Pseudomonas putida RT12 and application thereof

Abstract

The invention relates to a new saccharomycetes, in particular to a pseudomonas putida RT12 strain and an application of the pseudomonas putida RT12 strain in degradation of juglone in walnut green seedcases. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 19, 2025, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.36686. A strain of Pseudomonas putida RT12 is obtained through separation, screening and identification in Aksu Red-flag slope region in Xinjiang, and experiments on growth characteristics, physiology and biochemistry, ester production capability and tolerance of the strain verify that the strain has the activity of efficiently degrading juglone, which indicates that the Pseudomonas putida RT12 has a good application prospect, and can be used for preparing a high-efficiency degradation product of juglone. The method can be widely applied to the agricultural solid waste resource treatment industry.

Description

Technical Field

[0001] This invention relates to the technical field of microbial strains and their applications, specifically to a novel strain of *Pseudomonas putida* (…). Pseudomonas putida RT12 and its application in agricultural solid waste resource treatment. Background Technology

[0002] Walnut husks (the green outer skin of the walnut fruit), a major byproduct of initial walnut processing, present a significant challenge to the sustainable development of the walnut industry. The annual production of walnut husks is enormous; in Xinjiang alone, over 5.78 million tons of husk waste are generated annually. Traditional disposal methods primarily involve indiscriminate dumping. However, the allelochemicals in the husks, such as juglone, release toxic components during natural decomposition, polluting soil and water bodies, and even inhibiting crop growth or causing fish mortality. This problem is particularly severe in walnut-producing areas of Xinjiang, such as Hotan, Kashgar, and Aksu.

[0003] Juglone (5-hydroxy-1,4-naphthoquinone) is a naturally occurring quinone compound widely found in plants of the Juglandaceae family. It is an important secondary metabolite in plants such as black walnut (Juglans nigra), white walnut (Juglans scinerea), and walnut (Juglans regia). It is primarily stored in plants as a glucosinolate, but is released into the surrounding environment in a free state when plant tissues are damaged or decomposed. Juglone exhibits strong chemical activity and biotoxicity, reacting with various cellular components in redox reactions. It has significant inhibitory effects on plant, microbial, and animal cells, making it a typical allelopathic compound.

[0004] Juglone acts as a competitive factor in plants in nature, inhibiting seed germination and root growth of surrounding plants and helping Juglandaceae plants form allelopathic zones. However, this allelopathic effect brings new problems in artificial environments. With the expansion of walnut planting areas, timber processing, the stockpiling of walnut shells and leaves, and the production of industrial extracts, juglone has gradually accumulated in agricultural ecosystems and surrounding environments. Juglone is persistent and lipid-soluble, easily adsorbed onto the surface of soil organic matter and particles, and is not easily degraded naturally. In localized areas, it can cause soil microecological imbalance, decreased microbial diversity, and plant growth disorders. For water bodies, juglone can enter surface water or groundwater through runoff or infiltration, posing a potential toxic effect on aquatic organisms. Therefore, juglone is not only a plant-derived ecotoxin but also a pollutant that urgently needs to be addressed in the environmental remediation of certain areas.

[0005] Currently, the treatment of juglone pollution mainly focuses on physical and chemical methods. Physical adsorption methods, using materials such as activated carbon, bentonite, and biochar, can reduce juglone concentration to some extent, but their adsorption capacity is limited, and the regeneration process of these materials is complex and prone to secondary pollution. Chemical oxidation methods utilize strong oxidants such as ozone, persulfate, and Fenton's reagent to decompose juglone. While the degradation rate is relatively fast, the process conditions are harsh, the cost is high, and it may generate intermediate products with unknown toxicity. Furthermore, photocatalytic degradation technologies (such as TiO2 photocatalysis) have a certain removal effect on juglone, but their efficiency drops significantly in environments with low natural light intensity or turbid media, making large-scale application difficult. Therefore, existing physicochemical methods have significant shortcomings in terms of cost, operating conditions, environmental compatibility, and secondary pollution control.

