Rhodococcus globerulus and application thereof in relieving continuous cropping obstacles of crops

Abstract

The application provides a strain of rhodococcus qingshengii and application thereof in relieving continuous cropping obstacles of crops, and belongs to the technical field of microorganisms.The rhodococcus qingshengii JA11 is first isolated and screened from nature, can efficiently degrade phenolic acid autotoxic substances generated by continuous cropping of ginseng, the degradation rate of benzoic acid can reach 91.51%, can effectively degrade various phenolic acids such as vanillic acid and cinnamic acid, and has a wide degradation spectrum; and under field conditions, application of the rhodococcus qingshengii JA11 can effectively reduce the content of benzoic acid in the soil for continuous cropping of ginseng, can 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 certain yield-increasing effect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Rhodococcus qingsheng and its application in alleviating crop continuous cropping obstacles. Background Technology

[0002] Ginseng (Panax ginseng) Ginseng (CAMeyer) is a perennial herbaceous plant belonging to the Araliaceae family. As a traditional and precious Chinese medicinal herb, it is renowned as the "King of Herbs." Currently, to maintain ecological balance, ginseng cultivation has gradually shifted from deforestation to farmland cultivation. However, continuous cropping obstacles are a prominent problem in ginseng cultivation, severely restricting the promotion and development of farmland cultivation. Allelopathic autotoxicity is one of the main factors leading to continuous cropping obstacles in ginseng, and phenolic acids are the most common allelopathic autotoxic substances discovered so far. Therefore, reducing the content of phenolic acids in the rhizosphere soil of ginseng is crucial for alleviating continuous cropping obstacles. Summary of the Invention

[0003] The purpose of this invention is to address the aforementioned problems in the prior art and to find an effective solution to reduce the content of phenolic acid compounds in the rhizosphere soil of ginseng, thereby alleviating the continuous cropping obstacles of ginseng. To this end, this invention provides a strain of *Rhodococcus qingshengensis* and its application in alleviating continuous cropping obstacles.

[0004] This invention provides a strain of Rhodococcus qingshengense JA11, with accession number CGMCC No.33448.

[0005] The present invention also provides a method for culturing Rhodococcus qingshengense JA11 as described in the above technical solution, comprising the following steps: inoculating Rhodococcus qingshengense JA11 into a culture medium for culturing to obtain a culture of Rhodococcus qingshengense JA11;

[0006] The culture medium comprises the following components in weight percentage: yeast extract 0.3%~0.7%, soybean peptone 0.5%~1%, and K2HPO4·3H2O 0.5%~1%.

[0007] The present invention also provides a microbial inoculant, wherein the effective component of the microbial inoculant includes Rhodococcus qingshengii JA11 as described in the above technical solution or a culture of Rhodococcus qingshengii JA11 obtained by the culture method described in the above technical solution.

[0008] Preferably, the effective viable count of Rhodococcus qingshengense JA11 in the microbial agent is 1.1 × 10⁻⁶. 7 CFU / mL ~1.1×10 9 CFU / mL.

[0009] The present invention also provides the application of Rhodococcus qingshengense JA11 as described in the above technical solution, the culture of Rhodococcus qingshengense JA11 obtained by the culture method described in the above technical solution, or the microbial agent described in the above technical solution in alleviating crop continuous cropping obstacles.

[0010] Preferably, the mitigation of crop rotation obstacles includes degrading autotoxic substances produced by crops in the soil;

[0011] The self-toxic substances include at least one of benzoic acid, salicylic acid, cinnamic acid, and vanillic acid.

[0012] The present invention also provides a method for alleviating crop rotation obstacles, comprising the following steps:

[0013] The soil in continuously cropped fields is treated with the *Rhodococcus qingshengensis* JA11 described in the above technical solution, the culture of *Rhodococcus qingshengensis* JA11 obtained by the culture method described in the above technical solution, or the microbial agent described in the above technical solution.

[0014] Preferably, the treatment of the soil in the continuously cropped land includes mixing the *Rhodococcus qingshengensis* JA11 described in the above technical solution, the culture of *Rhodococcus qingshengensis* JA11 obtained by the cultivation method described in the above technical solution, or the microbial agent described in the above technical solution into the soil;

[0015] The concentration of the bacterial strain in the culture or microbial agent is 1.1 × 10⁻⁶. 7 CFU / mL ~1.1×10 9 CFU / mL;

[0016] The dosage of the culture or microbial agent added is 200~400 mL / m 2 .

