Application of acetyl ursolic acid in promoting growth of burkholderia

Abstract

The invention discloses an application of acetyl ursolic acid in promoting the growth of burkholderia. Eight Burkholderia strains are separated from soil, the separated eight Burkholderia strains are cultured in a nitrogen-free culture medium added with 50 mg / L of acetyl ursolic acid, and the result shows that the biomass of Burkholderia can be increased by 40%-95% compared with that of a control group without acetyl ursolic acid within 24 h through acetyl ursolic acid, the Burkholderia strain has a remarkable growth promoting effect on Burkholderia, and the Burkholderia strain has a good application prospect. The core functions such as nitrogen fixation are not influenced. The concentration gradient experiment further determines that the effective concentration range of the acetyl ursolic acid on the growth promotion of the burkholderia is 5-100 mg / L, and the optimal concentration of the acetyl ursolic acid is 25-75 mg / L. Acetyl ursolic acid is used as a specific growth promoting agent of burkholderia, not only are the problems of specificity, safety and stability in the prior art solved, but also the synergistic effect of promoting growth and improving crop quality is realized.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology. Specifically, it relates to the application of a natural compound, acetylsulolic acid, in promoting the growth of Burkholderia, as well as microbial culture media, inoculants, and soil conditioners containing this compound. Background Technology

[0002] Burkholderia ( Burkholderia As a type of beneficial soil bacteria with nitrogen-fixing, growth-promoting, and biocontrol functions, Burkholderia plays an irreplaceable role in green agriculture. Its nitrogen-fixing function converts atmospheric N2 into a usable nitrogen source for plants, reducing reliance on chemical nitrogen fertilizers; it improves crop nutrient absorption by secreting auxins and siderophores, thus promoting growth; and it inhibits soil-borne pathogens (such as Ralstonia solanacearum and Fusarium wilt) by competing for ecological niches and producing antimicrobial substances. Although the laboratory functional verification of Burkholderia is relatively mature, the transition from "laboratory trials" to "industrial mass production" still faces technical challenges such as low fermentation efficiency and poor formulation stability, resulting in high production costs and short product shelf life. In practical applications, it is limited by environmental factors, making it difficult to colonize and grow in soil or plants and exert its due effects.

[0003] How plants recruit Burkholderia and the mechanisms of plant-Burkholderia interaction are currently unclear. The development and practical application of Burkholderia growth promoters or inducers still face bottlenecks that are difficult to overcome with existing technologies: First, traditional growth promoters (such as yeast powder and glucose) are mostly general nutrient sources. While promoting the growth of Burkholderia, they may simultaneously stimulate the proliferation of other pathogens (such as Fusarium oxysporum), leading to an imbalance in the microbial community and increasing the risk of crop diseases. Second, some chemically synthesized inducers (such as naphthaleneacetic acid derivatives) have certain growth-promoting effects, but they degrade slowly in the soil and are prone to residue accumulation, which not only pollutes groundwater and agricultural products, but may also be toxic to native beneficial microorganisms in the soil (such as mycorrhizal fungi). Third, plant root exudates contain thousands of metabolites, and currently no single, highly effective Burkholderia growth promoter has been accurately isolated and identified. Most studies rely on complex extracts, which have problems such as unstable activity and large batch-to-batch variations. In addition, most existing methods only focus on increasing the biomass of Burkholderia, neglecting the synergistic activation of its core functions (such as nitrogen fixation), resulting in "quantity without function" and failing to effectively improve soil fertility and crop quality. Summary of the Invention

[0004] To address the aforementioned shortcomings, this invention precisely identifies acetylursolic acid, a natural metabolite, as a specific growth promoter for Burkholderia from chestnut-tea intercropping soil. This not only solves the specificity, safety, and stability issues of existing technologies but also achieves a synergistic effect of "promoting growth and improving crop quality".

