Endophytic bacteria for promoting the growth of atractylodes macrocephala and application thereof
Patent Information
- Application Number
- CN202611073151.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-20
- Publication Date
- 2026-10-27
AI Technical Summary
目前，已有文献报道关于白术内生细菌的分离及多样性研究，且公开白术内生细菌主要有荧光假单胞菌、沙雷氏菌、丛枝菌根真菌等，但关于微生物菌种促进白术生物量积累的研究不多
[0015]本发明筛选到一种可固氮、溶解无机磷的细菌菌株，Pantoea sp.EB7菌株溶解无机磷能力达到47.34 ± 2.87mg/L，并能够产生植物生长激素-吲哚乙酸。本发明提供的Pantoea sp.EB7菌株能显著提高白术生长，株鲜重和根鲜重均明显增加。
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Abstract
Description
Technical Field
[0001] This invention relates to the field of agricultural microbial technology, and in particular to an endophytic bacterium that promotes the growth of Atractylodes macrocephala and its application. Background Technology
[0002] Atractylodes macrocephala is a commonly used bulk medicinal herb with high medicinal value and good tonic effects, and is known as the "first essential herb for tonifying qi and strengthening the spleen". Among them, the root is the medicinal part of Atractylodes macrocephala, which contains a large number of pharmacologically active ingredients, including biomass, sesquiterpenes and phenolic acids, and has a variety of physiological activities such as immunomodulation, anti-tumor, antioxidant and organ protection.
[0003] Traditionally, fertilizers are applied to enhance the biomass accumulation of Atractylodes macrocephala. Currently, Atractylodes macrocephala is mainly fertilized with single-element and compound fertilizers. However, long-term application of these fertilizers can lead to reduced plant resistance, physiological imbalances, yield reduction, and quality decline. Therefore, utilizing microbial fertilizers or inoculants to promote biomass accumulation in Atractylodes macrocephala and protect the ecological environment is of great significance.
[0004] Endophytic bacteria, which reside within plants for extended periods, typically form stable, mutually beneficial relationships with them and can influence plant growth and development. Endophytic bacteria primarily promote plant growth and development by producing indoleacetic acid (IAA), dissolving inorganic phosphorus, and enhancing plant absorption of soil nutrients. Currently, there are literature reports on the isolation and diversity of endophytic bacteria in Atractylodes macrocephala, revealing that the main endophytic bacteria include Pseudomonas fluorescens, Serratia marcescens, and arbuscular mycorrhizal fungi. However, research on how microbial strains promote biomass accumulation in Atractylodes macrocephala is limited.
[0005] Screening endophytic bacteria with growth-promoting effects in Atractylodes macrocephala and developing corresponding inoculants can help reduce the dependence on chemical fertilizers in the artificial cultivation of Atractylodes macrocephala, improve its growth, promote the utilization of nutrients, and provide innovative strategies for the sustainable development of Chinese medicinal materials. Summary of the Invention
[0006] Based on the technical problems existing in the background art, the present invention proposes an endophytic bacterium that promotes the growth of Atractylodes macrocephala and its application.
[0007] In a first aspect, the present invention provides an endophytic bacterium that promotes the growth of Atractylodes macrocephala. The endophytic bacterium is isolated and purified from the rhizome of Atractylodes macrocephala. The endophytic bacterium is Pantoea dispersa, which is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC No. M20261159, accession name Pantoea sp. EB7, and deposit date June 1, 2026.
[0008] The endophytic bacteria provided by this invention have the characteristics of nitrogen fixation, inorganic phosphorus dissolution, and indoleacetic acid production, which can significantly promote the biomass accumulation of Atractylodes macrocephala seedlings and therefore play an important role.
[0009] The solid culture characteristics of the endophytic bacteria described in this invention are as follows: the endophytic bacteria grow well when cultured on LB medium at 20-38°C, and the colonies are round, yellow, smooth and opaque with neat edges.
[0010] Furthermore, the nucleotide sequence of the 16S rDNA of the endophytic bacteria is shown in SEQ ID NO. 1.
