Gordonia rubropigmentata and application thereof in relieving continuous cropping obstacles of crops

Abstract

The application provides a Gordonia rubripertincta and application thereof in relieving continuous cropping obstacles of crops, and belongs to the technical field of microorganisms.The Gordonia rubripertincta GrJYB4-4 is obtained by screening and isolating from the rhizosphere soil of continuous ginseng cropping for the first time, can efficiently degrade phenolic acid autotoxic substances generated by continuous cropping of ginseng, and the degradation rate of p-benzoic acid can reach 98.5%.Meanwhile, the Gordonia rubripertincta GrJYB4-4 has degradation abilities of different degrees to various phenolic acid autotoxic substances such as cinnamic acid, salicylic acid and p-hydroxybenzoic acid, and has a wide degradation spectrum.Under field conditions, applying the Gordonia rubripertincta GrJYB4-4 can effectively reduce the content of p-benzoic acid in the soil for continuous cropping of ginseng, and can also control the occurrence of root diseases of ginseng to a certain extent, significantly improves the survival rate, root length and other growth indexes of ginseng, and has a significant yield-increasing effect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a species of *Gordonella rubescens* and its application in alleviating crop rotation obstacles. Background Technology

[0002] Ginseng ( Panax ginseng Ginseng (CA Meyer) is a traditional and precious Chinese medicinal herb with huge market demand. However, severe continuous cropping obstacles exist in ginseng cultivation, becoming a core bottleneck restricting the sustainable development of the industry. Existing research confirms that the accumulation of phenolic acid autotoxic substances, represented by benzoic acid, produced by ginseng root secretions and plant residue decomposition, is a key inducing factor for continuous cropping obstacles. These substances directly inhibit ginseng root growth, induce oxidative stress, and enrich soil-borne pathogens, ultimately leading to reduced yield, frequent root diseases, and quality deterioration.

[0003] Currently, among the remediation technologies for ginseng continuous cropping obstacles, chemical remediation suffers from pesticide residues and secondary pollution, failing to meet the requirements of green cultivation; physical / agronomical remediation is hampered by long cycles, high costs, and unstable effects, making large-scale promotion difficult. Microbial remediation technology utilizing functional microorganisms to degrade autotoxic substances and regulate the rhizosphere microecology is a current research hotspot in green pest control. However, existing reports on benzoic acid-degrading bacteria are mostly Bacillus and Pseudomonas species, generally exhibiting low degradation efficiency, lack of systematic optimization of degradation characteristics, and unstable field effects, failing to meet the actual field application needs of major ginseng-producing areas. Summary of the Invention

[0004] The purpose of this invention is to address the current situation where existing technologies for remediating continuous cropping obstacles are insufficient to meet the practical application needs in ginseng-producing areas. This invention seeks an effective solution to reduce the content of phenolic compounds in the rhizosphere soil of ginseng, thereby alleviating continuous cropping obstacles. To this end, this invention provides a strain of *Gordonella rubrum* and its application in alleviating continuous cropping obstacles.

[0005] This invention provides a strain of *Gordonella rubescens* (…). Gordonia rubripertincta GrJYB4-4, with accession number CGMCC No.38412.

[0006] The present invention also provides a method for culturing Gordonella rubescens GrJYB4-4 as described in the above technical solution, comprising the following steps: inoculating Gordonella rubescens GrJYB4-4 into a culture medium for culturing to obtain a culture of Gordonella rubescens GrJYB4-4; The culture medium is LB medium, and the culture conditions are: temperature 25~37℃, initial pH 6.0~8.0, and inoculum size 1~3%.

[0007] The present invention also provides a microbial inoculant, wherein the effective component of the microbial inoculant includes the *Gordonella rubescens* GrJYB4-4 described in the above technical solution or the culture of *Gordonella rubescens* GrJYB4-4 obtained by the culture method described in the above technical solution.

[0008] Preferably, the effective viable count of *Gordonella rubescens* GrJYB4-4 in the microbial agent is not less than 1.04 × 10⁻⁶. 9 CFU / mL.

[0009] The present invention also provides the application of the *Gordonella rubescens* GrJYB4-4 culture obtained by the above-described technical solution or the microbial agent described in the above-described technical solution in alleviating the obstacles of continuous ginseng cultivation.

[0010] Preferably, the method for alleviating ginseng continuous cropping obstacles includes degrading autotoxic substances produced by ginseng in the soil; The self-toxic substances include at least one of benzoic acid, cinnamic acid, gallic acid, vanillic acid, salicylic acid, p-hydroxybenzoic acid, and syringic acid.

[0011] The present invention also provides a method for alleviating the obstacle of continuous cropping of ginseng, comprising the following steps: The soil of ginseng-continuous cropping sites is treated with the culture of *Gordonella rubescens* GrJYB4-4 obtained by the cultivation method described in the above technical solution or the microbial agent described in the above technical solution.

[0012] Preferably, the treatment of the soil in the ginseng-continuous cropping area includes mixing the microbial agent described in the above technical solution into the soil; The dosage of the microbial agent is 200~400 mL / m 2 .

[0013] This invention also provides the application of *Gordonella rubescens* GrJYB4-4 as described in the above-described technical solutions, or the culture of *Gordonella rubescens* GrJYB4-4 obtained by the cultivation method described in the above-described technical solutions, or the microbial agent described in the above-described technical solutions, or the method described in the above-described technical solutions, in any one or more of the functions described in ① to ④: ① Reduce benzoic acid content; ② Reduce the occurrence of diseases in ginseng roots; ③ Improve ginseng growth indicators; ④ Increase ginseng production.

