacia S66 bacterial suspension from step 1 was inoculated at a 1% (v / v) inoculation rate. The plate was then incubated at 30°C for 24 h; this group was designated as Bc.

[0057] Using a 6-well plate (10 mL TSB medium per well), a 40 µm filter membrane was added to each well, and the compound bacterial solution from step 1 was inoculated at a rate of 1% (v / v). The plate was then incubated at 30°C for 24 hours and designated as the BcBv group.

[0058] After static culture is complete, remove the filter membrane and remove the moisture with absorbent paper, then weigh the fresh weight of the biofilm.

[0059] Phenotypic observation results are shown in Figure 1 The results of the fresh weight measurement are shown in Table 1 and Figure 2 Among them, Table 1 and Figures 1-2 In this context, Bc represents Burkholderia cepacia S66; Bv represents Bacillus belyssus SQR9; and BcBv represents a compound microbial agent. Figure 2 Different letters in the text indicate significant differences.

[0060] Table 1 Fresh weight of each treatment group (unit: mg)

[0061] The results showed that the biofilm formed by the BcBv combination had a significant increase in fresh weight.

[0062] 3. Metabolic symbiotic experiment of compound microbial agent Bacillus belyssus SQR9 culture was inoculated at 1% (v / v) into 100 mL of M9 glucose medium (M9 medium with 10 g / L glucose added), and cultured at 30°C with shaking at 180 rpm for 4 days. 80 mL of SQR9 fermentation broth was collected, centrifuged at 6000 rpm for 5 min, and the supernatant was filtered and sterilized to obtain the SQR9 metabolite, which was then stored at 4°C. Burkholderia cepacia S66 culture was inoculated at 1% (v / v) into 10 mL of SQR9 metabolite, and cultured at 30°C with shaking at 170 rpm for 4 days. 8 mL of Burkholderia cepacia S66 fermentation broth was collected, centrifuged at 6000 rpm for 5 min, and the supernatant was filtered and sterilized to obtain the metabolite of Burkholderia cepacia S66 after growth using the Bacillus belyssus SQR9 metabolite.

[0063] Metabolites were sent to Beijing Novogene Biotech Co., Ltd. for non-targeted metabolomics analysis. Differential metabolites were compared, and the results are shown in Tables 2-1 to 2-3. Figure 3 As shown in Tables 2-1 to 2-3 and... Figure 3Bv in the middle section is the fermentation broth of Bacillus belye (control), and Bv_Bc is the fermentation broth of Bc after utilizing Bv.

[0064] Table 2-1 Comparison of Differential Metabolites (1)

[0065] Table 2-2 Comparison of Differential Metabolites (2)

[0066] Table 2-3 Comparison of Differential Metabolites (3)

[0067] The results showed that *Burkholderia cepacia* S66 can utilize a variety of metabolites from *Bacillus belyssus* SQR9: valeric acid, 5-aminovaleric acid, pentadecanoic acid, levulinic acid, cinnamic acid, valine, 2,3,5,6-tetramethylpyrazine, retinoic acid, N-α-L-acetyl-arginine, guanidine, N-acetyl-DL-valine, N-acetylvaline, valine-proline, prolylleucine, N-acetyl-D-isoleucine, nicotinic acid, and 2-isopropylmalic acid. In other words, the metabolites secreted by *Bacillus belyssus* SQR9 can be metabolized and utilized by *Burkholderia cepacia* S66.

[0068] 4. Testing of growth-promoting indicators Phosphate-solubilizing ability determination: 5 µL of bacterial suspension (i.e., Bacillus berberis SQR9 suspension, Burkholderia cepacia S66 suspension, and compound bacterial suspension from step 1 of this example) was spotted onto solid culture media containing inorganic phosphorus (calcium phosphate) and organic phosphorus (lecithin), respectively. After incubation at 30°C for 5 days, the phosphate-solubilizing zone was observed. Results are shown below. Figure 4 . Figure 4 In the text, Bc represents Burkholderia cepacia S66; Bv represents Bacillus belyssus SQR9; and BcBv represents a compound microbial agent.