[0017] This invention also provides the application of *Rhodococcus qingshengense* JA11 as described in the above technical solutions, the culture of *Rhodococcus qingshengense* JA11 obtained by the culture method described in the above technical solutions, the microbial inoculant described in the above technical solutions, or the method described in the above technical solutions in any one or more of the functions described in ① to ④:

[0018] ① Reduce benzoic acid content;

[0019] ② Reduce the occurrence of crop root diseases;

[0020] ③ Improve crop growth indicators;

[0021] ④ Increase crop yield.

[0022] Preferably, the crop is ginseng.

[0023] Beneficial effects:

[0024] This invention provides a strain of *Rhodococcus qingsheng* JA11, with the preservation number CGMCC No. 33448. This *Rhodococcus qingsheng* JA11 is the first strain isolated and screened from nature. It can efficiently degrade phenolic acid autotoxic substances caused by continuous cropping obstacles in ginseng, achieving a degradation rate of 91.51% for benzoic acid. It can also effectively degrade various phenolic acids such as vanillic acid and cinnamic acid, exhibiting a broad degradation spectrum. Furthermore, under field conditions, applying *Rhodococcus qingsheng* JA11 can effectively reduce the benzoic acid content in soil from continuous ginseng cropping, while also controlling the occurrence of ginseng root diseases to a certain extent, significantly improving ginseng survival rate, root length, and other growth indicators, and exhibiting a certain yield-increasing effect.

[0025] Biological Preservation Information

[0026] Rhodococcus qingshengense JA11, classified and named Rhodococcus qingshengii It was deposited on January 17, 2025, 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. 33448. Attached Figure Description

[0027] 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.

[0028] Figure 1 This is a statistical graph showing the degradation effect of strain JA11 on different phenolic acids.

[0029] Figure 2 The images show the colony morphology, Gram staining, and cell morphology of strain JA11; where A is the colony morphology of strain JA11, B is the Gram staining result of strain JA11, and C is the electron micrograph of strain JA11.

[0030] Figure 3 This is a schematic diagram of the phylogenetic tree of strain JA11 constructed using the maximum likelihood method based on the 16S rRNA sequence.

[0031] Figure 4 Figure 1 shows the effect of different concentrations of yeast powder on the growth of the strain.

[0032] Figure 5 Figure 1 shows the effect of different concentrations of soybean peptone on the growth of the bacterial strain.

[0033] Figure 6 The graph shows the effect of different concentrations of K2HPO4·3H2O on the growth of the strain.

[0034] Figure 7The response surface plot and contour plot show the interaction between carbon and nitrogen sources; where a is the contour plot of the interaction and b is the response surface plot of the interaction.

[0035] Figure 8 The response surface plot and contour plot show the interaction between carbon source and inorganic salt; where a is the contour plot of the interaction and b is the response surface plot of the interaction.

[0036] Figure 9 The response surface plot and contour plot show the interaction between nitrogen source and inorganic salt; where a is the contour plot of the interaction and b is the response surface plot of the interaction.

[0037] Figure 10 Figure 1 shows the effect of initial pH on the growth of strain JA11 and its degradation of benzoic acid.

[0038] Figure 11 The figure shows the effect of substrate concentration on the growth of strain JA11 and its degradation effect on benzoic acid.

[0039] Figure 12 Figure showing the effect of culture temperature on the growth of strain JA11 and its degradation of benzoic acid.

[0040] Figure 13 Figure showing the effect of inoculum size on the growth of strain JA11 and its degradation effect on benzoic acid.

[0041] Figure 14 The figure shows the interaction effect of culture temperature and initial pH on the benzoic acid degradation efficiency of strain JA11; where a is the contour plot of the interaction effect and b is the response surface plot of the interaction effect.

[0042] Figure 15 The figure shows the interaction effect of inoculum size and initial pH on the benzoic acid degradation efficiency of strain JA11; where a is the contour plot of the interaction effect and b is the response surface plot of the interaction effect.

[0043] Figure 16 The figure shows the interaction effect of inoculum size and culture temperature on the benzoic acid degradation efficiency of strain JA11; where a is the contour plot of the interaction effect and b is the response surface plot of the interaction effect.

[0044] Figure 17 The graph shows the changes in benzoic acid content in the soil after different treatment times. Detailed Implementation

