A bacterium of the genus porphyromonas and its use

Abstract

The application provides a luteimonas sp. and application thereof, and belongs to the technical field of microbial engineering. The luteimonas sp. DCA-T2 (Luteimonas sp. DCA-T2) has been preserved in the China General Microbiological Culture Collection Center on July 19, 2023, and the preservation number is CGMCC No. 27950. When the luteimonas sp. DCA-T2 strain and the microbial inoculant thereof in the application are used in soil rich in ferulic acid, the ferulic acid in the soil can be degraded, the inhibition of high-concentration ferulic acid on crops such as cucumbers is significantly reduced, and the biomass of cucumbers and other plants is increased. The luteimonas sp. DCA-T2 strain and the microbial inoculant thereof can be used to solve the problem of crop yield reduction caused by excessive accumulation of phenolic acid compounds represented by ferulic acid in the soil.

Description

Technical Field

[0001] This application belongs to the field of microbial engineering technology, and relates to a strain of *Xanthomonas camara* DCA-T2, as well as to a microbial agent produced using *Xanthomonas camara* DCA-T2, a method for preparing the microbial agent, and the application of *Xanthomonas camara* DCA-T2 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, widely present in the environment. Continuous cropping can lead to severe accumulation of certain single types of phenolic acids in the soil. High concentrations of single types 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] Environmental bioremediation is a technology that utilizes the life activities of microorganisms to repair damaged environments. It boasts advantages such as high remediation efficiency, low cost, and no drug residues or secondary 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 acids in the soil. Isolating highly efficient phenolic acid-degrading strains from the environment and preparing them into microbial agents for direct application to contaminated soil can remove key phenolic acid compounds, solving the problem of continuous cropping obstacles and promoting sustainable agricultural development. Summary of the Invention

[0004] This invention primarily involves screening bacteria from soil capable of degrading phenolic acid compounds, aiming to degrade these compounds and thus promote plant growth. The *Xanthomonas glaucus* strain and its microbial inoculant of this invention are simple to use, low in cost, and can be applied to soils containing phenolic acid compounds. The strains can degrade phenolic acid compounds in the soil, significantly promoting the germination rate and biomass of plants such as cucumbers, thereby promoting the growth of these plants.

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

[0006] On the one hand, the present invention provides a strain of *C. sarcodactylus* DCA-T2, which was deposited on July 19, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27950.

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

[0008] Preferably, the *Xanthomonas glaucus* strain DCA-T2 is applied to soil in which phenolic acid compounds have accumulated, meaning soil containing phenolic acid compounds. *Xanthomonas glaucus* strain DCA-T2 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 *Xanthomonas camara* DCA-T2, and the active ingredient of the microbial agent is *Xanthomonas camara* DCA-T2.

[0011] Preferably, the microbial agent contains more than 1 billion CFU / ml of the aforementioned *Cyclocarya paliurus* DCA-T2.

[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] Preferably, the phenolic acid compound is ferulic acid.

[0015] In another aspect, the present invention provides a method for preparing the microbial agent, the method comprising the following steps:

[0016] Step 1: Plate culture: After activating the *Gastrodinium glabra* DCA-T2 strain, inoculate it onto a plate to obtain the plate culture.

[0017] 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.

[0018] 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.

[0019] The present invention has the following beneficial effects:

[0020] 1. The *Gynostemma pentaphyllum* DCA-T2 and its microbial agent provided by this invention can be applied to soil containing phenolic acid compounds. The strain can degrade phenolic acid compounds in the soil, significantly promoting the germination rate and biomass of plants such as cucumber, thereby promoting the growth of plants such as cucumber.

[0021] 2. Using the *Gynostemma pentaphyllum* DCA-T2 and its microbial inoculant of this invention can expand the planting range of cucumbers and increase crop yield;

[0022] 3. This invention provides a microbial inoculant containing *Gastrodinium glabra* DCA-T2, which can be used in soils rich in phenolic compounds to degrade phenolic compounds in the soil and thus promote plant growth. It can also be used as a microbial fertilizer in farmland rich in phenolic compounds to improve the seed germination rate, biomass, or crop yield.

[0023] 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

[0024] 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:

[0025] Figure 1 The growth curve of ferulic acid degradation by *Gastromonas vaginalis* strain DCA-T2 is shown.

[0026] Figure 2 The bar chart shows the degradation rates of different phenolic acid compounds by the *Glaucoma glucosinolates* strain DCA-T2. Detailed Implementation

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Preservation information:

[0032] The *Luteimonas* strain DCA-T2 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. 27950; deposit date July 19, 2023.

[0033] The *Gynostemma pentaphyllum* strain DCA-T2 of the present invention has the following properties:

[0034] 1. Morphological characteristics: The bacteria are rod-shaped.

[0035] 2. Physiological and biochemical characteristics: Gram staining reaction negative, oxidase and catalase positive; after 3 days of culture in LB medium, it forms 0.5 mm in diameter, opaque, glossy, smooth and moist, with raised pale yellow colonies.