[0005] In order to achieve the above object, the following technical solutions are adopted in the present application:

[0006] The present application isolates 8 strains of Burkholderia from soil, and the isolated 8 strains of Burkholderia are cultured in nitrogen-free medium added with 50 mg / L acetyl ursolic acid, and the results show that acetyl ursolic acid can make the biomass of Burkholderia increase by 40%-95% within 24 h compared with the control group without addition, has a significant growth promoting effect on Burkholderia, and does not affect the core functions such as nitrogen fixation of Burkholderia.

[0007] Based on the concentration gradient experiment (0, 5, 10, 25, 50, 100 mg / L), it is preliminarily determined that the effective concentration range of acetyl ursolic acid for promoting the growth of Burkholderia is 5-100 mg / L, and within this range, the growth of Burkholderia can be promoted; the preferred concentration interval is 25-75 mg / L, which can balance the growth promoting effect and cost; the optimal concentration is 50 mg / L, at which the biomass of Burkholderia reaches the peak.

[0008] Compared with the prior art, the present application has the following beneficial effects:

[0009] Acetyl ursolic acid has the following advantages in promoting the growth of Burkholderia and agricultural application: (1) high growth promoting efficiency, solving the problems of slow cultivation and low bacterial amount: the laboratory cultivation period is shortened from the traditional 3-5 days to 2-3 days, and the large-scale production efficiency is increased by more than 40%; the abundance of Burkholderia in soil is increased by 1-2 orders of magnitude. (2) strong specificity, avoiding the risk of microbial imbalance: directional promotion of the growth of Burkholderia, no promotion effect on pathogenic bacteria, solving the defects of traditional nutrient sources "benefit and harm promotion"; maintaining soil microbial diversity, and having no significant effect on soil bacterial population after application. (3) environmentally friendly, meeting the needs of green agricultural development: natural source or green synthesis, no toxic residues, meeting the organic agricultural production standards; no adverse effects on soil physical and chemical properties (pH, organic matter). BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 : Colony morphology of Burkholderia isolated from soil on nitrogen-free medium.

[0011] Figure 2 : Evaluation of nitrogen fixation ability of Burkholderia isolated from soil.

[0012] Figure 3 : Growth promoting effect of acetyl ursolic acid on soil isolated nitrogen-fixing Burkholderia.

[0013] Figure 4 : Concentration screening of acetyl ursolic acid for promoting the growth of soil nitrogen-fixing Burkholderia.

[0014] Figure 5 The effects of Burkholderia on soil nitrogen content and tea quality. Detailed Implementation

[0015] Example 1: Isolation, Identification, and Nitrogen Fixation Assessment of Burkholderia

[0016] 1. Preparation and cultivation of Burkholderia culture medium in the laboratory

[0017] (1) Source and isolation of strains: The eight Burkholderia strains used were screened from non-rhizosphere soil between chestnut and tea plantations. 1 g of soil sample was weighed, 10 mL of sterile water was added, and the mixture was vortexed thoroughly and allowed to stand for 10 min. After sedimentation and filtration, 100 μL of the supernatant was diluted 50-fold with sterile water and mixed thoroughly. Then, 50 μL of the diluted bacterial suspension was spread onto a nitrogen-free medium plate containing 100 mg / L ampicillin and incubated at 28℃ for 48 h. Single colonies were picked and transferred to LB liquid medium for further incubation until the absorbance (OD) value of the bacterial suspension reached a certain level. 600 Once the concentration reaches 1.0, the strain is mixed with 30.0% glycerol and stored at -80°C.

[0018] (2) Culture medium formulation: LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L) or nitrogen-free medium (mannitol 10 g / L, KH2PO4 0.2 g / L, MgSO4·7H2O 0.2 g / L, CaSO4·2H2O 0.1 g / L, Na2MoO4·2H2O 0.005 g / L, pH 6.8), add acetylsolic acid stock solution (DMSO dissolved, concentration 10 mg / mL) to make the final concentration of acetylsolic acid in the culture medium 5, 10, 25, 50, 75, 100 mg / L.

