Prescottella bacterium and use thereof

Abstract

The application provides a kind of prescottella and its application, belongs to the field of microbial engineering technology.The strain is prescottella sp.DCR-T3, which has been preserved in China General Microbiological Culture Collection Center on July 19, 2023, and the preservation number is CGMCC No.27952.Prescottella sp.DCR-T3 strain and its bacterial agent can degrade cinnamic acid in soil when used in soil rich in cinnamic acid, significantly reduce the inhibition of high concentration cinnamic acid on cucumber and other crops, and increase the biomass of cucumber and other plants.The invention of the bacterial agent can solve the problem of reduced yield of cucumber and other plants caused by the accumulation of phenolic acid compounds represented by cinnamic acid in soil.

Description

Technical Field

[0001] This application belongs to the field of microbial engineering technology, and relates to a Prescottella sp. DCR-T3, as well as a microbial agent produced using the Prescottella sp. DCR-T3, a method for preparing the microbial agent, and the application of the Prescottella sp. DCR-T3 and the microbial agent in the degradation of phenolic acid compounds. Background Technology

[0002] Phenolic acids are common allelopathic substances in plants, exhibiting strong allelopathic activity. They are intermediate products of plant residue decomposition and important components of plant root exudates. Continuous cropping can lead to severe accumulation of certain types of phenolic acids in the soil. High concentrations of phenolic acids in the soil are a significant mechanism contributing to continuous cropping obstacles. High concentrations of phenolic acids severely reduce seed germination rates and inhibit plant growth and development, making them a major cause of severe yield reductions in sensitive crops.

[0003] Biological control utilizes the life activities of microorganisms to repair damaged environments, offering advantages such as high repair efficiency, low cost, and no pesticide residues or environmental pollution. Soils naturally contain a large number of bacteria, fungi, and actinomycetes. These strains not only do not cause plant diseases but can also promote plant growth by degrading phenolic compounds in the soil. Developing these soil microbial resources has significant practical implications for industrial and agricultural production. Summary of the Invention

[0004] This invention primarily involves screening soil bacteria capable of degrading phenolic acid compounds, aiming to promote plant growth by degrading these compounds. The Prescottella sp. DCR-T3 and its inoculant of this invention are simple to use and inexpensive.

[0005] Specifically, the present invention proposes the following technical solution.

[0006] On the one hand, the present invention provides a Prescottella strain, namely Prescottella sp. DCR-T3, which was deposited on July 19, 2023 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 27952.

[0007] On the other hand, the present invention provides an application of the aforementioned Prescottella bacteria in the degradation of phenolic acid compounds.

[0008] Preferably, the Prescottella sp. DCR-T3 strain is applied to soil in which phenolic acid compounds accumulate, meaning soil containing phenolic acid compounds. The Prescottella sp. DCR-T3 strain is capable of degrading phenolic acid compounds in the soil.

[0009] Preferably, the crop is cucumber.

[0010] On the other hand, the present invention provides a microbial agent, which is produced using the aforementioned Prescottella sp. DCR-T3, and the active ingredient of the microbial agent is the aforementioned Prescottella sp. DCR-T3.

[0011] Preferably, the microbial agent contains more than 1 billion Prescottella sp. DCR-T3 per ml.

[0012] In another aspect, the present invention provides an application of microbial agents in the degradation of phenolic acid compounds.

[0013] Preferably, the microbial agent is applied to soil in which phenolic acid compounds have accumulated, and the soil in which phenolic acid compounds have accumulated refers to soil containing phenolic acid compounds.

[0014] In another aspect, the present invention provides a method for preparing the microbial agent, the method comprising the following steps:

[0015] Step 1: Plate culture: After activating the Prescottella sp. DCR-T3, inoculate it onto a plate to obtain the plate culture;

[0016] Step 2, Erlenmeyer flask inoculation: Inoculate the plate culture from Step 1 into LB medium and culture at a constant temperature with shaking until the logarithmic growth phase to obtain the microbial inoculum.

[0017] Preferably, the LB medium comprises, by mass percentage, the following components: 1.0% tryptone, 0.5% yeast extract, 1.0% NaCl, with the remainder being water, and a pH of 6.8–7.2.

[0018] The present invention has the following beneficial effects:

[0019] 1. The Prescottella strain provided by this invention can degrade phenolic acid compounds in the soil, thereby promoting plant growth and improving the low yield of sensitive crops such as cucumbers in soil with accumulated phenolic acid compounds. It significantly promotes the germination rate and biomass of plants such as cucumbers.

[0020] 2. The microbial inoculant provided by this invention can be used as a microbial fertilizer in farmland where phenolic acid compounds accumulate, thereby improving crop germination rate, biomass, or grain yield.