[0011] Secondly, a microbial agent is also provided, comprising the aforementioned endophytic bacteria.
[0012] Thirdly, a microbial fertilizer is also provided, comprising the endophytic bacteria or the microbial agent.
[0013] The application of the aforementioned endophytic bacteria, the aforementioned microbial agent, or the aforementioned microbial fertilizer in promoting the growth of Atractylodes macrocephala.
[0014] The beneficial effects of this invention are as follows:
[0015] This invention screened a bacterial strain capable of nitrogen fixation and inorganic phosphorus dissolution, Pantoea sp. EB7, which has an inorganic phosphorus dissolution capacity of 47.34 ± 2.87 mg / L and can produce the plant growth hormone indoleacetic acid. The Pantoea sp. EB7 strain provided by this invention significantly improves the growth of Atractylodes macrocephala, with a marked increase in both plant and root fresh weight. Attached Figure Description
[0016] Figure 1 This is a normal growth colony diagram of the Pantoea sp. EB7 strain in Example 2.
[0017] Figure 2 This is a colony diagram of Pantoea sp. EB7 strain in Example 3 on Assumption medium.
[0018] Figure 3 This is a colony diagram of Pantoea sp. EB7 strain in Example 3 on a phosphate-solubilizing medium plate.
[0019] Figure 4 This is the phosphorus standard curve established in Example 3.
[0020] Figure 5 This is a graph illustrating the ability of Pantoea sp. EB7 strain in Example 3 to produce indoleacetic acid.
[0021] Figure 6 This is the IAA standard curve established in Example 3.
[0022] Figure 7 This is a growth chart of the potted seedlings in the treatment group (EB7) and the control group (CK) in Example 5. Detailed Implementation
[0023] The technical solution of the present invention will be described in detail through specific embodiments.
[0024] In the following examples and comparative examples, the specific information regarding the raw materials used is as follows:
[0025] Atractylodes macrocephala: Collected from a medicinal herb planting base in Bozhou City, Anhui Province. All materials were two-year-old artificially cultivated products with the same habitat and consistent growth. The collection time was November 10, 2023. It was identified by Professor Wang Dianlei of Anhui University of Traditional Chinese Medicine as Atractylodes macrocephala Koidz., a plant of the Asteraceae family.
[0026] Culture medium:
[0027] LB solid medium: 4g agar powder, 2.5g tryptone, 2.5g sodium chloride, 1.25g yeast extract, 250mL pure water.
[0028] LB liquid medium: 2.5g tryptone, 2.5g sodium chloride, 1.25g yeast extract, 250mL pure water.
[0029] Ashube medium: Qingdao Haibo Biotechnology Co., Ltd. (Product No.: HB8540).
[0030] Phosphate-solubilizing medium: Qingdao Haibo Biotechnology Co., Ltd. (Product No.: HB8549-2).
[0031] Note: All culture media were autoclaved at 121°C for 20 minutes before use.
[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0033] Example 1
[0034] Isolation of strains:
[0035] The Pantoea sp. EB7 strain provided by this invention was isolated and purified from the rhizome of Atractylodes macrocephala. The specific isolation steps are as follows:
[0036] Five planting areas were selected, and 10 disease-free Atractylodes macrocephala plants with uniform growth were randomly selected from each area. Excess soil was gently patted off, and the plants were placed in sterile sealed bags for later use. The surface of the Atractylodes macrocephala plants was rinsed with sterile water. The rhizomes were cut off with a sterile blade and tweezers, and the rhizomes were cut into pieces, with each piece approximately 0.5cm × 0.5cm in size. The Atractylodes macrocephala plant tissues were disinfected sequentially with ethanol and sodium hypochlorite. First, the plants were rinsed with 1% (v / v) sodium hypochlorite for 10 minutes, turning the tissue over continuously during this time. Then, they were immediately rinsed with sterile water for 2 minutes each time, for a total of 3 rinses. After rinsing, the plants were rinsed with 75% alcohol for 10 minutes. After disinfection, they were immediately rinsed with sterile water for 2 minutes each time, for a total of 3 rinses. Finally, the plants were placed on sterile filter paper to absorb excess water for later use.