[0014] Beneficial effects: This invention provides a strain of *Gordonella rubescens*, with accession number CGMCC No. 38412. This *Gordonella rubescens* GrJYB4-4, described in this invention, is the first strain isolated and screened from nature. It can efficiently degrade phenolic acid autotoxic substances produced by continuous cropping obstacles in ginseng, achieving a degradation rate of 96.6% for benzoic acid. After optimization of degradation conditions using response surface methodology, the degradation rate can be further increased to 98.5%, and the degradation rate exceeds 99% after 28 hours, demonstrating highly efficient and rapid benzoic acid reduction capabilities. Simultaneously, the strain exhibits varying degrees of degradation ability for other common phenolic acid autotoxic substances found in ginseng continuous cropping soils, such as cinnamic acid, salicylic acid, and p-hydroxybenzoic acid. It has a broad substrate spectrum and can effectively address the complex phenolic acid stress in ginseng continuous cropping soils, filling a gap in the application of this strain in the degradation of ginseng continuous cropping autotoxic substances and the alleviation of continuous cropping obstacles.

[0015] Especially under field conditions, the application of the *Gordonella rubescens* GrJYB4-4 described in this invention can effectively reduce the benzoic acid content in soil where ginseng is continuously cropped (degradation rate reaches 41.17%), and increase the underground fresh weight of ginseng by 50.10% compared with the control group. The control effects on ginseng root rot and rust rot reach 24.40% and 25.73%, respectively. The strain described in this invention has multiple functions including reducing autotoxic substances, promoting growth and increasing yield, controlling diseases, and regulating soil microecology, resulting in outstanding comprehensive benefits. More importantly, the pathogenicity test of this invention has verified that the strain is non-pathogenic to ginseng and common fruits and vegetables such as potatoes, radishes, and tomatoes. It is an environmentally friendly and safe strain, with no chemical residues and does not damage the original soil microecology, meeting the industry requirements for green ginseng cultivation, and posing no biosafety risks in field application.

[0016] Biological Preservation Information Gordon's dark red GrJYB4-4, classified and named Gordonia rubripertincta It was deposited on April 23, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 38412. Attached Figure Description To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The images show the morphological characteristics of strain GrJYB4-4; where A is the colony morphology of GrJYB4-4, B is the Gram staining result, and C is the transmission electron microscopy observation result (scale bar: 200 nm). Figure 2 This is a schematic diagram of the phylogenetic tree of the degrading strain GrJYB4-4 constructed based on the 16S rRNA sequence. Figure 3This image shows the results of a pathogenicity test of strain GrJYB4-4 on ginseng roots; where A-D are longitudinal sections of ginseng roots, and E-H are transverse sections of ginseng roots (corresponding to A-D); more specifically, A and E are the results of treatment with sterile water (negative control, CK-), B and F are the results of treatment with LB medium (blank control), and C and G are the results of treatment with Burkholderia jojoba (…). Burkholderia gladioli The results of the positive control (CK+) treatment are shown in Figure 1; Figures D and H are the results of the GrJYB4-4 treatment. Figure 4 The graphs show the pathogenicity test results of strain GrJYB4-4 on potatoes, radishes, and tomatoes; where A represents the potato test results, B represents the radish test results, and C represents the tomato test results; a represents the sterile water control (negative control, CK). - The results of the treatments are as follows: b is the result of the LB medium control (blank control), and c is the result of the treatment with Burkholderia gladioli (…). B. gladioli Positive control (CK) + The processing result is d, where d is the processing result of GrJYB4-4. Figure 5 The figure shows the degradation rate of different phenolic acid autotoxic substances by strain GrJYB4-4; different lowercase letters indicate significant differences, and the same letter indicates no significant difference.

[0018] Figure 6 Figure 1 shows the effect of different temperatures on the growth and benzoic acid degradation characteristics of strain GrJYB4-4; where A is the result of strain growth detection and B is the result of relative benzoic acid content detection. Figure 7 Figure 1 shows the effect of different initial pH values ​​on the growth and benzoic acid degradation characteristics of strain GrJYB4-4; where A represents the strain growth detection results and B represents the relative benzoic acid content detection results. Figure 8 Figure 1 shows the effect of different substrate concentrations on the growth and benzoic acid degradation characteristics of strain GrJYB4-4; where A represents the strain growth detection results and B represents the relative benzoic acid content detection results. Figure 9 Figure 1 shows the effect of different inoculum amounts on the growth and benzoic acid degradation characteristics of strain GrJYB4-4; where A represents the strain growth detection results and B represents the relative benzoic acid content detection results. Figure 10 The graph shows the effect of different metal ions on the degradation of benzoic acid. Figure 11 The graph shows the interaction effect of inoculum amount (A, %) and temperature (B, ℃) on the degradation rate of benzoic acid; where A is the contour plot of the interaction effect and B is the surface plot of the interaction effect. Figure 12 The graph shows the interaction effect of inoculum amount (A, %) and pH (C) on the degradation rate of benzoic acid; where A is the contour plot of the interaction effect and B is the surface plot of the interaction effect. Figure 13 The figure shows the interaction effect of temperature (B, °C) and pH (C) on the degradation rate of benzoic acid; where A is the contour plot of the interaction effect and B is the response surface plot of the interaction effect. Figure 14 The graphs show the growth-degradation coupling curves; where A is the strain growth curve and B is the result of the relative benzoic acid content detection. Figure 15 The graph shows the degradation effect of different treatments on benzoic acid in rhizosphere soil at different growth stages of ginseng. Among them, 40 d, 80 d, and 140 d represent the green fruit stage, red fruit stage, and harvest stage of ginseng, respectively. There is no significant difference in the average values ​​of the same letters among the different treatment groups at the same growth stage.