[0021] 3. The Prescottella strain and microbial agent of this invention have the advantages of low production and use costs and ease of use, which can expand the planting range of crops such as cucumbers and increase crop yields;

[0022] 4. The present invention also provides a method for preparing a microbial inoculant, which has the advantages of wide applicability and strong practicality. Attached Figure Description

[0023] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0024] Figure 1 The graph shows the degradation of Prescottella sp. DCR-T3 strain after inoculation into an inorganic salt medium;

[0025] Figure 2 The bar chart shows the degradation rate of different phenolic acids by the Prescottella sp. DCR-T3 strain. Detailed Implementation

[0026] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0027] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0028] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0029] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related areas.

[0030] Preservation information:

[0031] The Prescottella sp. DCR-T3 provided by this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China; accession number CGMCC No. 27952, deposit date July 19, 2023.

[0032] The Prescottella sp. DCR-T3 strain of the present invention has the following properties:

[0033] 1. Morphological characteristics: The bacterial cells are rod-shaped.

[0034] 2. Physiological and biochemical characteristics: After 3 days of culture on LB medium, it forms pale yellow colonies with a diameter of 0.5 mm, which are opaque, smooth and moist, and have raised surfaces.

[0035] 3.16S rRNA sequence identification

[0036] PCR amplification was performed using the following primers: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGACTTAACCCCAATCGC-3′). The PCR products were analyzed by gel electrophoresis and other methods. After verification, the products were sent to BGI Genomics (Beijing) Co., Ltd. for bidirectional sequencing. The 16S rRNA sequence is shown in SEQ ID No: 1 of the sequence listing. Alignment with sequences in GenBank revealed that the 16S rRNA sequence of strain DCR-T3 showed the highest homology (over 99%) with sequences from strains of the genus *Prescottella*.

[0037] This invention primarily involves screening bacteria from rhizosphere soil capable of degrading phenolic acid compounds, aiming to degrade these compounds in the soil and thus promote plant growth. The invention provides Prescottella sp. DCR-T3 and its microbial inoculant, which can grow in culture media containing 200 mg / L of phenolic acid (cinnamic acid) and other inorganic salt media. In soil containing 250 mg / L of phenolic acid compounds, it can significantly improve the germination rate of cucumber seeds. Adding this inoculant to soil containing 250 mg / L of phenolic acid compounds can significantly improve the germination rate of crops such as cucumbers and significantly increase crop biomass, thereby promoting plant growth.

[0038] The specific preparation steps for Prescottella sp. DCR-T3 are as follows:

[0039] Potato rhizosphere soil samples were collected. 1 g of pure potato rhizosphere soil was added to an inorganic salt medium containing 100 mg / L cinnamic acid. The medium was incubated on a shaker at 150 rpm and 30°C for 7 days. After serial dilution, the samples were plated onto LB agar plates (5.0 g / L yeast extract, 10.0 g / L peptone, 10.0 g / L NaCl, 20.0 g / L agar powder, diluted to 1 L with water, pH approximately 7.0). The plates were incubated at 30°C in the dark for 7 days. Colonies grown on the LB agar plates were collected, streaked for purification, and then stored.

[0040] The present invention also provides a microbial inoculant produced using the above-mentioned Prescottella sp. DCR-T3, the active ingredient of which is Prescottella sp. DCR-T3 cells.

[0041] The preparation method of the microbial inoculant is as follows:

[0042] The process for producing microbial agents is as follows: plate seeding – conical flask seeding – product, wherein the product is packaged as a liquid microbial agent or a solid adsorbent microbial agent.

[0043] In a preferred embodiment, the preparation of the microbial inoculant can specifically take the following steps:

[0044] S1. Plate culture: The Prescottella sp. DCR-T3 strain (original strain) was cultured on LB medium plates (composition as follows: yeast extract 5g / L, peptone 10g / L, NaCl 10g / L, agar powder 20g / L, pH 7.0-7.2) for later use.

[0045] S2. Erlenmeyer flask culture: Inoculate the plate culture into 200mL LB liquid medium (5g / L yeast extract, 10g / L peptone, 10g / L NaCl, the rest is water, pH 7.0-7.2), and culture at a constant temperature with shaking until the logarithmic growth phase. After centrifugation and resuspending in sterile water, directly dispense into liquid form using plastic packaging buckets or bottles, or dispense into solid form using peat adsorption packaging bags.

[0046] This invention relates to the Prescottellasp. DCR-T3 strain and microbial agent, which can degrade phenolic acid compounds in soil and thus promote cucumber growth. These components offer advantages such as low production and usage costs and ease of use. By degrading phenolic acid compounds in the soil, they significantly promote cucumber growth. The strain and agent provided by this invention can be used as microbial fertilizer in farmland with phenolic acid accumulation, improving crop germination rate, biomass, or yield in phenolic acid-rich soils. Using this agent can expand the planting area of ​​cucumbers and increase the total planted area.