[0037] After sterilization, the plant material was cut with an inoculation knife and inoculated onto LB agar plates, which were then incubated at 37°C. Once colonies appeared on the plates, vigorous single colonies with varying sizes and morphologies were selected and streaked onto fresh LB agar plates. The plates were incubated at 37°C for 48 hours. This process was repeated three times. After confirming the absence of contaminating bacteria, single colonies were inoculated into LB liquid medium and incubated on a shaker at 37°C and 150 rpm for 48 hours. Finally, the bacterial strain was preserved in a 50% glycerol suspension at -80°C to obtain glycerol tubes containing the Pantoea sp. EB7 strain for later use.
[0038] Example 2
[0039] Identification of Pantoea sp. EB7 strain:
[0040] When Pantoea sp. EB7 strain is grown on LB solid medium, such as Figure 1 As shown, the colonies are yellow, with neat edges, smooth and moist surfaces, and are opaque. Genomic DNA was extracted from the Pantoea sp. EB7 strain, and then PCR amplification was performed using universal primers 27F and 1492R for the bacterial 16S rRNA gene. The obtained PCR products were sequenced. BLAST analysis was performed based on the strain's 16S rDNA sequence using the NCBI nucleic acid database. The analysis results showed that the Pantoea sp. EB7 strain belongs to Pantoea dispersa, and the 16S rDNA sequence of Pantoea dispersa showed a homology of 99.42%. Pantoea sp. EB7 is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number CCTCC No. M20261159. The 16S rDNA sequence of the Pantoea sp. EB7 strain is shown in SEQ ID NO. 1, totaling 1422 bp, and the specific sequence is as follows:
[0041]
[0042] Example 3
[0043] Determination of nitrogen fixation, inorganic phosphorus solubility, and indoleacetic acid (IAA) production by Pantoea sp. EB7 strain:
[0044] (1) Nitrogen fixation capacity of Pantoea sp. EB7 strain
[0045] The nitrogen-fixing ability of Pantoea sp. EB7 was tested using Assumption medium. Activated colonies were placed in a clean bench and transferred to fresh LB agar plates for further activation and recovery. Single colonies of the activated strain were then inoculated onto Assumption medium using a sterile inoculation loop in a zigzag pattern. The inoculated plates were then incubated in a biochemical incubator at a constant temperature for three consecutive subcultures, with daily observation of the strain's growth. The ability to fix nitrogen was determined based on the strain's ability to grow normally, as well as the typical morphological characteristics of nitrogen-fixing bacteria, such as neat colony edges and a smooth, moist surface. The test results are as follows: Figure 2 As shown in the figure, Pantoea sp.EB7 can grow normally on the culture medium. Its cell edges are relatively round, the cell is viscous and moist, and its properties remain stable after three passages, with relatively stable nitrogen fixation ability.
[0046] (2) Determination of dissolved inorganic phosphorus in Pantoea sp. EB7 strain
[0047] Qualitative detection of bacterial phosphate-solubilizing ability was performed using the filter paper disc method: Colonies of activated bacteria were picked from the edge using an inoculation loop and inoculated into LB broth (5 g / L yeast extract, 10 g / L tryptone, 5 g / L sodium chloride). The culture was then placed on a shaker and incubated until the logarithmic growth phase was reached. 50 μL of the solution was added to a sterile filter paper placed on the surface of a phosphate-solubilizing medium plate and incubated at 37°C. Observations were made daily. The appearance of a clear zone around the colony indicated that the strain possessed the ability to solubilize inorganic phosphorus. The test results are as follows: Figure 3 As shown in the figure, there is a clear phosphorus-solubilizing zone, indicating that the Pantoea sp. EB7 strain has the ability to dissolve inorganic phosphorus.