[0006] In contrast, biodegradation methods offer advantages such as environmental friendliness, low energy consumption, and thorough treatment, making them a hot research topic in recent years. Some microorganisms in the natural environment possess the ability to metabolize quinones, converting them into low-toxicity or non-toxic products through reactions such as oxidation-reduction, hydroxylation, and decarboxylation. Previous studies have shown that some fungi (such as white-rot fungi and Aspergillus niger) and bacteria (such as Bacillus and Rhodococcus) can degrade naphthoquinones. However, specific and highly efficient degrading strains targeting juglone are still rarely reported, especially in complex environmental systems where microbial resources capable of stable degradation under neutral or weakly alkaline conditions are extremely limited.

[0007] Pseudomonas are a group of Gram-negative bacteria with extremely high metabolic diversity, playing an important role in environmental bioremediation. Many Pseudomonas strains can decompose recalcitrant organic pollutants such as polycyclic aromatic hydrocarbons, phenols, ketones, and quinones, possessing strong oxidoreductase systems and adaptive metabolic networks. Their extracellular and intracellular enzyme systems (such as monooxygenases, dioxygenases, and quinone reductases) can catalyze ring-opening and detoxification reactions of various aromatic compounds. Furthermore, Pseudomonas are widely distributed in water bodies, soil, and plant rhizospheres, requiring simple cultivation conditions and exhibiting rapid growth, making them suitable for industrial and field applications. Therefore, screening for Pseudomonas strains with highly efficient juglone degradation capabilities and establishing stable and controllable biodegradation methods will provide a novel and sustainable biotechnological approach for the control of juglone pollution. Summary of the Invention

[0008] In view of the current situation where traditional physicochemical treatment methods for walnut green husks, such as high-temperature incineration, easily generate secondary pollutants, this invention aims to provide a *Pseudomonas putida* strain, specifically involving a novel *Pseudomonas putida* strain and its application. This novel strain was isolated and screened from soil samples from the Hongqipo area of ​​Aksu, Xinjiang. Pseudomonas putidaRT12 was validated through hemolysis experiments, functional enzyme experiments, ester production capacity, and tolerance. The bacterium exhibits non-hemolytic properties, low or no H2S production, and good resistance to high temperatures, high sugars, and low pH. This bacterium is a novel *Pseudomonas putida* strain. Pseudomonas putida The colonies of RT12 in the ester-producing medium are bright yellow, indicating that the new strain RT12 provided by this invention has good ester-producing performance and tolerance characteristics, and can be applied to the walnut green skin processing industry, showing good application prospects.

[0009] This application aims to utilize microbial technology to decompose and utilize juglone, eliminating the allelopathic effects and hazards of juglone within the walnut husk during the on-site harmless treatment of walnut husks. By researching and improving composting methods, the potentially harmful husks can be transformed into usable bio-fertilizers for agriculture, thereby protecting the environment, implementing ecological agriculture and a circular economy, and increasing the economic value of walnut byproducts. The core purpose of microbial decomposition of juglone is environmental risk control. Juglone has high chemical stability, and traditional physicochemical treatment methods (such as high-temperature incineration) easily generate secondary pollutants. Microbial decomposition, through enzymatic reactions, degrades juglone into non-toxic or low-toxic products (such as carbon dioxide and water), significantly reducing environmental toxicity. Specific strains can destroy the quinone ring structure of juglone through redox pathways. Microbial decomposition not only eliminates the toxicity of juglone but also transforms the organic matter in walnut husks into high-value-added products. In terms of bio-fertilizers: through composting, the husk residue after microbial decomposition is rich in humic acid and nitrogen, phosphorus, and potassium elements, which can replace chemical fertilizers and improve soil structure. Microbial treatment of juglone offers high cost-effectiveness; compared to traditional processes such as solvent extraction and chromatographic purification, microbial treatment is low-cost, energy-efficient, and requires no complex equipment. By screening for highly efficient degrading strains or constructing complex microbial communities, the treatment cycle can be shortened and product yield increased, without the need for large machinery and complex equipment.