[0047] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores, as shown in Table 1. The cucumber seeds used were conventional cucumber seeds. All experiments were conducted in triplicate, and the results were averaged.

[0048] Table 1. Information on the commercial sources of each reagent.

[0049]

[0050]

[0051] Example 1: Obtaining and identifying Prescottella sp. DCR-T3 strain

[0052] I. Obtaining the Prescottella sp. DCR-T3 strain

[0053] Potato rhizosphere soil samples were collected from potato fields on the South Campus of Inner Mongolia University in September 2021. 1g of pure potato rhizosphere soil was added to an inorganic salt medium containing 100mg / L cinnamic acid. The medium was incubated in a shaker at 150rpm and 30℃ for 7 days. After serial dilution, the samples were plated onto LB agar plates (5.0g / L yeast extract, 10.0g / L peptone, 10.0g / L NaCl, 20.0g / L agar powder, diluted to 1L with water, pH approximately 7.0). The plates were incubated in a 30℃ incubator in the dark for 7 days. Single colonies grown on the LB agar plates were collected, streaked for purification, and then preserved.

[0054] II. Identification of Prescottella sp. DCR-T3 strain

[0055] (1) Morphological characteristics: The bacterial cells are rod-shaped.

[0056] (2) Physiological and biochemical characteristics: After 3 days of culture in LB medium, it forms pale yellow colonies with a diameter of 0.5 mm, which are opaque, smooth and moist, and have raised surfaces.

[0057] (3) 16S rRNA sequence identification

[0058] The following primers were used for PCR amplification: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGACTTAACCCCAATCGC-3′). The PCR products were analyzed by gel electrophoresis and other methods. After confirmation, the samples were sent to BGI Genomics (Beijing) Co., Ltd. for bidirectional sequencing. The 16S rRNA sequence is shown in SEQ ID No: 1 of the sequence listing. Comparison with sequences in GenBank revealed that the 16S rRNA sequence of strain DCR-T3 showed the highest homology with that of strain Prescottella. Physiological and biochemical indicators were consistent with those of the standard strain of Prescottella. Therefore, strain Prescottella sp. DCR-T3 was identified as a strain of the genus Prescottella.

[0059] III. Preservation of Prescottella sp. DCR-T3 strain

[0060] Based on the above identification results, the strain was confirmed to be a new strain of Prescottella, named Prescottella sp.DCR-T3, and deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No.27952.

[0061] Example 2: Degradation of cinnamic acid and other acidic compounds by Prescottella sp. DCR-T3 strain in inorganic salt culture medium.

[0062] DCRT3 seed culture was inoculated at a rate of 1% (v / v) into an inorganic salt medium with a cinnamic acid concentration of 100 mg / L. The inoculated culture was then placed in a shaker at 30°C and 150 rpm for incubation. The bacterial cell concentration (OD) of the culture was measured periodically. 600 The concentration of cinnamic acid and its value were determined. Samples with only cinnamic acid and samples inoculated with the bacterial strain were used as control groups, respectively, under the same culture conditions. Each group had three replicates. The formula for calculating the degradation rate of the compound by the bacterial strain is as follows:

[0063] Degradation rate = (Concentration of compound in control group - Concentration of compound in treatment group) / Concentration of compound in control group × 100%.

[0064] Figure 1 The degradation of Prescottella sp. DCR-T3 strain after inoculation with inorganic salt medium is shown. Figure 1 As shown, the concentration of cinnamic acid in the uninoculated culture medium did not change significantly throughout the experimental period. In the samples inoculated with Prescottella sp. DCR-T3, the concentration of cinnamic acid decreased rapidly, reaching a degradation rate of 98.8% at 52 h.

[0065] Figure 2 The degradation rates of different phenolic acids by the strains are shown. For example... Figure 2 As shown, the Prescottella sp. DCR-T3 strain can degrade p-hydroxybenzoic acid, but cannot degrade syringic acid, vanillic acid, coumaric acid, and ferulic acid. After 48 hours of cultivation, the remaining amount of p-hydroxybenzoic acid was almost zero, indicating that the strain was able to almost completely degrade p-hydroxybenzoic acid.

[0066] Example 3: Inoculating soil containing high concentrations of phenolic acids with Prescottella sp. DCR-T3 strain to increase cucumber biomass.