[0045] This invention provides a strain of *Rhodococcus qingsheng* JA11, with the preservation number CGMCC No. 33448. This *Rhodococcus qingsheng* JA11 is the first strain isolated and screened from nature. It can efficiently degrade phenolic acid autotoxic substances caused by continuous cropping obstacles in ginseng, achieving a degradation rate of 91.51% for benzoic acid. It can also effectively degrade various phenolic acids such as vanillic acid and cinnamic acid, exhibiting a broad degradation spectrum. Furthermore, under field conditions, applying *Rhodococcus qingsheng* JA11 can effectively reduce the benzoic acid content in soil from continuous ginseng cropping, while also controlling the occurrence of ginseng root diseases to a certain extent, significantly improving ginseng survival rate, root length, and other growth indicators, and exhibiting a certain yield-increasing effect. The *Rhodococcus qingshengense* JA11 strain described in this invention grows on NA medium with colonies approximately 3-4 mm in size. The colony is raised, pale pink, opaque, with a relatively smooth and glossy surface, regular edges, and no flagella. It is a Gram-positive bacterium, measuring 1.0-3.0 µm in length and 0.5-1.5 µm in width under a transmission electron microscope. The *Rhodococcus qingshengense* JA11 strain described in this invention is positive for Gram staining, nitrate reduction, nitrite reduction, citrate utilization, and malonic acid utilization tests. The 16S rRNA sequence of *Rhodococcus qingshengense* JA11 described in this invention is shown in SEQ ID NO.3.

[0046] Rhodococcus qingshengii .

[0047] This invention also provides a method for culturing *Rhodococcus qingshengense* JA11 as described in the above-mentioned technical solution, comprising the following steps: inoculating *Rhodococcus qingshengense* JA11 into a culture medium for cultivation to obtain a culture of *Rhodococcus qingshengense* JA11; the culture medium comprises the following components in weight percentage: yeast extract 0.3%~0.7%, soybean peptone 0.5%~1%, and K2HPO4·3H2O 0.5%~1%. This invention involves inoculating *Rhodococcus qingshengense* JA11 into a culture medium for cultivation to obtain a culture of *Rhodococcus qingshengense* JA11. As one embodiment, the culture medium of this invention comprises the following components in weight percentage: yeast extract 6.32 g / L, soybean peptone 7.44 g / L, and K2HPO4·3H2O 7.58 g / L. As one embodiment, the cultivation temperature of *Rhodococcus qingshengense* JA11 in this invention is 25℃~31℃, the initial pH is 5~7, and the initial inoculum size is 1%~3%. In another embodiment, the culture temperature of strain JA11 of the present invention is 31°C, the initial pH is 6.2, the initial inoculum size is 2%, the rotation speed is 180 r / min, and the bottling volume is 300mL / 500mL.

[0048] This invention also provides a microbial inoculant, the effective component of which includes *Rhodococcus qingshengense* JA11 as described in the above-described technical solution or a culture of *Rhodococcus qingshengense* JA11 obtained by the cultivation method described in the above-described technical solution. As one embodiment, the effective viable count of *Rhodococcus qingshengense* JA11 in the microbial inoculant of this invention is 1.1 × 10⁻⁶. 7 CFU / mL ~1.1×10 9 CFU / mL. As another embodiment, the effective viable count of *Rhodococcus qingshengense* JA11 in the microbial agent of the present invention is 1.1 × 10⁻⁶. 9 CFU / mL.

[0049] This invention also provides the application of *Rhodococcus qingsheng* JA11, the culture of *Rhodococcus qingsheng* JA11 obtained by the cultivation method described in the above-described technical solutions, or the microbial inoculant described in the above-described technical solutions in alleviating crop continuous cropping obstacles. As one embodiment, the method for alleviating crop continuous cropping obstacles according to this invention includes degrading autotoxic substances produced by crops in the soil; the autotoxic substances include at least one of benzoic acid, salicylic acid, cinnamic acid, and vanillic acid. As one embodiment, *Rhodococcus qingsheng* JA11 of this invention can achieve a degradation rate of 91.51% for benzoic acid at 200 µg / mL, and can also effectively degrade various phenolic acids such as vanillic acid and cinnamic acid, exhibiting a broad degradation spectrum.

[0050] The present invention also provides a method for alleviating crop rotation obstacles, comprising the following steps:

[0051] The soil in continuously cropped fields is treated with the *Rhodococcus qingshengensis* JA11 described in the above technical solution, the culture of *Rhodococcus qingshengensis* JA11 obtained by the culture method described in the above technical solution, or the microbial agent described in the above technical solution.

[0052] This invention utilizes the *Rhodococcus qingshengensis* JA11 described in the above technical solution, the culture of *Rhodococcus qingshengensis* JA11 obtained by the cultivation method described in the above technical solution, or the microbial inoculant described in the above technical solution to treat the soil of continuously cropped fields.

[0053] As one embodiment, the treatment of soil in continuously cropped fields according to the present invention includes mixing *Rhodococcus qingshengense* JA11, a culture of *Rhodococcus qingshengense* JA11 obtained by the cultivation method described in the above-described technical solution, or a microbial agent described in the above-described technical solution into the soil; the concentration of the strain in the culture or microbial agent is 1.1 × 10⁻⁶. 7 CFU / mL ~1.1×10 9 CFU / mL; the dosage of the culture or microbial agent added is 200~400 mL / mL. 2 In another embodiment, the concentration of the bacterial strain in the culture or microbial agent of the present invention is 1.1 × 10⁻⁶. 9 CFU / mL; the dosage of the culture or microbial agent added is 400 mL / mL. 2 .

