Atractylodes lancea endophytic bacterium ALX01 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to an atractylodes lancea endophytic bacterium ALX01 and application thereof, the atractylodes lancea endophytic bacterium ALX01 is named as Pseudomonas sp. ALX01, the preservation number is CCTCC NO: M20252198, the preservation place is China Center for Type Culture Collection, Wuhan University, Wuhan, China, on October 11, 2025, and the preservation number is CCTCC NO: M20252198. By utilizing the atractylodes lancea endophytic bacterium ALX01, the problem that high-content and high-stability production of aceratrol and beta-eudesmol in non-genuine production areas is difficult to realize in the prior art is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to Atractylodes lancea endophytic bacteria ALX01 and its application. BACKGROUND

[0002] Atractylodes lancea (Thunb.) DC. is a perennial herb of the family Asteraceae, and the rhizome is an important traditional Chinese medicine material. Atractylodes lancea is rich in volatile oil, which has the pharmacological effects of drying dampness, invigorating the spleen, dispelling wind and cold, and clearing away pathogens. It is a key drug for treating dampness obstruction in the middle-jiao and abdominal fullness. Among them, hinesol and beta-eudesmol play a synergistic role in invigorating the spleen and drying dampness, anti-inflammatory, regulating gastrointestinal motility, etc., so the content directly determines the grade of medicinal materials and clinical efficacy. The accumulation is not only dominated by its own genetic characteristics, but also closely related to the specific ecological environment (such as soil and climate) in which Atractylodes lancea grows. Atractylodes lancea can be divided into Dabie Mountain chemical type and Mao Mountain chemical type according to the proportion of volatile oil components, and the proportion of beta-eudesmol in Dabie Mountain chemical type is high, and the proportion of atractylone and atractylodin in Mao Mountain chemical type is high.

[0003] Microbial fertilizer does not pollute the environment, has no residue, and is harmless to humans and animals, and has important application prospects in promoting plant growth. Among them, plant endophytic bacteria, because their life cycle is all or part of the colonization in the plant body, co-evolve with plants, and select each other, form a complex and diverse symbiotic relationship, are an important resource library of biological fertilizer. At present, the strain resources of Atractylodes lancea endophytic bacteria for promoting the accumulation of volatile oil components of Atractylodes lancea are relatively scarce. Therefore, it has important theoretical and practical significance to isolate endophytic bacteria from healthy Atractylodes lancea plants and screen efficient growth-promoting bacteria for promoting the accumulation of hinesol and beta-eudesmol in Atractylodes lancea.

[0004] At present, the quality of Atractylodes lancea is improved mainly by selecting native varieties, optimizing cultivation management, and standardizing harvesting and processing. However, due to the strict requirements of native medicinal materials for specific habitats, it is difficult to produce high content and high stability of hinesol and beta-eudesmol in non-native production areas by using existing technologies. In addition, the wild resources are increasingly depleted and the quality of artificially cultivated products fluctuates, and there is a lack of a universal high-quality production technology system that can widely guarantee high hinesol and beta-eudesmol content and nativity. Excessive reliance on chemical fertilizers or improper harvesting and processing may lead to a decrease in effective ingredients, excessive agricultural residues, and pressure on the ecological environment. The sustainable use of Atractylodes lancea resources and quality assurance urgently need to develop an ecological-friendly, safe-controllable, efficient and stable comprehensive strategy.

[0005] In view of the above defects, the present inventors have finally obtained the present application after a long period of research and practice. SUMMARY

[0006] The application aims to solve the problem that the prior art is difficult to produce high content and high stability of valerenol and beta-eudesmol in non-native production areas, and provides a rhizome atractylodis inner bacteria ALX01 and application thereof.

[0007] In order to achieve the above-mentioned purpose, the application discloses a rhizome atractylodis inner bacteria ALX01, which is named Pseudomonas sp. ALX01, the preservation number is CCTCC NO: M20252198, the preservation place is China, Wuhan, Wuhan University, China Center for Type Culture Collection, and the preservation date is October 11, 2025.

[0008] The ALX01 bacteria belong to a kind of bacteria in the bacterial kingdom (Eubacteria), gram-negative bacteria (Gram-negative bacteria), proteobacteria (Proteobacteria), pseudomonadaceae (Pseudomonadaceae) and pseudomonas (Pseudomonas).

[0009] The application further discloses application of the rhizome atractylodis inner bacteria ALX01 in increasing content of valerenol and beta-eudesmol in rhizome of rhizome atractylodis.

[0010] Compared with the prior art, the application has the following beneficial effects:

[0011] 1. The content of effective components is significantly improved and the targeting is strong: the application first uses the rhizome atractylodis inner bacteria ALX01 isolated from the rhizome atractylodis to perform co-culture, and can specifically and efficiently promote synthesis of core pharmacodynamic substances valerenol and beta-eudesmol of the rhizome atractylodis. Experimental data show that the content of valerenol and beta-eudesmol of the rhizome atractylodis treated by the method of the application is 7 times and 2 times higher than that of a conventional cultivation control group, and the improvement effect is much higher than that of using a chemical inducer or a conventional method.