[0019] (3) Culture conditions: The isolated Burkholderia ( Burkholderia ), such as BKHEDT8 bacterial culture (OD) 600 =0.5) was inoculated at 1% (v / v) into the above-mentioned LB or nitrogen-free medium supplemented with acetylsurfural, and cultured at 28℃ and 200 rpm for about 24 h with shaking. After the culture was completed, the concentration was measured using a McFarland turbidimeter (model YP-MNTU, Youyunpu, Yantai, Shandong).

[0020] 2. Identification and nitrogen-fixing capacity analysis of Burkholderia

[0021] (1) 16S ribosomal RNA gene sequencing: Single colonies were picked from nitrogen-free medium and transferred to LB liquid medium for further culture until the absorbance value (OD) of the bacterial culture was reached. 600The PCR score reached 1.0. For strain classification and identification, primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') were used to amplify the 16S rRNA gene fragment of *Burkholderia ursenii*. After sequencing, the obtained sequences were compared with the 16S ribosomal RNA gene sequences in the National Center for Biotechnology Information (NCBI) database using BLAST. A phylogenetic tree was constructed using MEGA7 software based on the neighbor-joining method. In the bootstrap test (1000 replicates), the percentage of replicates where related taxa clustered together was indicated next to the branch. The results showed that BKHEDC2 and BKHEDT2 belong to *Burkholderia ursenii* (…). Burkholderia ubonensis BKHEDT1 belongs to Burkholderia pyrrole ( ); Burkholderia pyrracinia BKHEDT3, BKHEDT4, BKHEDT5, and BKHEDT8 strains belong to the stable Burkholderia species. Burkholderia stabilis BKHEDT7 belongs to Burkholderia unidentified strains. Burkholderia sp.).

[0022] (2) Growth test on nitrogen-free medium: Under aseptic conditions, the above 8 Burkholderia strains were inoculated using a sterile inoculation loop. Burkholderia Bacterial strains (using *Escherichia coli* DH5α as a negative control and CK1, CK2, and CK3 as positive nitrogen-fixing controls) were inoculated onto nitrogen-free agar medium and cultured at 28°C for 48 h. Bacterial growth was assessed by observing colony morphology and biomass accumulation. All strains grew normally on nitrogen-free medium. Specifically, BKHEDT2 and BKHEDT2 formed white, opaque, dry colonies; BKHEDT1, BKHEDT3, BKHEDT4, and BKHEDT8 formed white, translucent, moist colonies; and the remaining strains formed white, opaque, moist colonies. BKHEDT4 and BKHEDT8 showed the fastest growth rates (Figure 1).

[0023] (3) Nitrogen fixation ability evaluation: 100 μL of the Burkholderia bacteria liquid was inoculated into 6 mL of nitrogen-free liquid medium and cultured at 28°C and 200 rpm for 24 h. After the culture, 2 mL of the bacterial suspension was centrifuged at 12000 rpm for 10 min. Then, 1 mL of the centrifugal supernatant was mixed with 100 μL of Nessler's reagent (purchased from Shanghai Maikelin Company, China) and reacted for 10 min. The ammonia content was determined according to the color change of the solution. In addition, 0.05 g of the Burkholderia bacteria was collected from the nitrogen-free solid medium, and the glutamine synthetase (GS) activity of the bacteria was determined using a GS assay kit (purchased from Comin, Suzhou, China). The spectrophotometer was used to determine the absorbance peak at 540 nm, and the enzyme activity was calculated accordingly. The results showed that BKHEDC2, BKHEDT2, BKHEDT3, and BKHEDT8 had high nitrogen fixation ability, and BKHEDT8 had the strongest nitrogen fixation ability (Fig. 2A and 2B). BKHEDT8 had the highest glutamine synthetase (GS) activity, indicating that it had stronger ammonia assimilation ability (Fig. 2C). Figure 2 A, 2B). BKHEDT8 had the highest glutamine synthetase (GS) activity, indicating that it had stronger ammonia assimilation ability (Fig. 2C).