[0054] This invention also provides the application of *Rhodococcus qingshengense* JA11 as described in the above technical solutions, the culture of *Rhodococcus qingshengense* JA11 obtained by the culture method described in the above technical solutions, the microbial inoculant described in the above technical solutions, or the method described in the above technical solutions in any one or more of the functions described in ① to ④:

[0055] ① Reduce benzoic acid content;

[0056] ② Reduce the occurrence of crop root diseases;

[0057] ③ Improve crop growth indicators;

[0058] ④ Increase crop yield.

[0059] In one embodiment, the crop described in this invention is ginseng. In another embodiment, the *Rhodococcus qingshengensis* JA11 described in this invention can improve the survival rate of ginseng at harvest. In yet another embodiment, *Rhodococcus qingshengensis* JA11 described in this invention can alleviate the inhibition of ginseng growth, promote ginseng growth indicators such as root length, root diameter, and fresh root weight, and significantly increase ginseng yield, with a yield increase rate of up to 64.89%.

[0060] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a strain of Rhodococcus qingsheng and its application in alleviating crop continuous cropping obstacles, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0061] Example 1

[0062] Isolation and screening of strain JA11

[0063] 1. Isolation and initial screening of bacteria in soil samples

[0064] Ninety-eight soil samples collected from different ginseng-growing areas were isolated using an enrichment-acclimatization-re-isolation method. A total of 132 bacterial strains were obtained that could stably grow in an inorganic salt culture medium containing benzoic acid. These 132 bacterial strains were purified, numbered, and preserved. Ultraviolet spectrophotometry was used for initial screening of the 132 bacterial strains, and bacteria with a degradation rate of over 75% were further screened.

[0065] 2. Secondary screening of bacteria capable of degrading phenolic acids

[0066] High-performance liquid chromatography (HPLC) was used to re-screen the bacteria obtained from the initial screening, further determining the degradation effect and degradation spectrum of benzoic acid by the benzoic acid-degrading bacteria. The isolated degrading strains were cultured in LB liquid medium to prepare seed cultures, and inoculated at 2% (v / v) into MSM medium containing 200 mg / L benzoic acid, vanillic acid, cinnamic acid, salicylic acid, palmitic acid, and gallic acid, respectively. MSM culture without added bacteria served as a control. The cultures were incubated at 28℃ with shaking at 180 r / min for 3 days, and samples were collected. The culture solutions were 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 Aichrom Bond-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 water, 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 rates of different phenolic acids by the degrading strains were calculated. D The formula for calculating ) is:

[0067] , Formula I;

[0068] In the formula, C o represents the initial concentration of the sample (mg / L). C t represents the concentration (mg / L) of the sample after treatment with degrading bacteria.

[0069] After further screening, a strain named JA11 was obtained, exhibiting certain degradation activities against benzoic acid, vanillic acid, cinnamic acid, and salicylic acid. The calculated degradation rates of different phenolic acids by strain JA11 are shown below. Figure 1 As shown, strain JA11 exhibits degradation rates of 91.51%, 40.58%, 99.64%, and 49.65% for benzoic acid, vanillic acid, cinnamic acid, and salicylic acid at 200 mg / L, respectively. Besides its high degradation efficiency for benzoic acid, strain JA11 also demonstrates significant degradation effects on salicylic acid, cinnamic acid, and vanillic acid—three phenolic acids that are major autotoxic substances produced in ginseng-continuously cropped soils.

[0070] Example 2

[0071] Identification of strain JA11

[0072] 1. Morphological identification

[0073] The colony morphology of the degrading strain JA11 on NA plates was observed, and the strain was subjected to Gram staining and transmission electron microscopy. The colony morphology of strain JA11 was obtained as follows: Figure 2 As shown in A, the Gram staining results of strain JA11 are as follows: Figure 2 As shown in B, the electron microscopic observation results of strain JA11 are as follows: Figure 2 As shown in C.

[0074] It can be seen that the colonies of strain JA11 grown on NA medium are about 3-4 mm in size, with raised bacterial growth, light pink color, opaque colonies, relatively smooth and glossy surface, regular edges, no flagella, Gram-positive bacteria, and 1.0-3.0 µm in length and 0.5-1.5 µm in width under transmission electron microscopy.