[0012] 2. The production mode is green and sustainable: the application is completely based on biological interaction, does not introduce any chemical synthesis inducer or hormone, has no residue and no pollution, and meets the development direction of green and ecological traditional Chinese medicinal materials. Meanwhile, the method provides a new strategy for efficient production of effective components of the rhizome atractylodis in a controllable environment, and helps to protect the increasingly exhausted wild atractylodes resources.

[0013] 3. The technology has good affinity and is easy to implement: the strain used in the application is a plant endophyte, which has a symbiotic relationship with the host, is easy to colonize and play a role in the plant body. The co-culture method is simple, can be seamlessly connected with tissue rapid propagation technology or field cultivation management, is easy to operate, has low cost, and is easy to popularize and apply in large-scale production.

[0014] 4. Great industrial and economic value: the present application can directly produce high-content active ingredient of Atractylodes lancea medicinal materials or raw materials, significantly improving the market competitiveness of the product. At the same time, as the core intellectual property, the strain can be used to develop a special Atractylodes lancea yield-increasing bacterial agent, creating a new economic growth point. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 For endophytic bacteria ALX01 activation;

[0016] Figure 2 For the phylogenetic tree of strain ALX01 based on 16S rDNA gene sequence;

[0017] Figure 3 For the content changes of cymoterpene and beta-eudesmol in the interaction experiment of Atractylodes lancea endophytic bacteria ALX01-tissue culture seedlings. DETAILED DESCRIPTION

[0018] The above and other technical features and advantages of the present application will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings.

[0019] The Atractylodes lancea endophytic bacteria used in the present application is isolated and identified by Anhui University of Chinese Medicine, which has been preserved in China Center for Type Culture Collection (CCTCC for short; address: Wuhan University, Wuhan, China, China Center for Type Culture Collection), preservation date: October 11, 2025, preservation number: CCTCC NO: M20252198. The Atractylodes lancea endophytic bacteria in the present application is named Pseudomonas sp. ALX01.

[0020] Example 1

[0021] Isolation of Atractylodes lancea endophytic bacteria:

[0022] (1) The fresh rhizome part of Atractylodes lancea is first cleaned with running water to remove the soil attached to the surface, then washed with 75% ethanol for 30 s in a clean bench, then soaked with 2.5% sodium hypochlorite for 1 min, and finally washed with sterile water for 5-6 times. The pure water used for the last washing is placed on the LB medium, and observed daily. No bacteria grow in a short time, indicating that the disinfection is clean.

[0023] (2) After the clean and disinfected rhizome is cut into small cubes of 0.5 cm x 0.5 cm x 0.5 cm after the outer epidermis is removed, it is placed on LB medium and PDA culture plates containing chloramphenicol, and observed in a constant temperature incubator at 28°C. After the bacteria grow, the bacteria are picked to clean culture medium for purification. After purification for 2 times, it can be considered as a single colony. The obtained endophytic bacteria are preserved in a -80°C refrigerator using 30% glycerol.

[0024] Example 2

[0025] Isolation of endophytic bacteria in Atractylodes lancea:

[0026] (1) The total DNA of each strain was extracted by using the bacterial genomic DNA extraction kit produced by Applied Biosystems (USA) according to the operation process in the instruction manual. The 16S rRNA gene universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGTTACCTTGTTACGACTT-3') were used for PCR amplification. The PCR amplification reaction system and the PCR reaction conditions: the reaction system (50 μL) contained 2×Hieff PCR Master Mix 25 μL, 10 μmol·L -1 -1) 2 μL each of forward and reverse primers, 2 μL of DNA template (concentration of 26.0 ng / µL), and supplemented with ddH2O 19 μL (to a final volume of 50 μL). The PCR amplification program was set as follows: initial 94 ℃ pre-denaturation for 5 min; followed by 35 cycles of amplification (94 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, and 72 ℃ extension for 1 min); and finally 72 ℃ terminal extension for 10 min.

[0027] (2) The DNA of the bacteria was extracted by using the kit, and the 16S rDNA was amplified by using the primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGTTACCTTGTTACGACTT-3'). The obtained sequence was subjected to BLAST homology comparison in the National Center for Biotechnology Information (NCBI) database (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the sequences with higher homology were downloaded. The MEGA 11.0 software was used to construct a phylogenetic tree by using the Neighbor-joining method, so as to analyze the phylogenetic relationship of the strains.