[0036] 3.16S rRNA sequence identification

[0037] 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 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 DCA-T2 showed the highest homology (over 99%) with the sequence of *Luteimonas* strains.

[0038] This invention primarily involves screening bacteria from rhizosphere soil capable of degrading phenolic acid compounds, aiming to degrade these compounds and thus promote plant growth. The *Gastrodinium sarmentosum* DCA-T2 and its microbial inoculant provided by this invention can grow in culture media containing 200 mg / L of phenolic acid (ferulic acid) in inorganic salts, and can significantly improve the germination rate of cucumber seeds in soil containing 250 mg / kg of phenolic acid compounds. Adding this inoculant to soil containing 250 mg / kg of phenolic acid compounds can significantly improve the germination rate of crops such as cucumbers and significantly increase crop biomass, thereby promoting plant growth.

[0039] The specific preparation steps for *Vibrio vulnificus* DCA-T2 are as follows:

[0040] Potato rhizosphere soil samples were collected. 1 g of pure potato rhizosphere soil was added to an inorganic salt medium containing 100 mg / L ferulic 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.

[0041] The present invention also provides a microbial agent produced using the above-mentioned *Gastromonas gargarizans* strain DCA-T2, the active ingredient of which is *Gastromonas gargarizans* strain DCA-T2.

[0042] The preparation method of the microbial inoculant is as follows:

[0043] 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.

[0044] In a preferred embodiment, the preparation of the microbial agent may specifically take the following steps:

[0045] S1. Plate culture: The strain DCA-T2 (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.

[0046] 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 dosage form using plastic packaging buckets or bottles, or dispense into solid dosage form using peat adsorption packaging bags.

[0047] This invention relates to *Xanthomonas cambogia* DCA-T2 and its microbial inoculant, which can degrade phenolic acid compounds in soil and thus promote cucumber growth. These inoculants have the advantages of low production and usage costs and ease of use. By degrading phenolic acid compounds in the soil, they can significantly promote cucumber growth. The strains and microbial inoculants provided by this invention can be used as microbial fertilizers in farmland where phenolic acid compounds accumulate, improving the germination rate, biomass, or crop yield in phenolic acid-rich soils. Using this inoculant can expand the planting area of ​​cucumbers and increase the total planted area.

[0048] 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.

[0049] Table 1. Information on the commercial sources of each reagent.

[0050]

[0051]

[0052] Example 1: Obtaining and identifying *Cyclocarya glaucus* DCA-T2

[0053] I. Obtaining *Cyclocarya glaucus* DCA-T2

[0054] 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 100g / L ferulic acid. The medium was incubated on 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 at 30℃ in the dark for 7 days. Single colonies grown on the LB agar plates were collected, streaked for purification, and then preserved.

[0055] II. Identification of *Cyclocarya glaucus* DCA-T2

[0056] (1) Morphological characteristics: The bacteria are rod-shaped.

[0057] (2) Physiological and biochemical characteristics: Gram staining reaction negative, oxidase and catalase positive; after 3 days of culture in LB medium, it forms 0.5 mm in diameter, opaque, shiny, smooth and moist, with raised pale yellow colonies.

[0058] (3) 16S rRNA sequence identification

[0059] 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 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. Alignment with sequences in GenBank revealed that the 16S rRNA sequence of strain DCA-T2 showed the highest homology with that of *Luteimonas* strains. Physiological and biochemical indicators were consistent with those of the standard strain of *Luteimonas*. Therefore, strain DCA-T2 was identified as *Luteimonas*.

[0060] III. Preservation of *Gastromonas vulgaris* strain DCA-T2

[0061] Based on the above identification results, the strain was confirmed to be a new strain of *C. sarcodactylum*, named *C. sarcodactylum* DCA-T2, and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27950.

[0062] Example 2: Degradation of ferulic acid by *Cyclocarya glaucus* strain DCA-T2 in inorganic salt culture medium.

[0063] DCA-T2 seed culture was inoculated at a 1% inoculation rate into inorganic salt medium containing 100 mg / L ferulic acid. The culture was then incubated at 30°C and 150 rpm with shaking. Samples with only ferulic acid and samples inoculated with the strain were used as controls, with three replicates for each group. Ferulic acid and cell concentrations in the culture medium and cell OD were measured periodically. 600 The growth curve of the strain degrading ferulic acid was determined by the value. The degradation rate of ferulic acid by the strain was calculated using the following formula:

[0064] Degradation rate = (Ferulaic acid concentration in control group - Ferulaic acid concentration in treatment group) / Ferulaic acid concentration in control group × 100%.

[0065] Figure 1 The growth curve of ferulic acid degradation by *Gastrodinium glabra* strain DCA-T2 is shown. Figure 1 As shown, in the experimental group, the concentration of ferulic acid decreased slowly during the period of 0-32h; the concentration of ferulic acid decreased rapidly during the period of 32-52h; at 64h, ferulic acid was almost completely degraded, and then the concentration of ferulic acid remained basically stable. At 72h, the degradation rate of ferulic acid was 97.57%, and the degradation amount reached the maximum.