[0075] 2. Physiological and biochemical identification

[0076] The morphological characteristics and physiological and biochemical properties of the strain were determined according to Bergey's Manual of Bacterial Identification and the Manual of Systematic Identification of Common Bacteria. The physiological and biochemical results of strain JA11 are shown in Table 1, where "+" indicates a positive reaction and "−" indicates a negative reaction. The physiological and biochemical results showed that strain JA11 was positive in Gram staining, nitrate reduction, nitrite reduction, citrate utilization, and malonic acid utilization tests. Based on the morphological characteristics and physiological and biochemical test results, and referring to Bergey's Manual of Bacterial Identification, strain JA11 was preliminarily identified as... Rhodococcus sp.

[0077] Table 1. Results of physiological and biochemical assays for strain JA11

[0078]

[0079] 3. Molecular biological identification

[0080] Bacterial DNA was extracted using a molecular biology identification kit for bacteria (Beijing Solarbio Science & Technology Co., Ltd. D1600-100) for molecular biological identification of the degrading bacteria. The nucleotide sequences of the PCR primers (27F / 1492R) are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0081] SEQ ID NO.1: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0082] SEQ ID NO. 2: 5'-TACGGTTACCTTGTTACGACTT-3'.

[0083] The PCR products were sent to bioengineering sequencing facilities for sequence analysis and homology comparison using MEGA 11.0 software. The Bootstrap assay (1000 replicates) was used for verification. Corynebacterium diphtheriae NCTC 11397(X84248) is an outgroup. A phylogenetic tree was constructed using the maximum likelihood method to obtain the phylogenetic tree for strain JA11, as shown below. Figure 3 As shown, the measured 16S rRNA sequence was submitted to GenBank.

[0084] The 16S rRNA sequence of strain JA11 is shown in SEQ ID NO.3.

[0085] Using DNA from the degrading strain JA11 as a template, PCR amplification was performed using universal primers for the bacterial 16S rRNA gene. The sequence was compared with NCBI data, and the results showed that the degrading strain JA11 was similar to *Rhodococcus qingshengense* (a type of bacterium). Rhodococcus qingshengii YL-3 OK136187) belonged to the same branch, and finally, based on morphological observation, physiological and biochemical index determination, and 16S rRNA sequence analysis, strain JA11 was identified as a strain of Rhodococcus qingshengense (YL-3 OK136187). Rhodococcus qingshengii It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 17, 2025, with accession number CGMCC NO.33448.

[0086] Example 3

[0087] Optimization of culture medium formulation and benzoic acid degradation characteristics of strain JA11

[0088] 1. Optimization of culture medium formulation

[0089] LB medium was used as the basal fermentation medium, and subsequent medium optimization experiments were conducted based on this medium formulation. The following different carbon sources were used to replace the original optimal fermentation medium with equal masses: yeast extract, sucrose, glucose, maltose, and soluble starch; the carbon source concentration gradients were 0.1%, 0.3%, 0.5%, 0.7%, and 0.9%, respectively. The following different nitrogen sources were used to replace the optimal fermentation medium with equal masses: ammonium sulfate, soybean peptone, potassium nitrate, and ammonium chloride; the nitrogen source concentration gradients were 0.5%, 0.75%, 1%, 1.25%, and 1.5%, respectively. The following different inorganic salts were used to replace the optimal fermentation medium with equal masses: NaCl, Na₂HPO₄·12H₂O, K₂HPO₄·3H₂O, MgSO₄, and CaCl₂; the inorganic salt concentration gradients were 0.5%, 0.75%, 1%, 1.25%, and 1.5%, respectively.

[0090] After 24 hours of cultivation, the effects of each single factor on cell yield were finally clarified, and the optimal culture medium formulation was determined to be: yeast extract, peptone, and K₂HPO₄·3H₂O. The effects of different concentrations of yeast extract, peptone, and K₂HPO₄·3H₂O on bacterial growth under single-factor conditions are shown below. Figures 4-6 As shown. According to Figures 4-6 The results show that the highest absorbance was measured when the yeast powder concentration was 0.5%, the soybean peptone concentration was 0.75%, and the K2HPO4·3H2O concentration was 0.75%, indicating the highest cell growth.

[0091] 2. Optimization of the addition amounts of yeast powder, peptone, and K2HPO4·3H2O

[0092] Continuing with yeast powder additions of 0.3%, 0.5%, and 0.7%, soybean peptone additions of 0.5%, 0.75%, and 1%, and K₂HPO₄·3H₂O additions of 0.5%, 0.75%, and 1%, a Box-Behnken design was used to optimize the response surface methodology for these three factors. A three-factor, three-level experiment was designed, and the experimental factors and levels are shown in Table 2. The experimental design is shown in Table 3. The same fermentation medium without added bacterial solution was used as a control. After 1 day of cultivation, the results were measured. OD 600 .