[0010] Specifically, the present invention provides the following technical solutions:

[0011] This invention provides a strain of *Pseudomonas putida* RT12, which was deposited on November 19, 2025, 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, with accession number CGMCC No. 36686.

[0012] The *Pseudomonas putida* RT12 provided by this invention was isolated from a natural compost sample of green walnut husks collected in Hongqipo area, Aksu, Xinjiang. Phylogenetic and morphological analyses of its 16S rRNA gene sequence confirmed that strain RT12 belongs to *Pseudomonas putida*. Gene sequencing of this strain was performed, and the resulting sequences were compared using BLAST on the NCBI website. The 16S rRNA gene sequence of strain RT12 was consistent with that of standard strains of the same genus. Pseudomonas sp. The highest similarity (99.58%) was found with BR9109. A phylogenetic tree of 16S rRNA gene sequences was constructed using sequences with high homology. Strains RT12 and... Pseudomonas putida LZH-X8 Closer kinship groups clustered on a single branch. After polymorphic taxonomic identification, strain RT12 and... Pseudomonas sp. BR9109 and Pseudomonas putida LZH-X8 The strain Pseudomonas putida RT12 is clearly different from other strains and can be identified as a new species, exhibiting the typical characteristics of a new species.

[0013] The strain *Pseudomonas putida* RT12 provided by this invention has been identified at the molecular level by the well-known and recognized bacterial strain system, combined with identification and analysis based on morphological and physiological and biochemical characteristics. The colony morphology of *Pseudomonas putida* RT12 on LB solid medium is round, with neat edges, rough, flat surface, and opaque white color.

[0014] The above-mentioned strain identification, verified by well-known and recognized molecular-level identification and physiological and biochemical systematic tests, confirms that the obtained strain, numbered RT12, belongs to a typical new strain, *Pseudomonas putida* RT12. This strain was deposited prior to the application date at the Budapest Treaty International Collection Unit for Microbiology: China General Microbiological Culture Collection Center (CGMCC). Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China; Accession Number: CGMCC No. 36686; Deposit Date: November 19, 2025.

[0015] The 16S rRNA gene sequence of the above-mentioned strain Pseudomonas putida RT12 is shown in SEQ ID NO: 1.

[0016] The isolation medium for the strain Pseudomonas putida RT12 described in this invention is: PDA medium and juglone selection medium.

[0017] The juglone culture medium formula is as follows: 750 μg / L potassium dihydrogen phosphate, 480 μg / L disodium hydrogen phosphate, 40 μg / L ammonium chloride, 10 μg / L magnesium sulfate, 10 μg / L calcium chloride, 0.2 μg / L ferric chloride, and 15 μg / L juglone.

[0018] Meanwhile, the present invention also provides a microbial agent comprising Pseudomonas putida RT12 and its fermentation culture, which can be a liquid or solid microbial agent, and can be prepared by conventional technical means and by adding carriers or other excipients permitted in the field of microbial preparations.

[0019] Meanwhile, this application also provides a walnut green husk fermentation agent containing the aforementioned *Pseudomonas putida* (…). Pseudomonas putrid RT12, its fermentation culture can be a liquid inoculum or a solid inoculum, and can be prepared by conventional technical means and by adding carriers or other excipients permitted in the field of microbial preparations.

[0020] Furthermore, this application provides a juglone degradation agent comprising the aforementioned *Pseudomonas putida*. (Pseudomonas putida RT12, its fermentation culture can be a liquid inoculum or a solid inoculum, and can be prepared by conventional technical means and by adding carriers or other excipients permitted in the field of microbial preparations.