[0019] Figure 16 Figure 1 shows the results of the detection of the effects of different treatments on the enzyme activities in the rhizosphere soil of ginseng at different growth stages; where A is the result of the detection of the effect on urease activity, B is the result of the detection of the effect on sucrase activity, C is the result of the detection of the effect on phosphatase activity, and D is the result of the detection of the effect on catalase activity. Figure 17 The following figures show the results of the detection of the effects of different treatments on the activity of defensive enzymes in ginseng roots; where A is the result of the detection of the effect on peroxidase (POD) activity, B is the result of the detection of the effect on superoxide dismutase (SOD) activity, and C is the result of the detection of the effect on catalase (CAT) activity. Figure 18 The images show a comparison of root systems under different treatments at harvest time. 1-3 represent low, medium, and high dose treatments of GrJYB4-4 inoculant, respectively; 4 represents the treatment group treated with commercially available anti-continuous cropping inoculant; 5 represents the biochar treatment group; and 6 represents the aseptic fermentation broth control group. The numbers followed by "-1 / -2 / -3" represent three biological replicates of the corresponding treatment group. Detailed Implementation

[0020] This invention provides a strain of *Gordonella rubescens* (…). Gordonia rubripertinctaGrJYB4-4, with accession number CGMCC No. 38412, is the first strain of *Gordonella rubescens* isolated and screened from nature. It can efficiently degrade phenolic acid autotoxic substances caused by continuous cropping obstacles in ginseng. The degradation rate of benzoic acid reaches 96.6%, and after optimization of degradation conditions using response surface methodology, the degradation rate can be further increased to 98.5%. After 28 hours, the degradation rate exceeds 99%, demonstrating highly efficient and rapid benzoic acid reduction capabilities. Simultaneously, the strain also exhibits varying degrees of degradation ability against other common phenolic acid autotoxic substances found in ginseng continuous cropping soils, such as cinnamic acid, salicylic acid, and p-hydroxybenzoic acid. With a broad substrate spectrum, it can effectively address the complex phenolic acid stress in ginseng continuous cropping soils, filling a gap in the application of this strain in the degradation of ginseng continuous cropping autotoxic substances and the alleviation of continuous cropping obstacles. The *Gordonella rubescens* GrJYB4-4 described in this invention, after being cultured on LB solid medium at 37°C for 72 h, exhibits orange-red colonies that are round or nearly round with irregular, spiky edges. The surface is dry, rough, dense, highly raised, and opaque; the colony diameter is 1–3 mm. The *Gordonella rubescens* GrJYB4-4 cells are short rod-shaped or branched, Gram-positive, and lack flagella, spores, and capsules. They typically exist singly, in pairs, or in short chains, with a cell size of (0.3–0.6) μm × (1.0–3.0) μm, and further, (0.4–0.5) μm × (1.5–2.0) μm. Transmission electron microscopy reveals blunt, rounded ends, a relatively thick and clearly defined cell wall, high cytoplasmic electron density, and the absence of spores and capsules. The *Gordonella rubescens* strain GrJYB4-4 described in this invention is Gram-positive, negative for methyl red, negative for VP assay, negative for starch hydrolysis, negative for fluorescent dye production, negative for malonic acid utilization, positive for salt tolerance in 2% and 5% NaCl, negative for salt tolerance in 7% and 10% NaCl, negative for indole, positive for heptyl alcohol hydrolysis, negative for gelatin liquefaction, and positive for citrate utilization. The *Gordonella rubescens* strain GrJYB4-4 described in this invention has been verified through in vitro inoculation pathogenicity tests to be non-pathogenic to ginseng and common fruits and vegetables such as potatoes, radishes, and tomatoes. It is an environmentally friendly and safe strain, leaving no chemical residues and not disrupting the original soil microecology, meeting the industry requirements for green ginseng cultivation, and posing no biosafety risks in field application.

[0021] The 16S rRNA sequence of *Gordonella rubescens* GrJYB4-4 described in this invention is shown in SEQ ID NO.1: Gordonia rubripertincta .

[0022] This invention also provides a method for culturing *Gordonella rubescens* GrJYB4-4 as described in the above-mentioned technical solution, comprising the following steps: inoculating *Gordonella rubescens* GrJYB4-4 into a culture medium for cultivation to obtain a culture of *Gordonella rubescens* GrJYB4-4; the culture medium is LB medium, and the cultivation conditions are: temperature 25~37℃, initial pH 6.0~8.0, and inoculum size 1~3%. This invention inoculates *Gordonella rubescens* GrJYB4-4 into a culture medium for cultivation to obtain a culture of *Gordonella rubescens* GrJYB4-4. As one embodiment, the LB culture medium of this invention includes LB liquid medium or LB solid medium. As one embodiment, the LB liquid culture medium of this invention comprises 10~15 g / L tryptone, 5~8 g / L yeast extract and 5~10 g / L NaCl, with distilled water as the solvent, and pH 7.0~7.5. In another embodiment, the LB liquid culture medium of the present invention comprises 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl, with a pH of 7.2. In another embodiment, the LB solid culture medium of the present invention is based on the above LB liquid culture medium with the addition of 15-20 g / L agar. In another embodiment, the amount of agar added to the LB solid culture medium is 18 g / L. In another embodiment, the culture temperature of *Gordonella rubescens* GrJYB4-4 of the present invention is 25-37℃, the initial pH is 6.0-8.0, and the inoculum size is 1%-3%. In a preferred embodiment, the culture temperature of *Gordonella rubescens* GrJYB4-4 of the present invention is 36.71℃, the initial pH is 7.12, and the inoculum size is 2.60%. In another embodiment, the culture rotation speed is 180 r / min, and the culture time is 16-24 h. As one embodiment, the *Gordonella rubescens* GrJYB4-4 described in this invention can maintain more than 80% degradation activity within a pH range of 6.0 to 8.0 and a temperature range of 25 to 37°C.

[0023] This invention also provides a microbial inoculant, the effective component of which includes *Gordonella rubescens* GrJYB4-4 as described in the above-described technical solution, or a culture of *Gordonella rubescens* GrJYB4-4 obtained by the cultivation method described in the above-described technical solution. As one embodiment, the effective viable count of *Gordonella rubescens* GrJYB4-4 in the microbial inoculant of this invention is not less than 1.04 × 10⁻⁶. 9 CFU / mL. As one embodiment, the microbial agent described in this invention is stored at 4°C for later use.