[0093] Table 2 Factors and levels in Box-Behnken design experiments

[0094]

[0095] Table 3 Box-Benhnken Experimental Design

[0096]

[0097] Response surface methodology was used to optimize the response surface of carbon source, nitrogen source, and inorganic salt at three levels. The fitted quadratic polynomial regression equation is as follows:

[0098] Y JA11 A B C AB AC BC A 2 B 2 C 2 Formula II.

[0099] In Formula II, A, B, and C represent the coding values ​​for the amounts of yeast powder, soybean peptone, and K2HPO4·3H2O added, respectively, and Y is the response value for the growth of strain JA11. OD 600 .

[0100] Table 4 Results of Response Surface Design Variance Analysis

[0101]

[0102] The results of the response surface design variance analysis are shown in Table 4. As can be seen from Table 4, this model... R 2 =93.53%; P =0.0021 < 0.01, which is highly significant; the lack-of-fit term 0.3137 > 0.05 is not significant, indicating that the model is meaningful and fits the experimental data well. The probability of the experimental data not fitting the model is very small, and it can be used for theoretical prediction. Software analysis was used to obtain the results of the interaction effects of carbon source, nitrogen source, and inorganic salt as follows: Figures 7-9 As shown, where Figure 7 The response surface plot (b) and contour plot (a) show the interaction between carbon and nitrogen sources. Figure 8 The response surface plot (b) and contour plot (a) show the interaction between carbon source and inorganic salt. Figure 9The response surface plot (b) and contour plot (a) show the interaction between nitrogen source and inorganic salts. The optimal values ​​for the three main factors were obtained as follows: yeast extract 6.32 g / L, soybean peptone 7.44 g / L, and K₂HPO₄·3H₂O 7.58 g / L. Under these conditions, the predicted... OD 600 The maximum value was 2.161, based on 3 actual tests. OD 600 The average value was 2.093, which was higher than the predicted value and basically consistent with the experimental value. The experimental value and the predicted value had a high degree of fit, indicating that the established model was effective.

[0103] 3. Optimization of degradation conditions for strain JA11

[0104] The tested strain JA11 was inoculated into LB liquid medium and cultured with shaking for 24 h (28℃, 180 r / min) to obtain a bacterial suspension. The bacterial suspension was then inoculated into an inorganic salt medium containing 200 mg / L benzoic acid for single-factor experiments. The effects of pH (4.0, 5.0, 6.0, 7.0, 8.0), temperature (25℃, 28℃, 31℃, 34℃, 37℃), inoculum size (1%, 2%, 3%, 4%, 5%), and substrate concentration (100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L) on the degradation efficiency of the strain were determined. After 3 days of culture, the growth rate and benzoic acid degradation efficiency of the strain were measured. The effects of each single factor on the degradation efficiency of the strain were finally clarified, and the results of the influence of initial pH, substrate concentration, culture temperature, and inoculum size on the growth and benzoic acid degradation ability of strain JA11 are as follows: Figures 10-13 As shown. Figures 10-13 The results showed that strain JA11 exhibited the best degradation effect on benzoic acid at pH 5-8, substrate concentration 100-300 mg / L, temperature 28℃-31℃, and inoculum amount 1%-3%, with a degradation rate greater than 70.00% and good cell growth.

[0105] Based on the single-factor experiments, three factors that play a major role in the degradation rate of benzoic acid were identified: culture temperature (25℃, 28℃, 31℃), initial pH (5, 6, 7), and initial inoculum size (1%, 2%, 3%). The Box-Behnken design was used to optimize the response surface methodology for the three key factors of pH, temperature, and inoculum size. A three-factor, three-level experiment was designed, and the experimental design and grouping are shown in Table 5.

[0106] Strains JA11 were added to MSM inorganic salt medium (benzoic acid content 200 µg / mL) with benzoic acid as the sole carbon source, and the same MSM medium without strain JA11 was used as a control. After 3 days of culture, the degradation of benzoic acid was detected and the degradation rate of benzoic acid was obtained, as shown in Table 5.

[0107] Table 5. Box-Benhnken experimental design based on 3 factors and 3 levels.

[0108]

[0109] The quadratic polynomial regression model established through fitting is shown in Equation III:

[0110] Y JA11 A B C AB AC BC A 2 B 2 C 2 Formula III.