[0067] Single colonies of *Prescottella* sp. DCR-T3 strain were picked and cultured in 3 mL LB liquid medium at 30°C and 150 rpm for 24 h to obtain fresh bacterial suspension. After centrifugation, the suspension was resuspended in sterile water, and the bacterial concentration (OD600) was adjusted to 1 to obtain the bacterial agent. This agent was added at a 7% inoculum to soil containing 250 mg / kg cinnamic acid, with soil without the agent serving as a control group. Uniformly sized cucumber seeds were divided into two groups: one group was planted in soil containing the bacterial agent and cinnamic acid, and the other group was planted in soil without the agent (*Ferula assa-foetida*). The plants were incubated in a 28°C light incubator, with regular watering to maintain a soil moisture content of 20%. Cucumber growth was observed after 10 days. Root length, plant height, and fresh weight were measured using a balance and a measuring tape. The results are shown in Table 2.

[0068] Table 2 shows that cucumbers in phenolic acid soil inoculated with Prescottella sp. DCR-T3 exhibited significantly better growth indicators, including root length, plant height, and fresh weight, compared to cucumbers in uninoculated soil. Root length increased by 32.8%, plant height by 22.6%, and fresh weight by 41.0%. The experiment demonstrates that inoculation with DCR-T3 in cinnamic acid-rich soil can significantly improve cucumber seedling growth and has a good growth-promoting effect.

[0069] Table 2. Effects of inoculated strains on soil-grown cucumbers (average values)

[0070] Prescottella sp. DCR-T3 strain not introduced 6.4 5.3 0.39 Inoculation with Prescottella sp. DCR-T3 strain 8.5 6.5 0.55 Increase the percentage (%) 32.8 22.6 41.0

[0071] Example 4: Production of microbial inoculants using the above-mentioned Prescottella sp. DCR-T3

[0072] Step 1: Culture the Prescottella sp. DCR-T3 strain (original strain) on LB medium (components as follows: yeast extract 5g / L, peptone 10g / L, NaCl 20g / L, agar powder 20g / L, pH 7.0-7.2) plates for later use.

[0073] Step 2: Pick a single colony from an LB agar plate and inoculate it into 100 mL of LB medium (5 g / L yeast extract, 10 g / L peptone, 20 g / L NaCl, the remainder being water, pH 7.0–7.2). Incubate at a constant temperature with shaking until the logarithmic growth phase. After centrifugation and resuspending in sterile water, directly dispense into liquid form using plastic containers or bottles, or dispense into solid form using peat adsorption bags.

[0074] As can be seen from the above embodiments, the Prescottella sp. DCR-T3 strain and its inoculant provided by this invention can improve the growth rate of plants such as cucumbers in soils where phenolic acid compounds accumulate. This invention successfully solves the problem that common crops such as cucumbers cannot grow or grow poorly in soils where phenolic acid compounds accumulate, significantly reducing the workload in production and use, resulting in low production and use costs and promoting sustainable agricultural development.

[0075] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be covered by the claims of the present invention.

Claims

1. A type of Prescottella ( Prescottella sp. DCR-T3, characterized in that, The Prescottella strain DCR-T3 was deposited on July 19, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27952.

2. The application of Prescottella DCR-T3 as described in claim 1 in the degradation of phenolic acid compounds, characterized in that, The phenolic acid compounds are cinnamic acid and / or p-hydroxybenzoic acid.

3. A microbial agent containing Prescottella DCR-T3 as described in claim 1.

4. The microbial agent according to claim 3, characterized in that, The microbial agent is produced using Prescottella DCR-T3, and the active ingredient of the microbial agent is Prescottella DCR-T3.

5. The microbial agent according to claim 4, characterized in that, The microbial agent contains more than 1 billion Prescottella DCR-T3 cells / ml.

6. The application of the microbial agent according to any one of claims 3-5 in the degradation of phenolic acid compounds, characterized in that, The phenolic acid compounds are cinnamic acid and / or p-hydroxybenzoic acid.

7. The application of the microbial agent according to claim 6 in the degradation of phenolic acid compounds, characterized in that, The microbial agent is applied to soils where cinnamic acid and / or p-hydroxybenzoic acid accumulate.

8. A method for preparing a microbial inoculant as described in any one of claims 3-5, characterized in that, The preparation method includes the following steps: Step 1: Plate culture: After activating the Prescottella DCR-T3 strain, inoculate it onto a plate to obtain the plate culture; Step 2, Erlenmeyer flask inoculation: Inoculate the plate culture from Step 1 into LB medium and culture at a constant temperature with shaking until the logarithmic growth phase to obtain the microbial inoculum.

9. The method for preparing the microbial inoculant according to claim 8, characterized in that, The LB medium contains the following components by mass percentage: 1.0% tryptone, 0.5% yeast extract, 1.0% NaCl, and the remainder is water, with a pH of 6.8–7.2.

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