[0024] This invention also provides the application of *Gordonella rubescens* GrJYB4-4 as described in the above-mentioned technical solutions, or the culture of *Gordonella rubescens* GrJYB4-4 obtained by the cultivation method described in the above-mentioned technical solutions, or the microbial agent described in the above-mentioned technical solutions, in alleviating the obstacle of continuous ginseng cropping. As one embodiment, the method of alleviating the obstacle of continuous ginseng cropping in this invention includes degrading autotoxic substances produced by ginseng in the soil; the autotoxic substances include at least one of benzoic acid, cinnamic acid, gallic acid, vanillic acid, salicylic acid, p-hydroxybenzoic acid, and syringic acid. As one embodiment, the *Gordonella rubescens* GrJYB4-4 described in this invention achieves a degradation rate of 96.6% for 200 mg / L benzoic acid in 24 h, and after response surface methodology optimization, the degradation rate can reach 98.5%. Simultaneously, it exhibits varying degrees of degradation ability for multiple phenolic acid autotoxic substances such as cinnamic acid, salicylic acid, and p-hydroxybenzoic acid, demonstrating a broad degradation spectrum.

[0025] The present invention also provides a method for alleviating the obstacle of continuous cropping of ginseng, comprising the following steps: The soil of ginseng-continuous cropping sites is treated with the culture of *Gordonella rubescens* GrJYB4-4 obtained by the cultivation method described in the above technical solution or the microbial agent described in the above technical solution.

[0026] This invention utilizes the *Gordonella rubescens* GrJYB4-4 culture obtained by the above-described technical solution or the microbial agent described in the above-described technical solution to treat the soil of ginseng-continuously cropped fields.

[0027] As one embodiment, the treatment of soil in ginseng-continuous cropping fields according to the present invention includes mixing the microbial agent described in the above-mentioned technical solution into the soil; the dosage of the microbial agent is 200~400 mL / m³. 2 In another embodiment, the dosage of the culture or microbial agent mixed in according to the present invention is 400 mL / m 2 As one embodiment, the microbial agent of the present invention is supplemented with sterile water to a concentration of 1 L / m³ before mixing. 2 In one embodiment, the tillage depth of the mixture in the soil is 10-20 cm. In another embodiment, after treating the soil in ginseng-continuously cropped fields, the soil moisture content is maintained at 60%-70% of field capacity, and the soil is left to stand for 3-5 days before ginseng transplanting.

[0028] This invention also provides the application of *Gordonella rubescens* GrJYB4-4 as described in the above-described technical solutions, or the culture of *Gordonella rubescens* GrJYB4-4 obtained by the cultivation method described in the above-described technical solutions, or the microbial agent described in the above-described technical solutions, or the method described in the above-described technical solutions, in any one or more of the functions described in ① to ④: ① Reduce benzoic acid content; ② Reduce the occurrence of diseases in ginseng roots; ③ Improve ginseng growth indicators; ④ Increase ginseng production.

[0029] The *Gordonella rubiginii* GrJYB4-4 described in this invention can improve the survival rate of ginseng during the harvest period. This *Gordonella rubiginii* GrJYB4-4 can alleviate the inhibition of ginseng growth, promoting growth indicators such as root length, root diameter, and fresh root weight, and significantly increasing ginseng yield, with an increase rate of up to 50.10%. As one embodiment, the *Gordonella rubiginii* GrJYB4-4 described in this invention has a control effect on ginseng root diseases, with a control effect of up to 24.40% on root rot and up to 25.73% on rust rot.

[0030] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a strain of *Gordonella rubrum* and its application in alleviating crop rotation obstacles, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 Screening and identification of strain GrJYB4-4 1. Enrichment and initial screening of bacteria in soil samples Fifty-four ginseng rhizosphere soil samples collected from major ginseng-producing areas in Jilin Province, including Changchun, Ji'an, and Fusong, were isolated using an enrichment-acclimatization-reseparation method. Aseptically, 10 g of soil sample was weighed and added to 90 mL of sterile physiological saline to prepare a soil suspension. The supernatant was inoculated into MSM liquid medium containing 50 mg / L benzoic acid. The MSM liquid medium formula was: (NH4)2SO4 2.0 g, K2HPO4 1 g, NaH2PO4 1.0 g, MgSO4·7H2O 0.35 g, CaCl2 0.1 g, and distilled water 1000 mL. The culture was carried out at 28℃ and 180 r / min for 7 days to complete the first enrichment. The benzoic acid concentration was then increased sequentially to 100 mg / L and 200 mg / L, and the enrichment culture was repeated twice. The third enrichment culture was then serially diluted to 10 mg / L. -4 10 -5 10 -6Take 0.1 mL of the solution and spread it on an MSM solid plate containing 200 mg / L benzoic acid (add 17 g agar to the above liquid culture medium). Incubate at 28°C for 72 h. Select single colonies with different morphologies and streak them repeatedly for purification. A total of 75 pure strains were obtained and stored at 4°C for later use.

[0032] 2. Secondary screening of bacteria capable of degrading benzoic acid The 75 pure strains obtained from the initial screening were inoculated into LB liquid medium and cultured at 37℃ and 180 r / min for 16 h to obtain OD. 600 =0.6~0.8% activated seed culture. Inoculate 2% (v / v) into MSM medium containing 200 mg / L benzoic acid, with uninoculated MSM medium as a blank control. Culture at 37℃ and 180 r / min for 24 h. Benzoic acid residue was determined by high-performance liquid chromatography (HPLC). The specific steps are as follows: the culture medium was extracted three times with equal volumes of ethyl acetate. The upper ethyl acetate layer was evaporated to dryness by rotary evaporation and reconstituted in acetonitrile. The solution was filtered through a 0.22 µm organic filter membrane. Chromatographic analysis was performed using an AichromBond-AQ C18 column (4.6 mm × 250 mm, 5 µm). The detection method was as follows: mobile phase A was 50% acetonitrile, mobile phase B was 0.1% formic acid aqueous solution, flow rate was 0.4 mL / min, injection volume was 20 µL, column temperature was 30℃, gradient elution was used, and UV detection wavelength was 230 nm. The degradation rate of benzoic acid by the degrading strain was calculated. The degradation rate (D) was calculated using the following formula: , Formula I; In the formula, Co is the initial concentration of the sample (mg / L), and Ct is the concentration of the sample after treatment with degrading bacteria (mg / L).