[0021] Furthermore, this application also provides the aforementioned *Pseudomonas putida* (…). Pseudomonas putida RT12, the application of its fermentation products or fermentation agents in the degradation fermentation of juglone.

[0022] By implementing the specific technical solutions provided by this invention, the following beneficial effects can be obtained: This invention provides a strain of *Pseudomonas putida* RT12, which has been systematically identified and is therefore required to be deposited in accordance with legal requirements. This strain of *Pseudomonas putida* RT12 was obtained through isolation, screening, and identification from the Hongqipo area of ​​Aksu, Xinjiang. Pseudomonas putrid RT12, through experiments verifying its growth characteristics, physiological and biochemical properties, ester production capacity, and tolerance, demonstrated that the strain possesses excellent fermentation characteristics and highly efficient activity in degrading juglone. Its fermentation broth, with an OD600 of 0.6–2, achieved a juglone degradation efficiency exceeding 85%, significantly higher than existing juglone-degrading bacteria such as Bacillus subtilis. This indicates that *Pseudomonas putida* (…) Pseudomonas putidaRT12 has good application prospects and can be widely used in the agricultural solid waste resource treatment industry. Attached Figure Description

[0023] Figure 1 The image shows *Pseudomonas putida* (…). Pseudomonas putida The phylogenetic tree of RT12 is constructed based on the 16S rRNA gene sequence.

[0024] Figure 2 The image shows *Pseudomonas putida* (…). Pseudomonas putida RT12 colony morphology characteristics.

[0025] Figure 3 The image shows *Pseudomonas putida* (…). Pseudomonas putida RT12 growth curve.

[0026] Figure 4 The figure shows the effect of culture temperature on *Pseudomonas putida* (…). Pseudomonas putida ) RT12 degradation capability verification.

[0027] Figure 5 The figure shows the constructed standard curve for juglone.

[0028] Figure 6 The image shows a comparison of the degradation effects of juglone. Detailed Implementation

[0029] The present invention will now be illustrated with examples; however, the present invention is not limited to the examples described below. All raw and auxiliary materials used in the present invention, as well as the selected microbial culture methods, are well known in the art. All percentages mentioned in the present invention are by weight and volume percentages unless otherwise specified.

[0030] To better explain the present invention, the main contents of the invention are further illustrated below with reference to specific embodiments, but the content of the present invention is not limited to the following embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0031] The PDA and LB media used in this invention are both conventional media.

[0032] The juglone culture medium formula of the present invention is: 750 μg / L potassium dihydrogen phosphate, 480 μg / L disodium hydrogen phosphate, 40 μg / L ammonium chloride, 10 μg / L magnesium sulfate, 10 μg / L calcium chloride, 0.2 μg / L ferric chloride, and 15 μg / L juglone.

[0033] The Bacillus subtilis used in this application ( Bacillus subtilisThis is a common strain, which can be obtained by the general public through the China General Microbiological Culture Collection Center (CGMCC) or biotechnology companies.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0035] In this invention, the term "Pseudomonas putida" in the following embodiments refers to... Pseudomonas putida "RT12" is abbreviated as "strain RT12".

[0036] Example 1: Isolation, purification and identification of strain RT12 (1) Acquisition of strains 1. Samples of naturally decayed walnut husks were collected from five locations in the Hongqipo area of ​​Aksu, Xinjiang.

[0037] 2. Divide the samples into three groups: left, middle, and right. Take a large number of samples from each group and place them in a weighing pan.

[0038] 3. Sample detoxification pretreatment: The naturally decayed walnut samples taken from Aksu, Xinjiang were pretreated by soaking in 0.6% sodium hypochlorite for two minutes, rinsing with sterile water, and drying for later use.

[0039] 4. Weigh 5 g of dried sample from each of the three sampling sites and add it to a 50 ml centrifuge tube. Add 10 mL of 1×PBS and 10 sterile glass beads to the tube and vortex for 7 to 8 minutes to evenly disperse the microorganisms in the PBS buffer.

