Burkholderia cepacia and application thereof

Abstract

The morphological characteristics of the burkholderia cepacia are as follows: a bacterial strain grows in an LB solid culture medium at 37 DEG C for 12 hours to form a white protuberant irregular bacterial colony, the surface is smooth and wet, and the diameter of the bacterial colony is 3.8 mm. The burkholderia cepacia is used as a special strain for bioremediation, and compared with a physical remediation technology and a chemical remediation technology, the burkholderia cepacia is low in cost, environmentally friendly, efficient and convenient to operate. The burkholderia cepacia disclosed by the invention is applied to pepper planting to reduce Cd enrichment of fruits.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a Burkholderia cepacia strain and also to use of the Burkholderia cepacia strain in reducing heavy metal cadmium enrichment in pepper fruits. Background Art

[0002] my country is the world's largest producer and consumer of chili peppers, with Guizhou Province leading the nation in both chili cultivation and processing. Geochemical anomalies of the heavy metal cadmium (Cd) are observed in most of Guizhou's chili-growing areas. Multiple surveys have found that soil Cd levels exceed the standard in over 44.5% of pepper-growing areas province-wide, with over 83.7% of sites experiencing moderate to mild contamination. Currently, key measures to reduce heavy metal levels in chili peppers include cultivating low-accumulation chili varieties, using hyperaccumulator plants for passivation, and using microorganisms for passivation. Microbial remediation has attracted significant attention due to its low cost, safety, and low risk of secondary pollution. However, commercially available microbial agents specifically for Cd-excessive chili peppers are currently lacking.

[0003] Burkholderia has plant growth-promoting properties such as phosphate solubilization, nitrogen fixation, siderophore production, and indoleacetic acid production. It can reduce heavy metal accumulation in plants by forming soluble metal complexes with heavy metal ions through the secretion of extracellular polymers, biosurfactants, siderophores and other substances. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above-mentioned disadvantages and provide a Burkholderia cepacia strain that is simple to prepare, low in cost and easy to obtain.

[0005] Another object of the present invention is to provide an application of the Burkholderia cepacia strain in reducing Cd accumulation in pepper fruits during cultivation.

[0006] The invention discloses Burkholderia cepacia KL4-1-1, which has the following morphological characteristics: after the strain grows in an LB solid culture medium at 37° C. for 12 hours, it forms white protruding irregular colonies with a smooth and moist surface and a colony diameter of 3.8 mm.

[0007] This strain was deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) on April 15, 2025, with the accession number: CCTCC M 2025785, and the name: Burkholderia cepacia KL4-1-1.

[0008] The present invention provides a method for isolating Burkholderia cepacia KL4-1-1, comprising the following steps: (1) Isolation of Burkholderia cepacia KL4-1-1: The rhizosphere soil of peppers with excessive Cd was collected from Guiyang, Zunyi and Tongren, the main pepper producing areas in Guizhou Province. The soil samples were pretreated by gradient dilution method and then coated with Cd 2+ The LB solid medium with a Cd content of 500 mg / L was cultured at 37°C for 24 hours and then screened to gradually increase the Cd content of the LB solid medium. 2+ concentration, and finally screened out the strain with the strongest cadmium resistance.

[0009] (2) Culture medium: Solid culture medium: LB solid medium (yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, agar powder 15 g / L); (3) Preservation and subculture of bacterial strains: (1) Culture medium: liquid LB medium (yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L); (2) Glycerol storage method: After culturing in liquid LB medium, place in 30% glycerol tube and freeze at -80℃; (IV) Biological identification of bacterial species: The bacterial cells were collected and the genome of the strain was extracted. The strain was amplified and sequenced using 16s rDNA primers. The strain was identified to species by BLAST and phylogenetic tree construction. The strain was analyzed to be Burkholderia cepacia KL4-1-1. The invention discloses an application of Burkholderia cepacia in reducing Cd enrichment in pepper fruits during pepper cultivation.

[0010] Compared with the prior art, the present invention has obvious beneficial effects. It can be seen from the above technical solutions that the present invention utilizes microbial remediation to better meet the requirements of ecological environmental protection and sustainable development, and screens out the Cd-resistant strain Burkholderia cepacia. The Burkholderia cepacia KL4-1-1 of the present invention is a strain isolated from the rhizosphere soil of peppers with excessive Cd in Guizhou. As a special strain for bioremediation, it is low-cost, environmentally friendly, efficient and convenient to operate compared with physical remediation technology and chemical remediation technology. Burkholderia cepacia is a common plant rhizosphere growth-promoting bacterium in soil. It has the characteristics of promoting plant growth and the ability to tolerate Cd. It promotes plant growth under Cd stress and reduces the adsorption and aggregation of Cd by plants. The preparation of Burkholderia cepacia liquid is simple, low-cost and easy to obtain. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Colony morphology of Burkholderia cepacia KL4-1-1. Figure 2Gram stain of Burkholderia cepacia KL4-1-1. Figure 3 Phylogenetic tree of Burkholderia cepacia KL4-1-1. Figure 4 Electron micrograph of Burkholderia cepacia KL4-1-1 under Cd stress. Figure 5 . A diagram showing the period from pepper planting to flowering; Figure 6 . Picture of pepper fruit ripening; Figure 7 . Graph showing cadmium accumulation in mature pepper fruits. DETAILED DESCRIPTION