[0033] After secondary screening, a strain with the highest benzoic acid degradation rate was obtained and named GrJYB4-4. Under the basic conditions, this strain achieved a degradation rate of 96.6% ± 1.2% of 200 mg / L benzoic acid in 24 h, which was significantly higher than that of the other 74 initially screened strains.

[0034] 3. Identification of strain GrJYB4-4 1) Morphological identification The colony morphology of strain GrJYB4-4 on LB solid medium was observed, and the strain was subjected to Gram staining and transmission electron microscopy. The colony morphology of strain GrJYB4-4 is shown below. Figure 1 As shown in Figure A, the Gram staining results are as follows: Figure 1 As shown in Figure B, the transmission electron microscopy observation results are as follows: Figure 1 As shown in C.

[0035] As observed, after culturing strain GrJYB4-4 on LB solid medium at 37℃ for 72 h, the colonies were orange-red, round or nearly round, with irregular, spiky edges, a dry, rough, dense, highly raised, and opaque surface; the colony diameter was 1–3 mm. The bacteria were short rod-shaped or branched, Gram-positive, without flagella, spores, or capsules, and often existed singly, in pairs, or in short chains. The bacterial size was (0.3–0.6) μm × (1.0–3.0) μm, further decreasing to (0.4–0.5) μm × (1.5–2.0) μm. Transmission electron microscopy revealed blunt ends to the bacterial cells, a relatively thick and clearly defined cell wall, high cytoplasmic electron density, and the absence of spores and capsules.

[0036] 2) Physiological and biochemical identification The physiological and biochemical characteristics of strain GrJYB4-4 were determined according to Bergey's Manual of Bacterial Identification and the Manual of Systematic Identification of Common Bacteria. The results of the physiological and biochemical determination of strain GrJYB4-4 are shown in Table 1, where "+" indicates a positive reaction and "-" indicates a negative reaction.

[0037] Table 1. Results of physiological and biochemical characterization of strain GrJYB4-4

[0038] Note: "+" indicates a positive reaction or is tolerable; "-" indicates a negative reaction or is intolerable. The data represent typical results from three biological replicates.

[0039] Physiological and biochemical assays showed that strain GrJYB4-4 was Gram-positive, negative for methyl red, negative for VP, negative for starch hydrolysis, negative for fluorescent dye production, negative for malonic acid utilization, positive for salt tolerance in 2% and 5% NaCl, negative for salt tolerance in 7% and 10% NaCl, negative for indole, positive for aesculin hydrolysis, negative for gelatin liquefaction, and positive for citrate utilization. These characteristics perfectly matched the typical physiological and biochemical characteristics of the *Gordonella rubrum* type strain.

[0040] 3) Molecular biological identification The whole genome DNA of bacterial strain GrJYB4-4 was extracted using a bacterial genomic DNA extraction kit. Universal primers 27F (SEQ ID NO.2): 5'-AGAGTTTGATCMTGGCTCAG-3' and 1492R (SEQ ID NO.3): 5'-GGTTACCTTGTTACGACTT-3' were used to amplify the 16S rRNA sequence. The PCR amplification system consisted of 25 μL of 2×Taq Mix, 2 μL each of forward and reverse primers, 2 μL of template DNA, and water added to a final volume of 50 μL. The amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, for 32 cycles; and a final extension at 72℃ for 10 min. The PCR product was purified and sent to a biotechnology company for sequencing, yielding a 16S rRNA sequence with an effective sequence length of 1428 bp (as shown in SEQ ID NO.1). The obtained sequences were submitted to the NCBI database for BLAST comparison. The results showed that strain GrJYB4-4 was similar to the model strain of *Goldenella rubrum*. G. rubripertincta strain The sequence homology of CWB2 (CP022580) reached 100%. A neighbor-joining phylogenetic tree constructed based on the 16S rRNA sequence is shown below. Figure 2 As shown, the numbers at the nodes are Bootstrap values ​​(only values ​​≥70% are displayed); the scale represents a nucleotide substitution rate of 0.010. Mycobacterium simians It is an outgroup. It can be seen that strain GrJYB4-4 clusters with *Gordonella rubrum* in the same independent branch, with 100% bootstrap support.

[0041] Based on the comprehensive morphological characteristics, physiological and biochemical properties, and 16S rRNA sequence analysis, strain GrJYB4-4 was identified as a strain of *Gordonella rubrum*. Gordonia rubripertincta It was deposited at the China General Microbiological Culture Collection Center on April 23, 2026, with accession number CGMCC No. 38412.

[0042] 4. Biosafety testing Pathogenicity tests were conducted on ginseng and common fruits and vegetables such as potatoes, radishes, and tomatoes.

[0043] Pathogenicity was determined using an in vitro tissue inoculation method: Healthy, undamaged ginseng roots, potato tubers, radish roots, and tomato fruits were selected. After surface disinfection, fresh cut surfaces (cross and longitudinal sections) were created on the ginseng roots, and four sets of puncture wounds were created on the surfaces of the potatoes, radishes, and tomato fruits. Activated bacterial solution (OD) was then inoculated into the ginseng roots. 600=0.8) was evenly inoculated onto tissue sections or wounds, with sterile water as a negative control, and the known pathogen Burkholderia gladioli ( ) was used. B. gladioli ( ) served as a positive control. The cultures were maintained at a constant temperature and humidity of 25℃, and the tissue decay was observed periodically. The results showed that strain GrJYB4-4 was not pathogenic to ginseng or common fruits and vegetables such as potatoes, radishes, and tomatoes. Figure 3 , Figure 4 It is an environmentally friendly and safe strain.