[0040] 5. Perform serial dilution in a clean bench, using a pipette to draw 10... - ² ~ 10 -4 100 μL of the treatment sample was added to PDA medium and juglone screening medium, respectively, and spread evenly with a spreader. Each concentration was repeated three times.

[0041] 6. Select single colonies from the screening medium, pick out single colonies, spot them in LB medium in different areas, and incubate them in a 30℃ incubator.

[0042] (2) Identification of strains The 16S rRNA gene sequencing analysis of strain RT12 is as follows: Genomic DNA was extracted from strain RT12 and used as a template. The 16S rRNA gene fragment was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). The PCR reaction mixture consisted of 1 μL template DNA, 1 μL primer 27F, 1 μL primer 1492R, 12.5 μL 2×Taq Mixture, and 9.5 μL ddH2O. The PCR program was as follows: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, 30 cycles, and 72℃ for 5 min. The amplified product was detected by 1% agarose gel electrophoresis, purified, and sequenced. The sequencing results are shown in SEQ ID No. 1.

[0043] 3. Sequencing BLAST homology sequence retrieval was performed using NCBI, and a phylogenetic tree was constructed using the Neighbor-Joining method (with 1000 repeated samplings) with MEGA 7.0 software, a commonly used tool in this field. The results are attached. Figure 1 As shown, the obtained sequences were compared and analyzed on the NCBI website. Phylogenetic and morphological analyses of the 16S rRNA gene sequence revealed that strain RT12 belongs to *Pseudomonas putida*. Gene sequencing of this strain was performed, and BLAST alignment analysis on the NCBI website showed that the 16S rRNA gene sequence of strain RT12 was consistent with that of standard strains of the genus. Pseudomonas sp. The highest similarity (99.58%) was found with BR9109. A phylogenetic tree of 16S rRNA gene sequences was constructed using sequences with high homology. Strains RT12 and... Pseudomonas putida LZH-X8 Closer kinship groups clustered on a single branch. After polymorphic taxonomic identification, strain RT12 and... Pseudomonas sp. BR9109 and Pseudomonas putida LZH-X8 The strain exhibits significant differences, confirming that *Pseudomonas putida* RT12 is a novel species, possessing the typical characteristics of a new species. Through a comprehensive assessment of similarity and homology, it aligns with the conclusions of molecular-level typicality analysis for identifying new species in this field, confirming that the obtained strain, numbered RT12, belongs to a typical new species within the genus *Pseudomonas*.

[0044] Example 2: Physiological and biochemical characteristics of strain RT12 (I) Colony morphology characteristics of strain RT12 After inoculating strain RT12 onto LB solid medium and incubating at 30°C for 24 hours, round, smooth-edged, flat, opaque white colonies were observed. (See Appendix) Figure 2 As shown.

[0045] (II) Physiological and biochemical characteristics of strain RT12 The physiological and biochemical tests of strain RT12 were mainly conducted according to the "Handbook of Systematic Identification of Common Bacteria". This strain produces a clear hydrolysis zone on protein solid culture medium, and the catalase test result is positive. Strain RT12 does not produce a clear zone on solid plates containing cellulose, starch, inorganic phosphorus, or nitrogen; therefore, strain RT12 cannot secrete cellulase or amylase, cannot decompose inorganic phosphorus, and does not possess nitrogen-fixing ability.

[0046] Table 1: Results of physiological and biochemical characteristics of strain RT12

[0047] Based on the comprehensive analysis of 16S rDNA gene sequence homology, morphological identification, and physiological and biochemical characteristics provided in Example 1, this strain RT12 is significantly different from the standard type strain, consistent with the conclusions of typical analysis for identifying new bacterial species in this field. Therefore, this strain RT12 is identified as a *Pseudomonas putida*. Pseudomonas putida A new species of bacteria in ) possesses the characteristics of *Pseudomonas putida* ( Pseudomonas putida Significant characteristics of new species within the same genus.