[0048] Establishment of the phosphorus standard curve: Accurately weigh 0.2197 g of dried potassium dihydrogen phosphate, dissolve it in 100 mL of distilled water, add 5 mL of concentrated sulfuric acid for preservation, and dilute to 1 L to prepare a 50 mg / L phosphorus stock solution. Before use, dilute the stock solution to 5 mg / L as the working standard solution. Pipette 0, 2, 4, 6, 8, and 10 mL of the 5 mg / L working standard solution into 50 mL volumetric flasks, and add distilled water to approximately 30 mL. Add 2 drops of 2,4-dinitrophenol indicator, first adjusting the liquid to a slightly yellow color with 4 mol / L sodium hydroxide solution, then adjusting again with 1 mol / L sulfuric acid until the yellow color just disappears. Then add 5 mL of molybdenum-antimony colorimetric reagent (each 100 mL of molybdenum-antimony stock solution contains 1.50 g ascorbic acid; prepare fresh before use), dilute to the mark with distilled water, shake well, and develop the color at room temperature in the dark for 30 min. Using distilled water as a blank control, the absorbance values of the solutions in each volumetric flask were measured at a wavelength of 700 nm using a spectrophotometer. A phosphorus standard curve was plotted with phosphorus concentration on the x-axis and absorbance values on the y-axis. Figure 4 The ability of Pantoea sp. EB7 to dissolve inorganic phosphorus was calculated to be 47.34 ± 2.87 mg / L using a standard curve.
[0049] (3) Determination of IAA secretion by Pantoea sp. EB7 strain
[0050] The activated bacterial strain was placed in a clean bench and transferred to a new LB solid medium. After colonies had fully grown, colonies were picked from the edge using an inoculation loop and transferred to LB liquid medium. The culture was then incubated on a shaker until the logarithmic growth phase was reached. The culture was then centrifuged at 8000 rpm for 10 min, resuspended in sterile water, and the OD600 value was adjusted to approximately 1.0. The culture was then inoculated into LB liquid medium containing L-tryptophan (0.5 g / L) and incubated on a shaker in the dark for 3 days to obtain a bacterial suspension containing Pantoea sp. EB7. After the shaker incubation, the bacterial suspension was centrifuged at 10000 rpm for 10 min. 50 μL of the supernatant was mixed with an equal volume of Salkowski reagent (Feijing Biotechnology Co., Ltd.) and incubated in the dark for 30 min. The color change was observed; if IAA was produced, the sample turned pink, indicating a positive result; no color change indicated the absence of indoleacetic acid production, indicating a negative result. The test results are as follows: Figure 5 As shown, the sample is pink, indicating that the Pantoea sp. EB7 strain has the ability to produce IAA.
[0051] IAA Standard Curve Establishment: Accurately weigh 10 mg of indoleacetic acid (IAA) standard and dissolve it in 100 mL of anhydrous methanol to obtain an IAA standard stock solution with a concentration of 100 mg / L. After serial dilution, samples with concentrations of 80 mg / L, 60 mg / L, 40 mg / L, and 20 mg / L were obtained. The absorbance of the samples at 530 nm was measured using a UV spectrophotometer. An IAA standard curve was constructed with IAA concentration on the x-axis and OD530 nm value on the y-axis. The standard curve is shown below. Figure 6 As shown. The absorbance of the bacterial suspension containing Pantoea sp. EB7 strain at 530 nm was measured using a UV spectrophotometer. The results were repeated three times. Uninoculated LB liquid medium was used as a blank control group. The yield of indoleacetic acid produced by Pantoea sp. EB7 strain was calculated to be 51.74 ± 8.15 mg / L using a standard curve.
[0052] Example 4
[0053] Activation and suspension preparation of Pantoea sp. EB7 strain:
[0054] Activation of Pantoea sp. EB7 strain: A loopful of the inoculum was picked from the glycerol tube containing Pantoea sp. EB7 strain obtained in Example 1 and aseptically streaked onto LB agar plates. The culture was then incubated at 37°C for 48 h. Then, a single colony of a vigorous Pantoea sp. EB7 strain was streaked three times onto a fresh LB agar plate. After incubation, a single colony of Pantoea sp. EB7 was picked and inoculated into LB liquid medium. The culture was then incubated at 37°C and 150 rpm for 48 h with shaking.