[0024] Example 2 Effect of acetyl ursolic acid on the growth promotion of nitrogen-fixing Burkholderia

[0025] The isolated 8 strains of nitrogen-fixing Burkholderia were inoculated into liquid nitrogen-free medium containing 50 mg / L acetyl ursolic acid, and the bacterial biomass was quantitatively analyzed after 24 h of uniform culture. The results showed that the biomass of the 8 strains was significantly increased compared with the medium without the addition of acetyl ursolic acid (Fig. 3). To further analyze the effect of acetyl ursolic acid concentration on the growth of Burkholderia, strains BKHEDT4, BKHEDT7, and BKHEDT8 were selected and inoculated into nitrogen-free medium containing different concentrations of acetyl ursolic acid (0, 5, 10, 25, 50, and 100 mg / L), and the optimal concentration of acetyl ursolic acid was determined by detecting the bacterial growth after 24 h of culture. The bacterial density was determined by a McFarland turbidimeter (YP-MNTU, Youyunpu, Yantai, Shandong). The results showed that the acetyl ursolic acid concentration of 5-100 mg / L could promote the growth of the above 3 strains of nitrogen-fixing bacteria, and the growth reached a peak at 50 mg / L, while the growth at 100 mg / L was lower than that at 50 mg / L, indicating that the optimal concentration of acetyl ursolic acid for promoting the growth of Burkholderia was about 50 mg / L (Fig. 4).

[0026] To test the nitrogen-fixing ability of Burkholderia and its effect on tea quality, strain BKHEDT8 was inoculated onto a solid nitrogen-free medium. Single colonies were picked and cultured in a nitrogen-free liquid medium with an optimal concentration of 50 mg / L acetylsolic acid at 28°C for 24 h. Subsequently, the bacterial culture was diluted with sterile water to OD0.05. 600 Values ​​of 0.5 and 1.0 are reserved. Field trials were conducted at the tea garden of Wuhan Xinbifang Ecological Fruit and Tea Co., Ltd. in Jiangxia District (30.250°N, 114.583°E): In June 2024, OD... 600 BKHEDT8 bacterial solutions with concentrations of 0.5 and 1.0 were applied to the soil near the trunks of tea trees in the field (Figure 5A). Tea and soil samples were collected in April 2025 to determine the amino acid and caffeine content in the tea leaves and the nitrogen content in the soil. Each treatment was repeated at least three times. The results showed that the application of Burkholderia strain BKHEDT8 (bacterial solution OD...)... 600 Application of BKHEDT8 (values ​​of 0.5 or 1.0) significantly increased soil nitrogen content (Fig. 5B). Although the application of BKHEDT8 had no significant effect on tea polyphenol content (Fig. 5D), this strain could improve tea quality by increasing the content of caffeine and amino acids in tea (Fig. 5C, 5E).

Claims

1. Application of acetylsulolic acid in promoting the growth of Burkholderia.

2. A method for promoting the growth of Burkholderia, characterized in that, Add 5-100 mg / L of acetylsolic acid to Burkholderia culture medium.

3. A culture medium for Burkholderia, characterized in that, It contains acetylursolic acid.

4. A soil conditioner, characterized in that, It contains Burkholderia and acetylsulolic acid.

5. The soil conditioner according to claim 4, characterized in that, The concentration of acetylsuldoic acid used is 5-100 mg / L.

6. The soil conditioner according to claim 5, characterized in that, The concentration of acetylsuldoic acid used is 25-75 mg / L.

7. The soil conditioner according to claim 6, characterized in that, The concentration of acetylsuldoic acid used is 50 mg / L.

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