[0048] Based on the above biological characteristics, strain RT12 was identified as *Pseudomonas putida*. Pseudomonas putrid ) 。 This strain was deposited prior to the application date at the Budapest Treaty International Collection Unit for Microbial Cultures: China General Microbiological Culture Collection Center (CGMCC). Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. Accession number: CGMCC No. 36686. Deposit date: November 19, 2025.

[0049] Example 3: Growth Curve Measurement 1. Activation of microbial strains and preparation of seed culture Activation: Take one tube of RT12 strain from the slant or glycerol tube, streak it onto an LB agar plate, and incubate upside down at 30°C (or the optimal temperature for this strain) for 24-48 hours.

[0050] Seed culture preparation: Pick a single colony and inoculate it into an Erlenmeyer flask containing 50 mL of LB liquid medium.

[0051] Pre-culture: Place the Erlenmeyer flask in a constant temperature shaker and culture at 30°C and 150 rpm until the late logarithmic growth stage (usually about 12-16 hours, OD600 ≈ 0.8-1.0). This step is crucial to ensure that the inoculum is in a vigorous and synchronous growth state.

[0052] 2. Preparation and grouping of experimental culture media Basic culture medium: Prepare no less than 300 mL of inorganic salt culture medium and juglone screening medium, and autoclave (121℃, 20 minutes).

[0053] Grouping: In a laminar flow hood, dispense the sterilized basal culture medium into multiple 100 mL sterile Erlenmeyer flasks, 50 mL per flask. Then add the filtered and sterilized juglone stock solution according to the following grouping: Experimental group: Juglone culture group Positive control group: LB culture group Negative control group: Culture medium containing juglone (e.g., 50 mg / L), without bacterial inoculation, used to monitor the non-biodegradation of juglone and background values.

[0054] Blank control: Only sterile inorganic salt culture medium is used for zeroing the spectrophotometer.

[0055] 3. Inoculation and zero-time sampling (t=0) Take an appropriate amount of seed culture and, under aseptic conditions, add the above bacterial suspension to each experimental group and positive control group Erlenmeyer flask at an inoculation rate of 1% (v / v). For example, add 0.5 mL of bacterial suspension to 50 mL of culture medium.

[0056] Zero-hour sampling: Immediately after inoculation, transfer 2 mL of culture from each Erlenmeyer flask into a sterile centrifuge tube and label it as 0 hours. This sample is used to determine the initial OD600.

[0057] 4. Cultivation and periodic sampling Culture: All inoculated Erlenmeyer flasks and negative control flasks were placed in a constant temperature shaker and cultured at 30℃ and 150 rpm.

[0058] Sampling: At time points of 0, 2, 4, 6, 8, 12, 24, 36, and 48 after incubation (sampling intervals are short in the early stages and can be appropriately extended in the later stages), 2 mL of culture is aseptically taken from each Erlenmeyer flask.

[0059] Measurement: Vortex the extracted sample to mix thoroughly, pour it into a cuvette with a light path of 1 cm, zero the instrument with blank control medium, and measure the OD600 value. Record the data immediately. If the sample turbidity is too high (OD600>0.8), it needs to be appropriately diluted with sterile inorganic salt medium before measurement, and the result should be multiplied by the dilution factor when recording the data.

[0060] See the appendix for the measurement results. Figure 3 As shown, this indicates that the RT12 strain has good fermentation characteristics.