[0055] Preparation of bacterial suspension: Pantoea sp. EB7 strain was inoculated onto LB solid medium and activated at 37°C for 2 days. Bacterial suspension was prepared by picking the bacterial strain with an inoculation loop and transferring it to a 50 mL Erlenmeyer flask containing 20 mL of sterile water. The suspension was then placed on a shaking incubator and shaken at 37°C / 180 rpm for 15 min. Serial dilutions were then performed to a bacterial concentration of 1 × 10⁻⁶. 8 CFU / mL yields Pantoea sp. EB7 liquid bacterial agent.
[0056] Example 5
[0057] Verification of Pantoea sp. EB7 strain's promotion of Atractylodes macrocephala growth:
[0058] Pot experiment and biomass
[0059] Uniformly growing Atractylodes macrocephala seedlings were transplanted into pots filled with nutrient-rich garden soil. The nutrient soil used for Atractylodes macrocephala growth was mainly Tasha's Garden brand general-purpose substrate, which was mixed evenly with vermiculite at a mass ratio of 1:1 and then dispensed into the pots. 10 mL of the aforementioned Pantoea sp. EB7 liquid inoculant was applied near the roots of each seedling as the treatment group (EB7), while the control group (CK) received an equal volume of sterile water. Under natural conditions and with regular, appropriate watering, Atractylodes macrocephala was harvested 60 days after planting to measure plant height, fresh weight, fresh root weight, and root length. The test results are shown in Table 1.
[0060] Table 1
[0061]
[0062] Note: * indicates p<0.05, meaning there is a significant difference between treatment levels.
[0063] Biomass measurements are shown in Table 1: After inoculation with this strain, the growth indicators of Atractylodes macrocephala were enhanced. Compared with the control, plant height, plant fresh weight, root fresh weight and root length were significantly increased by 0.37%, 62.77%, 33.33% and 13.85%, respectively. Among them, plant fresh weight and root fresh weight showed significant differences (p<0.05).
[0064] The growth status of potted plants in the control group (CK) and the treatment group (EB7) is as follows: Figure 7 As shown in the figure, EB7 bacterial solution root irrigation treatment has a certain promoting effect on the growth of Atractylodes macrocephala.
[0065] In summary, the Pantoea sp. EB7 strain of this invention has the characteristics of nitrogen fixation, inorganic phosphorus dissolution, and indoleacetic acid production, which can significantly promote the growth of Atractylodes macrocephala, significantly increase the accumulation of fresh plant weight and fresh root biomass, and play an important role in improving the quality of Atractylodes macrocephala.
[0066] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. An endophytic bacterium that promotes the growth of Atractylodes macrocephala, characterized in that, The endophytic bacteria were isolated and purified from the rhizomes of Atractylodes macrocephala. The endophytic bacteria is Pantoea dispersa, which is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC No. M20261159, accession name Pantoeasp.EB7, and deposit date June 1, 2026.
2. The endophytic bacteria according to claim 1, characterized in that, The solid culture characteristics of the endophytic bacteria are as follows: when cultured on LB medium at 20-38℃, the endophytic bacteria grow well, with round, yellow colonies that are smooth, opaque, and have neat edges.
3. The endophytic bacteria according to claim 1, characterized in that, The nucleotide sequence of the 16S rDNA of the endophytic bacteria is shown in SEQ ID NO.
1.
4. A microbial agent, characterized in that, Includes the endophytic bacteria described in any one of claims 1 to 3.
5. A microbial fertilizer, characterized in that, It includes the endophytic bacteria as described in any one of claims 1 to 3 or the bacterial agent as described in claim 4.
6. The application of the endophytic bacteria according to any one of claims 1 to 3, the microbial agent according to claim 4, or the microbial fertilizer according to claim 5 in promoting the growth of Atractylodes macrocephala.