[0111] In Equation III, A, B, and C represent the encoded values ​​of factors pH, temperature (°C), and inoculum size (%), respectively, and Y represents the degradation rate of benzoic acid. This model... R 2 =97.53%, P =(0.0001)<0.05 is highly significant, and the lack-of-fit term =0.0918>0.05 is not significant, indicating that the model is meaningful and the experimental data fits well. The probability of the experimental data not fitting the model is very small, and it can be used for theoretical prediction. Software analysis was used to obtain the interaction effects of inoculum size, culture temperature, and initial pH on the benzoic acid degradation efficiency of strain JA11, as shown below. Figures 14-16 As shown, where Figure 14 The results of the interaction between culture temperature and initial pH on the benzoic acid degradation efficiency of strain JA11 are shown in the figure, including the interaction effect contour plot as shown in a and the response surface plot as shown in b. Figure 15 The results of the interaction between inoculum size and initial pH on the benzoic acid degradation efficiency of strain JA11 are shown in the figure, including the interaction contour plot as shown in a and the response surface plot as shown in b. Figure 16The results show the interaction between inoculum size and culture temperature on the benzoic acid degradation efficiency of strain JA11, including the interaction contour plot shown in a and the response surface plot shown in b. The optimal values ​​for the three main factors were obtained as follows: pH 6.208, temperature 31.007℃, and inoculum size 2.039%. Under these conditions, the predicted maximum degradation rate was 89.982%, and the average of the three actual test results was 91.34%, which was higher than the predicted value. The experimental values ​​and predicted values ​​showed a high degree of fit, indicating that the established model was effective (Table 6).

[0112] Table 6 Results of Response Surface Experiment

[0113]

[0114] In summary, the optimal fermentation medium formulation for strain JA11 was determined to be: yeast extract (6.32 g / L), soybean peptone (7.44 g / L), and K₂HPO₄·3H₂O (7.58 g / L). Under the culture conditions of pH 6.2, temperature 31℃, inoculum size 2%, rotation speed 180 r / min, and flask volume 300 mL / 500 mL, the degradation rate of benzoic acid at 200 µg / mL was 91.34%, and the optimized cell biomass reached [value missing]. It increased by 12.5 times.

[0115] Example 4

[0116] Evaluation of the field application effect of benzoic acid degrading bacteria JA11

[0117] The 2025 field trial was conducted in a ginseng continuous cropping plot in Fusong County, Baishan City, Jilin Province (5 years of continuous cropping). The application method is shown in Table 7. The culture used was prepared according to the above method (seed liquid cultured on LB medium for 24 h was inoculated into 300 / 500 mL of fermentation medium at a 2% volume fraction, and cultured at 31℃ and 180 r / min for 24 h). Commercially available biochar (Gongyi Shengxiang Activated Carbon Business Department) for alleviating continuous cropping obstacles was used at 75 g / m³. 2 The treatment dose and an equal volume of fermentation medium (CK) served as the control. A total of 5 treatments were administered, with 3 replicates per treatment, resulting in 15 plots of 5 m² each. 2Cultivation conditions (soil type, fertilization, and plant spacing) were consistent across all experimental plots in each experimental site. Healthy ginseng (large ginseng, three years old) with uniform growth were selected for transplanting. To avoid cross-influence between different treatments, only the middle portion was sampled during the survey. The planting density in the Fusong experimental site was 1 meter four rows, with 15 ginseng plants per row. The bacterial culture solution was added to 500 mL and then mixed with the soil before application. Soil samples were taken for testing at 30, 80, and 150 days after treatment. At harvest at 150 days, the agronomic traits of ginseng and the incidence of root diseases were investigated. The grading standards for ginseng root diseases are shown in Table 8. The calculation of control effects and disease index is shown in Equations IV and V.

[0118] Table 7. Field Trial Treatments and Dosages

[0119]

[0120] Table 8 Grading Standards for Ginseng Root Diseases

[0121]

[0122] Formula IV;

[0123] , formula V.

[0124] 1. Detection of benzoic acid content in soil

[0125] The changes in benzoic acid content in soil were detected using the Martens method. The results of changes in benzoic acid content in soil after different treatment times are shown below. Figure 17 As shown, Figure 17 The study measured the benzoic acid content in the rhizosphere soil of ginseng in the Fusong experimental site. The results showed that the benzoic acid content in the soil of the control treatment in the Fusong experimental site initially increased and then decreased over time. At 30 days post-treatment, treatment group 2 showed the best degradation effect. After 80 days of treatment with strain JA11, the degradation effect of the strain reached its maximum, with a degradation rate of 59.15%. After 150 days of treatment, the degradation effect of strain JA11 on benzoic acid decreased, but the degradation level was still higher than that of biochar treatment, with treatment group 1 showing the best effect, reaching the highest degradation rate of 43.17% in the Fusong experimental site. This may be due to strain loss over time. The benzoic acid concentration in the uninoculated biochar treatment decreased to some extent, but the rate of increase in benzoic acid degradation decreased over time, and the degradation effect was not as good as that of strain JA11 treatment. Therefore, strain JA11 treatment is superior to commercially available biochar.