[0012] A method for isolating Burkholderia cepacia KL4-1-1 (Burkholderia cepacia KL4-1-1) comprises the following steps: The rhizosphere soil of peppers with excessive Cd was selected from Guiyang, Zunyi and Tongren, the main pepper producing areas in Guizhou Province. The soil samples were pretreated by gradient dilution and then coated with Cd 2+ The LB solid medium with a Cd content of 500 mg / L was cultured at 37°C for 24 hours and then screened to gradually increase the Cd content of the LB solid medium. 2+ concentration, and finally the strain with the strongest cadmium resistance was screened out and inoculated into liquid cdcl2-LB medium (yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L, Cd 2+ 100 mg / L) for 48 h, and then observed under an electron microscope and preserved.

[0013] Strain identification method: The bacterial cells were collected and the genome of the strain was extracted. The 16s rDNA primers were used for PCR amplification and sequencing. The strain was identified to species by BLAST and phylogenetic tree construction. The strain was identified as Burkholderia cepacia KL4-1-1.

[0014] Colony appearance: Figure 1 From the figure, we can see that the bacteria of this strain are white and rod-shaped, and the colonies are round and convex and milky white. The surface is smooth and moist, with irregular edges.

[0015] Gram stain: Figure 2 , as can be seen from the figure, the strain was Gram-negative.

[0016] Phylogenetic tree: Figure 3 , as can be seen from the figure, the strain has 100% homology with Burkholderia cepacia.

[0017] Electron microscopic observation of Cd stress: Figure 4 ,As can be seen from the figure, under Cd stress, the strain ,underwent obvious morphological changes, and the strains ,were sticky to each other.

[0018] Test example: The method to reduce the adsorption of cadmium by pepper is as follows: Preparation of LB liquid medium: Mix 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 1000 mL of distilled water, adjust the pH to 7, and sterilize at 121°C for 30 min to obtain LB liquid medium; Activate Burkholderia cepacia: Remove the stored Burkholderia cepacia from a -80°C freezer, thaw on ice, and add 50 μL of the solution to 50 mL of LB liquid medium. Incubate at 37°C and 180 rpm for 24 hours to obtain activated Burkholderia cepacia. Soil Cd balance: The weight of each pot of soil is 5 kg, and the soil Cd 2+ The concentration was (1mg / kg, 2mg / kg, 3mg / kg), and Cd was added 2+ The soil was aged for 1 month and turned over once a day.

[0019] After the pepper seedlings grew 6 true leaves, they were divided into 9 groups with 5 plants in each group. 2+ Different amounts of Burkholderia cepacia liquid and sheep manure organic fertilizer were added to the soil. The specific addition amounts are as follows: Group 1: Cd 2+ 1 mg / kg, 50 mL of sterile water; Group 2: Cd 2+ 2 mg / kg, 50 mL of sterile water; Group 3: Cd 2+ 3 mg / kg, 50 mL of sterile water; Group 4: Cd 2+ 1mg / kg, sheep manure organic fertilizer 50g; Group 5: Cd 2+ 2mg / kg, sheep manure organic fertilizer 50g; Group 6: Cd 2+ 3mg / kg, sheep manure organic fertilizer 50g; Group 7: Cd 2+ 1mg / kg, 50g of sheep manure organic fertilizer, 50mL of Burkholderia cepacia liquid; Group 8: Cd 2+2mg / kg, 50g of sheep manure organic fertilizer, 50mL of Burkholderia cepacia liquid; Group 9: Cd 2+ 3mg / kg, 50g sheep manure organic fertilizer, 50mL of Burkholderia cepacia liquid.

[0020] After the pepper seedlings of the 9 groups have survived and turned green, during the flowering period of the peppers, 50 mL of sterile water was added to the soil of the 1-3 groups, and 50 mL of Burkholderia cepacia solution was added to the soil of the 7-9 groups. Figure 5 .

[0021] Analysis of Cd accumulation in pepper fruit: After 9 groups of pepper fruits mature (see Figure 6 ), wash the pepper fruits with ultrapure water.

[0022] The 9 groups of pepper fruits were placed in an oven at 105°C for 30 min, dried at 65°C to constant weight, and ground through a 20-mesh sieve.

[0023] Weigh 0.1 g of pepper fruit, add 4 mL of HNO3 and 1 mL of HClO4, pre-digest overnight, place it on a hot plate at 160°C to remove the acid until the liquid becomes the size of a soybean, transfer it to a 50 mL volumetric flask, dilute to the scale, and use ICP-MS for determination.

[0024] Figure 7 This is a diagram of the cadmium accumulation in pepper fruits under different treatments. It can be seen that the addition of Burkholderia cepacia to the treatment group significantly reduced the cadmium accumulation in pepper fruits.

[0025] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A Burkholderia cepacia, characterized in that: The strain is named as Burkholderia cepacia KL4-1-1, and the deposit number of the strain is CCTCC M 2025785.

2. Use of Burkholderia cepacia as claimed in claim 1 for reducing Cd accumulation in pepper fruits during cultivation.

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