[0044] Example 2 Degradation characteristics determination and response surface optimization of strain GrJYB4-4 1. Degradation profile determination of strain GrJYB4-4 A single colony of GrJYB4-4 was picked and inoculated into LB liquid medium and incubated at 37°C and 180 r / min for 16 h to obtain OD. 600 =0.6~0.8 activating seed solution.

[0045] Based on MSM medium, cinnamic acid, gallic acid, vanillic acid, salicylic acid, p-hydroxybenzoic acid, and syringic acid were added at 200 mg / L as the sole carbon source. The seed culture of strain GrJYB4-4 was activated (OD). 600 =0.6~0.8) were inoculated into the above culture medium at an inoculum rate of 2% (v / v) and cultured at 37℃ and 180 r / min for 24 h with constant temperature shaking. Each treatment was set up in triplicate, and a blank control group without inoculation was also set up. After the culture was completed, the supernatant was collected by centrifugation and filtered through a 0.22 μm organic filter membrane. The residual amount of each phenolic acid substance was determined by HPLC and the degradation rate was calculated. The detection conditions were as follows: the chromatographic analysis used an AichromBond-AQ C18 column (4.6 mm × 250 mm, 5 μm), the mobile phase A was 50% acetonitrile and B was 0.1% formic acid water, the flow rate was 0.4 mL / min, the injection volume was 20 μL, the column temperature was 30℃, and gradient elution was used; the UV detection wavelengths were: cinnamic acid 278 nm, gallic acid 272 nm, vanillic acid 260 nm, salicylic acid 230 nm, p-hydroxybenzoic acid 246 nm, and syringic acid 262 nm.

[0046] The degradation rates of different phenolic acid autotoxic substances by strain GrJYB4-4 were measured as follows: Figure 5 As shown.

[0047] The results showed that strain GrJYB4-4 had varying degrees of degradation ability for cinnamic acid (95.23%), salicylic acid (85.2%), and p-hydroxybenzoic acid (82.83%), as well as the other three phenolic acids. It has a broad substrate spectrum and can cope with the combined phenolic acid stress in soils where ginseng is continuously cropped.

[0048] 2. Single-factor test of degradation characteristics 1) Single-factor experiment on culture conditions Using MSM medium containing 200 mg / L benzoic acid as the basal system, with an inoculum size of 2%, the culture was carried out at 37℃ and 180 r / min for 24 h. The effects of culture temperature (25℃, 28℃, 31℃, 34℃, 37℃), initial pH (4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0), inoculum size (0.5%, 1%, 2%, 4%, 8%, v / v), and substrate concentration (100, 200, 300, 400, 500 mg / L) on the growth and benzoic acid degradation ability of strain GrJYB4-4 were investigated using a single-variable method (except for the single variable, all other conditions were the same as the basal system). Each treatment was replicated three times, and a blank control without inoculation was set up simultaneously.

[0049] After 24 h of cultivation, the benzoic acid degradation rate and bacterial OD of each single variable group were measured. 600 The effects of different culture conditions on the growth and benzoic acid degradation characteristics of strain GrJYB4-4 are as follows: Figures 6-9 As shown.

[0050] 2) The effect of metal ions on degradation ability In the basic system (MSM medium containing 200 mg / L benzoic acid, inoculation amount 2%, 37℃, 180 r / min), 0.5 mmol / L Mg was added. 2+ Zn 2+ Ca 2+ Fe 3+ Mn 2+ Cu 2+ Using a baseline system without added metal ions as a control, each treatment was repeated three times, and the benzoic acid degradation rate was measured after 24 h of incubation. Figure 10 ).

[0051] The results showed that the optimal degradation temperature of the strain was 36.71℃, and the degradation rate remained above 80% within the range of 25–37℃; the optimal initial pH was 7.12, and the degradation activity remained stable within the pH range of 6.0–8.0; the optimal inoculum size was 2.60%; it exhibited high degradation capacity for benzoic acid at concentrations of 100–300 mg / L, but the degradation activity significantly decreased above 500 mg / L; Mg 2+ Fe 3+ It can slightly promote degradation, Cu 2+ It exhibits a strong inhibitory effect on degradation.

[0052] 3. Degradation Conditional Response Surface Optimization 1) Selection of variables and levels Based on the results of the single-factor experiment, three core factors that have the most significant impact on the degradation rate of benzoic acid were selected: culture temperature (A), initial pH (B), and inoculum size (C). Each factor was set with three levels, coded as -1, 0, and +1, respectively. The values ​​of each factor level are shown in Table 2.

[0053] Table 2 Factor Level Coding Table

[0054] 2) Box-Behnken experimental design Using the Box-Behnken design principle, a three-factor, three-level response surface methodology experiment was designed with the benzoic acid degradation rate at 24 h as the response value. A total of 17 experimental points were set up (12 factorial points and 5 centroid points). The experimental design and results are shown in Table 3.

[0055] Table 3. Response surface methodology and results of benzoic acid degradation rate of strain GrJYB4-4

[0056] Note: Std is the standard order, and Run is the random order of the actual test run; to eliminate systematic errors, the 17 test points are executed randomly in the Run order.

[0057] 3) Model building and analysis of variance The quadratic polynomial regression equation was obtained by fitting the experimental data: Y =97.35 + 2.27* A + 3.36* B + 0.8375* C + 0.7800*AB + 0.7125* AC +0.2625* BC - 2.26* A 2 - 4.57* B 2 - 5.84* C 2 Formula II; Where Y is the predicted value (%) of benzoic acid degradation rate over 24 h, and A, B, and C are the encoded values ​​of inoculum amount (%), temperature (°C), and pH, respectively.

[0058] The results of the response surface variance analysis are shown in Table 4. The constructed quadratic regression model is highly significant ( P <0.0001, the lack-of-fit term is not significant ( P The model has a good fit (>0.05) and can accurately predict the benzoic acid degradation rate of the strain.