[0069] Siderogenic capacity determination: Supernatants from bacterial cultures treated with different methods were mixed with CAS detection solution at a volume ratio of 1:1, and OD was measured. 630 The absorbance value As was obtained. Uninoculated culture medium served as a control, and the measured value was Ar. The relative siderophore content (SU) was calculated as: SU = 1 - As / Ar. The results are shown in Table 3 and... Figure 5 . Figure 5 In this context, Bc represents Burkholderia cepacia S66; Bv represents Bacillus belyssus SQR9; and BcBv represents a compound microbial agent. Figure 5 Different letters in the text indicate significant differences.

[0070] Supernatant was obtained as follows: The *Bacillus belyssioides* SQR9 culture, *Burkholderia cepacia* S66 culture, and the compound bacterial culture from step 1 of this example were inoculated into MKB liquid medium (casein amino acids 5.0 g / L, glycerol 15 mL / L, dipotassium hydrogen phosphate 2.5 g / L, magnesium sulfate heptahydrate 2.5 g / L) at a 1% (v / v) inoculation rate for 3 days. The culture was then centrifuged at 6000 rpm for 5 min and sterilized by membrane filtration to obtain the supernatant.

[0071] CAS detection solution formulation: Mix 1.5 mL of 1 mM ferric chloride solution (0.2703 g ferric chloride hexahydrate dissolved in 1 L 10 mM hydrochloric acid), 2 mL of CAS stock solution (0.2421 g CAS dissolved in 200 mL water), 50 mL of HTDMA solution (0.0219 g HTDMA dissolved in 50 mL water), and 30 mL of piperazine buffer (4.3709 g anhydrous piperazine dissolved in 30 mL water, adjusted to pH=5.6), and bring the volume to 100 mL.

[0072] Table 3. Relative content of siderophores in each treatment group

[0073] From Table 3 and Figures 4-5 It can be seen that the compound microbial agent increases the degradation capacity of inorganic and organic phosphorus, enhances the iron-producing capacity, and improves the plant's ability to acquire phosphorus and iron from the environment.

[0074] 5. Experiment on plant growth promotion using compound microbial agents The Arabidopsis and cucumber experiments were conducted in pots at the Nanjing Agricultural University experimental base in July 2024. Arabidopsis and cucumber seeds were surface-sterilized with a 2% sodium hypochlorite solution for 20 minutes, then washed with sterile water and cultured on MS solid medium. After germination, Arabidopsis seedlings with uniform growth were transplanted into an alkaline substrate, while cucumber seedlings with uniform growth were transplanted into natural soil. The pH of the alkaline substrate used was adjusted to 8.50 with KOH. The physicochemical properties of the natural soil were as follows: pH 8.5, organic matter content 8.55 g / kg, organic carbon content 4.96 g / kg, total nitrogen 0.551 g / kg, hydrolyzable nitrogen 206 mg / kg, total phosphorus 0.069 mg / kg, available phosphorus 15.9 mg / kg, total potassium 16.9 g / kg, and available iron 0.7 mg / kg.

[0075] The potted plants were then placed in a greenhouse for cultivation and watered every 4 days. Experimental treatments included: (1) CTL treatment, no inoculation; (2) Bc treatment, inoculated with Burkholderia cepacia S66; (3) Bv treatment, inoculated with Bacillus belyssus SQR9; and (4) BcBv treatment, inoculated with a compound microbial agent. The inoculation standard for all agents was 10... 7 CFU / g soil; the standard for bacterial suspension at inoculation was OD. 600 The process for potted plants is as follows: transfer the Arabidopsis thaliana or cucumber seedlings to an alkaline substrate or natural soil and cultivate them normally for one week. After the plants have stabilized, add the bacterial solution for each treatment. The bacterial solution is added by watering.

[0076] The greenhouse conditions were as follows: Cucumber: constant temperature of 30℃, 16 hours of light, and 8 hours of dark cycle; Arabidopsis: 25℃, 16 hours of light, and 8 hours of dark cycle.

[0077] Four weeks after inoculation with the inoculum, the fresh weight or plant height of each treatment group was measured. Results are shown below. Figures 6-7 And Tables 4 and 5, where CTL corresponds to the non-inoculated treatment group; Bc corresponds to the Burkholderia cepacia S66 inoculation treatment group; Bv corresponds to the Bacillus belyssus SQR9 inoculation treatment group; and BcBv corresponds to the compound bacterial agent inoculation treatment group. Figure 6 The top-middle image shows the effects of each treatment group on the growth of potted Arabidopsis thaliana; the bottom image shows the effects of each treatment group on the fresh weight of Arabidopsis thaliana. Figure 7 The top-middle image shows the effects of each treatment group on the growth of potted cucumbers; the bottom image shows the effects of each treatment group on the height of cucumber plants.