[0028] (3) The 16S rDNA gene sequence with a length of 1401 bp was obtained after sequencing the PCR product, and the sequencing result is shown in the sequence table SEQ ID NO. 1. The Neighbor-joining method was used to construct a phylogenetic tree, and the phylogenetic tree result showed that the endophytic bacterial strain ALX01 of Atractylodes lancea was clustered into the same branch with Pseudomonas sp. (accession number: MG269625.1), and the phylogenetic relationship was closest Figure 2 ).

[0029] SEQ ID NO. 1:

[0030]

[0031] Example 3

[0032] Establishment of the co-culture system of A. lanceolatum endophytic bacteria ALX01 strain and tissue culture seedlings:

[0033] (1) The tissue culture seedlings of A. lanceolatum were 180-day-old tissue culture seedlings, and the seeds were from Yingshan County, Hubei Province. The seed culture medium formula was 2.75 g / L 1 / 2MS + 30 g / L sucrose + 8 g / L agar. After 30 days, the seedlings were subcultured into a liquid culture medium, and the liquid culture medium formula was 2.75 g / L 1 / 2MS + 30 g / L sucrose + 0.3 mL / L NAA. The seedlings were cultured in the tissue culture room for 180 days.

[0034] (2) After the endophytic bacteria were activated on LB plates, the bacteria were picked and cultured in LB medium at 28°C and 180 rpm for 3 days. The fermentation supernatant was collected by centrifugation at 12000 rpm for 30 min. The fermentation supernatant and LB liquid medium were prepared at a ratio of 5%. The 3-month-old tissue culture seedlings were transferred to the prepared medium and cultured in the artificial climate chamber for 30 days. Then the seedlings were dried at 40°C and ground into powder for use. Three treatment groups and three blank control groups were set up. The dried and powdered parts were the rhizome and root of the tissue culture seedlings.

[0035] (3) 0.1 g of the dried powder was placed in a 20 mL headspace bottle, 0.4 mL of saturated sodium chloride solution was added, and 100 µL of internal standard solution was added. Under the condition of 80°C water bath, the solid phase extraction head was extracted for 30 min. After extraction, the solid phase microextraction device was quickly inserted into the GC injector, and the analysis was performed at 250°C for 10 min under the mode of split ratio 20:1.

[0036] (4) Preparation of saturated sodium chloride solution: Take 4.56 g of sodium chloride solid and place it in a 50 mL centrifuge tube containing 12.5 mL of pure water. Shake well to melt it. Internal standard preparation method: Take 1 µL of internal standard solution and place it in 1 mL of filtered n-hexane solution (use 0.22 µm filter membrane). At this time, the internal standard concentration is 1 µL / mL. The relative content of each component is calculated by internal standard semi-quantitative method: Xi = Ii × Cs × Vs / Is × M × 1000. Xi: the content of the measured substance i (µg / g); Ii / ls: the peak area ratio of the measured substance to the internal standard; Cs × Vs / 1000: the total mass of the internal standard (µg); M: the mass of the measured substance (g), used for normalization, unit mass content.

[0037] GC conditions: DB-5MS (60 m x 0.25 mm x 0.25 µm) column was used, with helium as the carrier gas, and the flow rate was 1 mL / min; the temperature program of the column was as follows: the initial temperature was 85 ℃, and was kept for 5 min, then increased to 185 ℃ at a rate of 3 ℃ / min, and was kept for 10 min, then increased to 250 ℃ at a rate of 5 ℃ / min, and was kept for 5 min.

[0038] MS conditions: the source temperature of the electron impact source (EI) was 230 ℃; the electron energy was 70 eV, the connector temperature was 280 ℃, the solvent delay was 5 min, the scanning range was 33-350 amu; and the voltage of the electron multiplier was 2.4 kV.

[0039] (5) The results show that in the tissue culture seedlings inoculated with the endophytic bacteria ALX01, the average content of manoyl oxide is 41.9 ng / mg, and the average content of β-eudesmol is 65.6 ng / mg; in the blank control group, the average content of manoyl oxide is 5.0 ng / mg, and the average content of β-eudesmol is 17.2 ng / mg. The content of manoyl oxide is increased by 7 times, and the content of β-eudesmol is increased by more than 2 times.

[0040] The above merely describes the preferred embodiments of the present application, which are merely illustrative but not restrictive. It is understood by those skilled in the art that many changes, modifications, and even equivalents can be made to the present application within the spirit and scope defined by the claims of the present application, and all of them shall fall within the protection scope of the present application.

Claims

1. An endophytic bacteria ALX01 of Atractylodes lancea, characterized in that, The endophytic bacteria ALX01 of Atractylodes lancea is named Pseudomonas sp. ALX01, and the preservation number is CCTCC NO: M20252198, the preservation place is Wuhan, China, Wuhan University, China Center for Type Culture Collection, and the preservation date is October 11, 2025.

2. The endophytic bacteria ALX01 of Atractylodes lancea according to claim 1 is applied to increasing the content of atractylol and β-eudesmol in rhizomes of Atractylodes lancea.

Images