[0126] 2. Effects of strain JA11 on ginseng growth

[0127] Agronomic traits of ginseng were investigated and measured at harvest. The statistical results of ginseng agronomic traits after treatment with strain JA11 are shown in Table 9. It can be seen that adding different concentrations of JA11 fermentation broth promoted the growth of ginseng, with treatment group showing the best effect, achieving a yield increase of 64.89%. Furthermore, the number of surviving ginseng at harvest was significantly higher than in the control treatments. This indicates that treatment with strain JA11 in soil containing benzoic acid significantly alleviated the inhibition of ginseng growth, and all growth indicators were improved.

[0128] Table 9. Agronomic traits of ginseng after treatment with strain JA11 for 150 days.

[0129]

[0130] 3. Effects of strain JA11 on ginseng root diseases

[0131] Based on the grading method shown in Table 8, and formulas IV and V, an investigation was conducted on the occurrence of ginseng root diseases during the harvest period. The effects of strain JA11 treatment on ginseng root diseases after 150 days are shown in Table 10. According to the results in Table 10, treatment with strain JA11 has a certain ameliorative effect on the occurrence of ginseng root diseases. The treatment with strain JA11 at 200~400 mL / m... 2 After 150 days of treatment at the specified dosage, the efficacy against ginseng root diseases reached 14.61%~16.85%, which was significantly different from that of biochar treatment.

[0132] Table 10. Effects of strain JA11 on ginseng root diseases after 150 days of treatment.

[0133]

[0134] In summary, the fermentation broth of strain JA11 at 400 mL / m 2 and 300 mL / m 2 The degradation rate of benzoic acid at the applied dosage was high, significantly higher than that of the biochar control. Furthermore, the application of the degrading bacteria JA11 in the field significantly improved growth indicators such as root length and root diameter of ginseng, and while having a certain yield-increasing effect, it could also inhibit the occurrence of root diseases of ginseng to a certain extent.

[0135] 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 Rhodococcus globerulus (R. globerulus) JA11, characterized in that, Rhodococcus qingshengii ) JA11, characterized in that, The accession number is CGMCCNo.33448.

2. The culture method of Rhodococcus sp. JA11 according to claim 1, characterized in that, The steps include: inoculating Rhodococcus qingshengense JA11 into a culture medium for culture to obtain a culture of Rhodococcus qingshengense JA11; The culture medium comprises the following components in weight percentage: yeast extract 0.3%~0.7%, soybean peptone 0.5%~1%, and K2HPO4·3H2O 0.5%~1%.

3. A microbial inoculant, characterized in that, The effective components of the microbial agent include the *Rhodococcus qingshengense* JA11 as described in claim 1 or the culture of *Rhodococcus qingshengense* JA11 obtained by the culture method described in claim 2.

4. The microbial inoculant of claim 3, wherein, The effective viable cell number of Rhodococcus globerulus JA11 in the microbial inoculant is 1.1×10 7 CFU / mL~1.1×10 9 CFU / mL.

5. The application of Rhodococcus qingshengense JA11 as described in claim 1, the culture of Rhodococcus qingshengense JA11 obtained by the culture method described in claim 2, or the microbial agent as described in claim 3 or 4 in alleviating the obstacles of continuous ginseng cultivation.

6. Use 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, salicylic acid, cinnamic acid, and vanillic acid.

7. A method for alleviating ginseng continuous cropping obstacles, characterized in that, Includes the following steps: The soil of ginseng-continuous cropping land is treated with the culture of Rhodococcus qingshengense JA11 as described in claim 1, the culture of Rhodococcus qingshengense JA11 obtained by the culture method described in claim 2, or the microbial agent as described in claim 3 or 4.

8. The method of claim 7, wherein, The treatment of soil in ginseng-continuous cropping sites includes mixing the culture of Rhodococcus qingshengense JA11 as described in claim 1, the culture of Rhodococcus qingshengense JA11 obtained by the culture method described in claim 2, or the microbial agent described in claim 3 or 4 into the soil; The concentration of the bacterial strain in the culture or microbial agent is 1.1 × 10⁻⁶. 7 CFU / mL ~1.1×10 9 CFU / mL; The culture or microbial inoculant is mixed at a dosage of 200 to 400 mL / m 2 .

9. The application of *Rhodococcus qingshengense* JA11 as described in claim 1, the culture of *Rhodococcus qingshengense* JA11 obtained by the culture method described in claim 2, the microbial agent as described in claim 3 or 4, or the method described in claim 7 or 8, 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; The ginseng growth indicators are one or more of the following: root survival count, root length, root diameter, and underground fresh weight.

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