[0078] Table 4. Fresh weight of Arabidopsis thaliana in each treatment group (mg)

[0079] Table 5. Cucumber plant height (cm) in each treatment group

[0080] From Tables 4 to 5 and Figures 6-7 It was found that applying a compound microbial agent composed of Burkholderia cepacia S66 and Bacillus belyss SQR9 to the plant rhizosphere significantly improved plant growth and health under alkaline iron-deficient conditions. Inoculation with either Burkholderia cepacia S66 or Bacillus belyss SQR9 alone also significantly improved plant growth, but the compound microbial agent showed the best effect.

[0081] Example 3 1. The bacterial agent used in this embodiment: Acinetobacter baumannii ( Acinetobacter baumanniiXL380: Collection number ACCC 61689 (http: / / www.accc.org.cn / Column.asp?Column_ID=34929&Model=product_detail&P_ID=1966301645). Abbreviation: Ab.

[0082] Bacillus belesiensis ( Bacillus velezensis SQR9. Abbreviation: Bv.

[0083] Burkholderia cepacia ( Burkholderia contaminans S66. Abbreviation: Bc.

[0084] 2. Preparation of bacterial culture The preparation methods for Bacillus vesicles SQR9 and Burkholderia cepacia S66 bacterial suspensions are the same as in Example 2.

[0085] The preparation method of Acinetobacter baumannii XL380 bacterial suspension is as follows: Acinetobacter baumannii XL380 strain, preserved in glycerol tubes at -80℃, is streaked onto a TSB solid medium plate and incubated upside down at 30℃ for 2 days. Single colonies are then selected and cultured overnight at 30℃ with shaking at 170 rpm. The fermentation broth is centrifuged at 6000 rpm for 2 minutes to collect the bacterial cells. The culture medium is removed, and the cells are resuspended in 0.9% sodium chloride solution and the OD is adjusted. 600 The concentration was 2.0, and Acinetobacter baumannii XL380 bacterial suspension was obtained.

[0086] The preparation method of AbBc bacterial suspension is as follows: Equal volumes of Acinetobacter baumannii XL380 bacterial suspension and Burkholderia cepacia S66 bacterial suspension are mixed to obtain OD. 600 The compound bacterial agent solution is 1.0.

[0087] The preparation method of AbBv bacterial suspension is as follows: Equal volumes of Acinetobacter baumannii XL380 bacterial suspension and Bacillus belyssus SQR9 bacterial suspension are mixed to obtain OD. 600 The compound bacterial agent solution is 1.0.

[0088] The preparation method of BcBv bacterial suspension is as follows: Equal volumes of Bacillus belyeis SQR9 bacterial suspension and Burkholderia cepacia S66 bacterial suspension are mixed to obtain OD. 600 The compound bacterial agent solution is 1.0.

[0089] 3. The preparation method of the bacterial supernatant is as follows: The bacterial solutions of different treatments in step 2 of this example are inoculated into MKB liquid medium (casein amino acids 5.0 g / L, glycerol 15 mL / L, dipotassium hydrogen phosphate 2.5 g / L, magnesium sulfate heptahydrate 2.5 g / L) at an inoculation rate of 1% (v / v) and cultured at 30℃ and 180 rpm with shaking for 3 days. The bacterial solutions are then centrifuged at 6000 rpm for 5 min and sterilized by membrane filtration to obtain the supernatant.

[0090] 4. The phosphorus solubilization capacity of the bacterial solution obtained in step 2 of this embodiment was determined; the iron-producing capacity of the supernatant obtained in step 3 of this embodiment was determined. The specific methods are as follows: Phosphorus solubilization capacity test: 5 µL of bacterial culture was spotted onto solid culture medium containing inorganic phosphorus (calcium phosphate) and organic phosphorus (lecithin), respectively. After standing in an incubator at 30℃ for 5 days, the phosphorus solubilization zone was observed.