[0061] Example 4: Verification of the strain's degradation ability 1. Verification Experiment One Prepare the juglone culture medium needed for the experiment, and dispense it into ten 200ml Erlenmeyer flasks (protected from light by wrapping with aluminum foil). Record the color of the culture medium before the reaction. Juglone is unstable under light, therefore, during the experiment, the Erlenmeyer flasks containing the culture medium must be wrapped with aluminum foil to maintain a light-protected environment. The specific formula is as follows: Juglone culture medium formula: 750 μg / L potassium dihydrogen phosphate, 480 μg / L disodium hydrogen phosphate, 40 μg / L ammonium chloride, 10 μg / L magnesium sulfate, 10 μg / L calcium chloride, 0.2 μg / L ferric chloride, 15 μg / L juglone.

[0062] The RT12 strain was activated on LB plates and cultured at 30°C for 24 hours. It was then inoculated into liquid LB medium for expansion culture. An appropriate amount of seed culture was centrifuged at 4000 rpm for 5 minutes under aseptic conditions, and the supernatant was discarded. The purpose was to remove residual carbon sources in the LB medium.

[0063] Washing: Resuspend the bacterial cells in sterile inorganic salt medium, centrifuge, and repeat this step 2-3 times to wash thoroughly. Resuspend the washed bacterial cells in sterile inorganic salt medium and adjust the OD600 to 0.1 ± 0.01 as the inoculum stock solution.

[0064] Inoculation: Add the above bacterial suspension to the experimental group culture medium at an inoculation rate of 1% (v / v). Add 1 mL of bacterial suspension to 100 mL of culture medium. The control group was treated with 100 mL of juglone culture medium.

[0065] Cultivation: The culture medium containing the inoculated bacterial solution was incubated at 30℃ and 200 rpm in a shaker for 5-7 days. After 7 days, observation and testing were performed. The detection indicators were based on the Bornträger reaction. A variant of the Bornträger reaction is a classic chemical colorimetric reaction used to detect and identify hydroxyanthraquinone compounds. Its basic principle is that hydroxyanthraquinones exhibit a specific color change under alkaline conditions, typically from yellow or colorless to red or reddish-purple. This method can be used to detect the hydroxysubstituted quinone structure in naphthoquinone compounds, thereby determining the content of juglone.

[0066] Reaction steps: Place approximately 15 ml of juglone liquid culture medium in a test tube. Add 2 mL of 5% NaOH aqueous solution and shake well to dissolve. Heat in a boiling water bath for 2-3 minutes. Observe the color change. Observation: The solution gradually changes from yellow to reddish-brown or orange-red.

[0067] The results of the colorimetric reaction are shown in the appendix. Figure 4 As shown, juglone undergoes a color change after the addition of sodium hydroxide. In the control group, it first turns blue-purple and then orange-red after being heated in a water bath. The experimental groups showed no color reaction, indicating that the selected strain successfully decomposed and utilized juglone.

[0068] 2. Verification Experiment Two (1) Preparation of Juglone Standard Solution Preparation of stock solution: Accurately weigh 10 mg of juglone standard, dissolve it in ethanol or methanol and dilute to 10 mL to obtain a stock solution of 1 mg / mL.

[0069] Gradient dilution: The mother liquor was successively diluted with deionized water to obtain a series of standard solutions with concentrations of 0, 2, 3, 4, 5, 7.5, and 10 μg / mL.

[0070] (2) Color reaction: Prepare a 10 μg / mL juglone standard solution and dilute it to 2, 3, 4, 5, 7.5, and 10 μg / mL sample solutions, respectively. Label the solutions and add 2 mL of 5% NaOH solution to each sample solution. After color development, add the solution to a 75℃ water bath for 3 min. Once the solution turns orange-yellow, remove it and store it in the dark. Observe the absorbance at 412 nm to construct a standard curve.

[0071] (3) Absorbance measurement Blank control: A reagent mixture without juglone was used as a blank.

[0072] Measurement parameters: Wavelength: 412 nm (4) Plotting the standard curve Record the absorbance values ​​of the standard solutions at each concentration. Plot the concentration of juglone as the x-axis (X) and the absorbance value as the y-axis (Y), and fit the standard curve equation (e.g., Y = aX + b) using linear regression.