[0059] Table 4. Analysis of variance of the response surface regression model for benzoic acid degradation rate of strain GrJYB4-4

[0060] Note: A: Inoculum size (%); B: Temperature (°C); C: pH; R 2 =0.9748. P <0.05 indicates a significant difference. P <0.01 indicates a highly significant difference; lack of fit term P A value >0.05 indicates that the model fits well.

[0061] 4) Interaction analysis and determination of optimal conditions Response surface methodology for the pairwise interactions of various factors on the degradation rate of benzoic acid is as follows: Figures 11-13 As shown, the data are presented as mean ± standard deviation (n=3). The optimal degradation conditions obtained through model solving were: culture temperature 36.71℃, initial pH 7.12, and inoculum size 2.60%. Considering practical applications, the temperature can be adjusted to 37℃. Under these conditions, the theoretical predicted degradation rate of benzoic acid in 24 h is 97.51%. Three repeated validation experiments were conducted under optimized conditions, and the measured degradation rate of benzoic acid in 24 h was 98.5% ± 0.3%, which showed no significant difference from the predicted value, indicating that the model is stable and reliable.

[0062] 4. Determination of growth-degradation coupling curves Under optimized degradation conditions (temperature 36.71℃, pH 7.12, inoculum size 2.60%), strain GrJYB4-4 was inoculated into MSM medium containing 200 mg / L benzoic acid. Samples were taken every 2 hours from 0 to 30 h to determine the OD of the strain. 600 Dynamic coupling curves between strain growth and benzoic acid degradation were plotted based on benzoic acid residue levels, as shown in the figure. Figure 14 As shown in the figure. The results indicate that the growth of the strain is significantly positively correlated with the degradation of benzoic acid, and 200 mg / L benzoic acid can be completely degraded in 28 h.

[0063] Example 3 Field application efficacy verification of strain GrJYB4-4 in alleviating ginseng continuous cropping obstacles 1. Experimental Design The field trial was conducted in a ginseng planting base of Jilin Agricultural University, in a field with a three-year continuous cropping history. The viable count of the basic fermentation broth of the tested strain GrJYB4-4 was 1.04 × 10⁻⁴. 9 CFU / mL, the tested ginseng was a 2-year-old healthy seedling.

[0064] The experiment consisted of 6 treatment groups, with 3 replicates per group. The plots were randomly arranged, with a plot area of ​​2.4 m². 2 (1.2 m × 2 m), a 50 cm isolation strip will be set between the units, and the specific treatment is as follows: T1: Low-dose bacterial suspension treatment, 1.04 × 10⁻⁶ 9CFU / mL, application rate 200 mL / m 2 ; T2: Medium-dose bacterial suspension treatment, 1.04 × 10⁻⁶ 9 CFU / mL, application rate 300 mL / m 2 ; T3: High-dose bacterial suspension treatment, 1.04 × 10⁻⁶ 9 CFU / mL, application rate 400 mL / m 2 ; T4: Treatment with commercially available anti-continuous cropping fungicide (Greenstar "Continuous Crop 120", containing Trichoderma harzianum and Bacillus subtilis, viable count ≥ 5.0 × 10⁻⁶). 8 CFU / g), according to the product instructions (20 g / m 2 Recommended dosage; T5: Biochar treatment, application rate 100 g / m³ 2 After spreading evenly, till the soil into the soil; CK: Blank control, administered with an equal volume of sterile LB medium.

[0065] Before ginseng transplanting, sterile water was added to the T1-T3 bacterial suspensions and an equal volume of sterile LB medium (CK) to bring the concentration to 1 L / m³. 2 Then spray evenly onto the surface of continuously cropped soil; T4 commercially available anti-continuous cropping fungicide is diluted with water according to the instructions and sprayed in the same way; T5 biochar is applied at 100 g / m³. 2 Apply the fertilizer evenly. After all treatments are completed, till the soil thoroughly, mix well, and maintain a soil moisture content of 60%–70% of field capacity. Let it stand for 3 days before transplanting the ginseng seedlings. Select healthy 2-year-old ginseng seedlings and plant them at a row spacing of 10 cm × 10 cm, with 100 seedlings planted in each plot. After transplanting, watering, weeding, and other management measures should be completely consistent across all plots. No other microbial agents, pesticides, or soil conditioners should be applied throughout the entire process.

[0066] 2. Determination of benzoic acid content in soil Rhizosphere soil samples of ginseng were collected at 40 days (green fruit stage), 80 days (red fruit stage), and 140 days (harvest stage) after transplanting, and the benzoic acid content in the soil was determined by HPLC.

[0067] The degradation effects of different treatments on benzoic acid in rhizosphere soil at different growth stages of ginseng were measured as follows: Figure 15 As shown, the data are presented as mean ± standard deviation (n=3). According to Tukey's HSD (strictly significant difference) test (…),… P<0.05), there was no significant difference in the average values ​​of the same letters among different treatment groups at the same growth stage. The results showed that throughout the entire growth period, the degradation rate of benzoic acid in the high-dose bacterial solution treatment (T3) was consistently significantly higher than that in other treatments, reaching 41.17% at harvest; the degradation rate in the medium-dose group (T2) was 32.54%; and the degradation rate in the low-dose group (T1) was 21.38%.

[0068] 3. Determination of soil enzyme activity and root defense enzyme activity Soil samples from the rhizosphere and root systems of ginseng were collected at each growth stage. The activities of urease, sucrase, phosphatase, and catalase in the soil were determined using a colorimetric assay kit, following the instructions of the corresponding soil enzyme activity detection kits. The activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) in ginseng roots were determined using a plant physiological enzyme activity kit. After low-temperature grinding and extraction of the crude enzyme solution, the absorbance was measured at the corresponding characteristic wavelength using a microplate reader, and the values ​​of each enzyme activity were calculated.