[0091] Siderogenic capacity determination: Supernatants from bacterial cultures treated with different methods were mixed with CAS detection solution at a volume ratio of 1:1, and OD was measured. 630 The absorbance value As was obtained. The uninoculated culture medium served as a control, and the measured value was Ar. The relative content of siderophores (SU) was calculated as: SU = 1 - As / Ar.

[0092] The results of the comparison of the phosphorus solubility of various bacterial cultures are as follows: Figure 8 As shown. Figure 8 Ab represents Acinetobacter baumannii XL380; Bc represents Burkholderia cepacia S66; Bv represents Bacillus belyssus SQR9; AbBc represents a complex of Acinetobacter baumannii XL380 and Burkholderia cepacia S66; AbBv represents a complex of Acinetobacter baumannii XL380 and Bacillus belyssus SQR9; BcBv represents a complex of Bacillus belyssus SQR9 and Burkholderia cepacia S66.

[0093] Depend on Figure 8 It can be seen that, compared with BcBv, Ab did not significantly improve the phosphorus solubilization ability of either Bv or Bc.

[0094] The results of the siderophore production capacity tests for each bacterial culture are shown in Table 6 and... Figure 9 As shown in Table 6 and Figure 9 Ab represents Acinetobacter baumannii XL380; Bc represents Burkholderia cepacia S66; Bv represents Bacillus belyssus SQR9; AbBc represents a complex of Acinetobacter baumannii XL380 and Burkholderia cepacia S66; AbBv represents a complex of Acinetobacter baumannii XL380 and Bacillus belyssus SQR9; BcBv represents a complex of Bacillus belyssus SQR9 and Burkholderia cepacia S66. Figure 9 Different letters in the text indicate significant differences.

[0095] Table 6 Results of the determination of iron carrier production capacity of each bacterial culture

[0096] From Table 6 and Figure 9 It can be concluded that, compared with BcBv, Ab does not significantly improve the iron-producing capacity of Bv or Bc.

[0097] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Burkholderia cepacia ( Burkholderia contaminans S66, characterized in that, The preservation number of Burkholderia cepacia S66 is GDMCC No: 67740.

2. A compound microbial agent, characterized in that, include: Bacillus belye and Burkholderia cepacia S66 as described in claim 1.

3. The compound microbial agent according to claim 2, characterized in that, The viability ratio of *Bacillus belyssioides* and *Burkholderia cepacia* S66 in the compound microbial agent is 1:1; the OD of the compound microbial agent... 600 The value is 1.0 to 2.

0.

4. The method for preparing the compound microbial agent according to claim 2 or 3, characterized in that, include: A compound bacterial agent was obtained by mixing Bacillus bellis bacterial suspension and Burkholderia cepacia S66 bacterial suspension.

5. The preparation method according to claim 4, characterized in that, The method for preparing the Bacillus berberis bacterial suspension includes: inoculating Bacillus berberis into a culture medium for culture to obtain a fermentation broth, separating the bacterial cells and resuspending them to obtain the Bacillus berberis bacterial suspension; The method for preparing Burkholderia cepacia S66 bacterial suspension includes: inoculating Burkholderia cepacia S66 into a culture medium for cultivation to obtain a fermentation broth, separating the bacterial cells and resuspending them to obtain Burkholderia cepacia S66 bacterial suspension.

6. The preparation method according to claim 5, characterized in that, The culture medium includes TSB medium; the culture temperature is 25~37℃; the culture time is ≥24h; and the culture rotation speed is 170~180rpm.

7. The application of Burkholderia cepacia S66 of claim 1, the compound microbial agent of claim 2 or 3, or the compound microbial agent prepared by the preparation method of any one of claims 4 to 6 in improving soil, promoting plant growth, and enhancing plant stress resistance, or in any two or more of these applications.

8. The application according to claim 7, characterized in that, The plants include Arabidopsis thaliana and / or cucumber.

9. The application according to claim 7, characterized in that, The improvement of soil includes improving soil physicochemical properties; the improvement of soil physicochemical properties includes realizing the bioavailability conversion of iron and / or increasing the available phosphorus content in the soil.

10. A method for improving soil and / or promoting plant growth, characterized in that, include: The compound microbial agent prepared by the method described in any one of claims 4 to 6 is applied during plant growth.

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