[0066] Example 3: Degradation of other phenolic acid compounds by *Glaucoma glabra* strain DCA-T2 in inorganic salt culture medium.

[0067] p-hydroxybenzoic acid, coumaric acid, and syringic acid, each at a final concentration of 100 mg / L, were added as the sole carbon source to different inorganic salt culture media. Pre-prepared DCA-T2 seed culture was added to these liquid media at an inoculum rate of 1%. Samples containing these compounds but without the strain were used as a control group, with three replicates per group. The samples were cultured in a constant temperature shaker at 30℃ and 150 rpm for 46 h. The concentrations of various compounds in the culture medium were measured to analyze the ability of the strain to degrade other phenolic acid compounds. The degradation rate of phenolic acid compounds by the strain was calculated using the following formula:

[0068] Degradation rate = (Concentration of compound in control group - Concentration of compound in treatment group) / Concentration of compound in control group × 100%.

[0069] Figure 2 A bar chart showing the degradation rates of different phenolic acid compounds by *Glaucoma glucosinolates* strain DCA-T2 is presented. Figure 2 As shown, the *Gastrodinella lucida* strain DCA-T2 did not show significant degradation effects on syringic acid and coumaric acid, but it could degrade p-hydroxybenzoic acid. After 46 hours of cultivation, the degradation rate of p-hydroxybenzoic acid was 85.62%.

[0070] Example 4: Inoculating cucumber seedlings with strain DCA-T2 in soil containing high concentrations of phenolic acids to increase biomass.

[0071] Single colonies of strain DCA-T2 were picked and cultured in 3 mL of LB broth at 30°C and 150 rpm for 24 h with shaking to obtain fresh bacterial suspension. After centrifugation, the suspension was resuspended in sterile water to obtain the bacterial agent, and the OD was adjusted. 600 A control group was prepared by adding a fungicide to soil with accumulated phenolic acid, while the soil without the fungicide served as the control. Uniformly sized cucumber seeds were divided into two groups: one group was planted in soil with added ferulic acid, and the other group was planted in soil without the fungicide. The plants were placed in a 28℃ light incubator, and watered regularly to maintain a soil moisture content of 20%. The growth of the cucumbers was observed after 10 days. Root length, plant height, and fresh weight were measured using a balance and a measuring tape, and the average values ​​were recorded. The results are shown in Table 2.

[0072] Table 2 shows that the growth (including root length, plant height, and fresh weight) of cucumber seedlings in phenolic acid soil inoculated with *Fructus glaucus* DCA-T2 was significantly better than that of the uninoculated samples. Root length increased by 32.7%, plant height by 31.5%, and fresh weight by 31.4%. The experiment demonstrates that inoculation with *Fructus glaucus* DCA-T2 in soil rich in ferulic acid can significantly improve the growth of cucumber seedlings and has a good growth-promoting effect.

[0073] Table 2. Effects of inoculated strains on soil-grown cucumbers

[0074] Uninoculated strain DCA-T2 5.2 5.0 0.35 Inoculated strain DCA-T2 6.9 6.7 0.46 Increase the percentage (%) 32.7 31.5 31.4

[0075] Example 5: Production of microbial inoculants using *Cyclocarya pallida* DCA-T2

[0076] Step 1: Culture the original strain of *Gastrodinium glabra* DCA-T2 on LB agar plates (composition as follows: yeast extract 5g / L, peptone 10g / L, NaCl 20g / L, agar powder 20g / L, pH 7.0-7.2) for later use.

[0077] 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.

[0078] As can be seen from the above embodiments, the *Gynostemma pentaphyllum* DCA-T2 and its microbial 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, lowering production and use costs, and promoting sustainable agricultural development.

[0079] 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 *Gynostemma pentaphyllum* ( Luteimonas sp. DCA-T2, characterized in that, The *Glaucoma glabra* DCA-T2 strain was deposited on July 19, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 27950.

2. The application of *Gynostemma pentaphyllum* as described in claim 1 in the degradation of phenolic acid compounds, characterized in that, The phenolic acid compounds are ferulic acid and / or p-hydroxybenzoic acid.

3. A microbial inoculant containing *Gastrodinium glabra* as described in claim 1.

4. The microbial agent according to claim 3, characterized in that, The microbial agent is produced using *Gastromonas vulgaris* DCA-T2, and the active ingredient of the microbial agent is *Gastromonas vulgaris* DCA-T2.

5. The microbial agent according to claim 4, characterized in that, The microbial agent contains more than 1 billion CFU / ml of the aforementioned *Gynostemma pentaphyllum* DCA-T2.

6. The application of the microbial inoculant according to any one of claims 3-5 in degrading phenolic acid compounds to improve crop growth, characterized in that, The phenolic acid compounds are ferulic 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 ferulic acid and / or p-hydroxybenzoic acid accumulate.

8. A method for preparing a microbial inoculant according to any one of claims 3-5, characterized in that, The preparation method includes the following steps: Step 1: Plate culture: After activating the *Gastrodinium glabra* DCA-T2 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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