[0069] The effects of different treatments on rhizosphere soil enzyme activity of ginseng at different growth stages were measured as follows: Figure 16 As shown, the effects of different treatments on the activity of ginseng root defense enzymes are as follows: Figure 17 As shown. Data are presented as mean ± standard deviation (n=3). According to Tukey's HSD (strictly significant difference) test (…),… P <0.05), and there was no significant difference in the mean values ​​of the same letters among different treatment groups at the same growth stage. The results showed that the T3 treatment significantly increased the activities of soil urease, sucrase, phosphatase, and catalase, as well as the activities of SOD, POD, and CAT defense enzymes in ginseng roots, which were significantly better than the blank control and the low and medium dose treatment groups.

[0070] 4. Effects on ginseng agronomic traits The survival rate, root length, root diameter, and underground fresh weight of ginseng in each treatment plot were investigated and measured during the harvest period. The results are shown in Table 5.

[0071] Table 5 Effects of strain GrJYB4-4 on agronomic traits of ginseng underground parts

[0072] Note: Data are presented as mean ± standard deviation (n=3). According to Tukey's HSD (strictly significant difference) test (…),… P <0.05), there is no significant difference between the averages of the same letters in the same column; "-" indicates no increase in production data.

[0073] The results showed that the survival rate of ginseng treated with T3 was 77.00%, an increase of 7.94% compared with the control (CK); the root length was 13.11 cm and the root diameter was 7.10 mm, increases of 11.29% and 6.61% respectively compared with the CK; and the underground fresh weight was 307.50 g / m³. 2 It increased by 50.10% compared to CK, showing a significant effect in promoting growth and increasing production.

[0074] 5. Control effect on ginseng root diseases During the harvest period, the incidence of root rot and rust rot in ginseng in each treatment area was investigated. The disease classification standards are shown in Table 6. The control effect and disease index are calculated using Equations III and IV.

[0075] Table 6 Grading Standards for Ginseng Root Diseases

[0076] Formula III; Formula IV.

[0077] The control effects of different treatments on ginseng root diseases are shown in Table 7. Actual photos of ginseng roots under different treatments at harvest are also shown. Figure 18 As shown.

[0078] Table 7. Control efficacy of strain GrJYB4-4 against ginseng root diseases.

[0079] Note: Data are presented as mean ± standard deviation (n=3). According to Tukey's HSD (strictly significant difference) test (P<0.05), there is no significant difference between means with the same letter in the same column; "-" indicates no efficacy data.

[0080] The results showed that the T3 treatment was significantly more effective than other treatments in controlling root rot and rust rot in ginseng, with control rates of 24.40% and 25.73%, respectively.

[0081] In summary, field application results indicate that the fermentation broth of strain GrJYB4-4 can effectively alleviate the obstacles caused by continuous cropping of ginseng, especially at high doses (1.04 × 10⁻⁶). 9 CFU / mL, application rate 400 mL / m 2 Under the conditions of application, it can effectively degrade benzoic acid in the soil of ginseng continuous cropping, enhance soil enzyme activity and ginseng root defense enzyme activity, significantly promote ginseng growth (increase yield by 50.10%), and show good control effect on ginseng root rot and rust rot, with outstanding comprehensive benefits.

[0082] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of *Gordonella erythrorhizon* ( Gordonia rubripertincta GrJYB4-4, with accession number CGMCCNo.38412.

2. The method for culturing *Gordonella rubescens* GrJYB4-4 according to claim 1, characterized in that, Includes the following steps: Gordon's bacterium GrJYB4-4 was inoculated into a culture medium and cultured to obtain a culture of Gordon's bacterium GrJYB4-4; The culture medium is LB medium, and the culture conditions are: temperature 25~37℃, initial pH 6.0~8.0, and inoculum size 1~3%.

3. A microbial inoculant, characterized in that, The effective components of the microbial agent include the *Gordonella rubescens* GrJYB4-4 as described in claim 1 or the culture of *Gordonella rubescens* GrJYB4-4 obtained by the culture method described in claim 2.

4. The microbial agent according to claim 3, characterized in that, The effective viable count of *Gordonella rubrum* GrJYB4-4 in the microbial agent is not less than 1.04 × 10⁻⁶. 9 CFU / mL.

5. The application of the *Gordonella rubescens* GrJYB4-4 as described in claim 1, or the culture of *Gordonella rubescens* GrJYB4-4 obtained by the culture method described in claim 2, or the microbial agent as described in any one of claims 3 to 4, in alleviating the obstacles of continuous ginseng cultivation.

6. The application according to claim 5, characterized in that, The measures to alleviate the obstacles of continuous ginseng cropping include degrading the autotoxic substances produced by ginseng in the soil; The self-toxic substances include at least one of benzoic acid, cinnamic acid, gallic acid, vanillic acid, salicylic acid, p-hydroxybenzoic acid, and syringic acid.

7. A method for alleviating ginseng continuous cropping obstacles, characterized in that, Includes the following steps: The soil of ginseng-continuous cropping sites is treated with a culture of *Gordonella rubescens* GrJYB4-4 as described in claim 1, or a culture of *Gordonella rubescens* GrJYB4-4 obtained by the culture method described in claim 2, or a microbial agent as described in any one of claims 3 to 4.

8. The method according to claim 7, characterized in that, The treatment of soil in ginseng-continuous cropping sites includes mixing the microbial agent described in any one of claims 3 to 4 into the soil; The dosage of the microbial agent is 200~400 mL / m 2 .

9. The application of the *Gordonella rubescens* GrJYB4-4 as described in claim 1, or the culture of *Gordonella rubescens* GrJYB4-4 obtained by the culture method described in claim 2, or the microbial agent as described in any one of claims 3-4, or the method described in claim 7 or 8, in any one or more of the functions described in ①-④: ① Reduce benzoic acid content; ② Reduce the occurrence of ginseng root rot and / or rust rot; ③ Improve ginseng growth indicators; ④ Increase ginseng production.

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