[0073] See appendix for standard curve. Figure 5 As shown.

[0074] Example 5: Determination of degradation efficiency of the strain after culturing in juglone medium (1) Activation and inoculation of bacterial strains The RT12 strain, frozen at -80°C, and the control Bacillus subtilis were activated and streaked onto LB agar plates. The plates were incubated at 30°C for 24 hours, then inoculated into liquid LB medium for scale-up. An appropriate amount of seed culture was centrifuged at 4000 rpm for 5 minutes under aseptic conditions, and the supernatant was discarded. The cells were resuspended in sterile inorganic salt medium, centrifuged, and this step was repeated 2-3 times, followed by thorough washing. The washed cells were then resuspended in sterile inorganic salt medium. The cells were inoculated into six juglone media and cultured, with three uninoculated juglone media serving as blank controls. The media were incubated at 30°C and 200 rpm for 7 days.

[0075] (2) Detection of juglone content in juglone culture medium Take 5 ml of culture medium from 9-juglone culture medium after 7 days of culture and centrifuge at 8000 rpm for 3 minutes. Use sterile water to zero the spectrophotometer at 412 nm. Place the supernatant in a cuvette. Repeat the measurement three times and take the mean value for calculation.

[0076] (3) Determination of strain degradation efficiency Degradation efficiency (%) = (Juglone content before reaction) Juglone content after reaction ÷ Juglone content before reaction × 100% The average residual content of juglone in the experimental group was 1.432 μg, and the degradation efficiency was 85.68%, determined using the degradation efficiency formula. See the appendix for the measurement results. Figure 6 As shown, the experimental group of the strain showed significant differences compared with the control group and the Bacillus subtilis treatment group, and the experimental group with added strain RT12 showed obvious juglone degradation ability.

[0077] 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. A strain of *Pseudomonas putida* ( Pseudomonas putida RT12, characterized in that, The strain was deposited on November 19, 2025, 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, with accession number CGMCC No. 36686.

2. The *Pseudomonas putida* strain as described in claim 1 ( Pseudomonas putida RT12, characterized in that, The 16S rRNA gene sequence is shown in SEQ ID NO:

1.

3. A type of putrid Pseudomonas ( Pseudomonas putida The isolation medium for RT12 is characterized by, The isolation medium used is either PDA medium or juglone screening medium.

4. The *Pseudomonas putida* as described in claim 3 ( Pseudomonas putida The isolation medium for RT12 is characterized by, The juglone culture medium formula is as follows: 750 μg / L potassium dihydrogen phosphate, 480 μg / L disodium hydrogen phosphate, 40 μg / L ammonium chloride, 10 μg / L magnesium sulfate, 10 μg / L calcium chloride, 0.2 μg / L ferric chloride, and 15 μg / L juglone.

5. A microbial agent, characterized in that, The microbial agent contains *Pseudomonas putida* as described in claim 1. Pseudomonas putida RT12, its fermentation culture can be a liquid inoculum or a solid inoculum, and can be prepared by conventional technical means and by adding carriers or other excipients permitted in the field of microbial preparations.

6. A walnut green husk fermentation agent, characterized in that, The fermentation agent comprises *Pseudomonas putida* as described in claim 1. Pseudomonas putida RT12, its fermentation culture can be a liquid inoculum or a solid inoculum, and can be prepared by conventional technical means and by adding carriers or other excipients permitted in the field of microbial preparations.

7. A juglone degradation agent, characterized in that, The formulation contains *Pseudomonas putida* as described in claim 1. (Pseudomonas putida RT12, its fermentation culture can be a liquid inoculum or a solid inoculum, and can be prepared by conventional technical means and by adding carriers or other excipients permitted in the field of microbial preparations.

8. A *Pseudomonas putida* strain as described in claim 1 (… Pseudomonas putida RT12, the application of its fermentation products or fermentation agents in the degradation fermentation of juglone.

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