Pseudomonas monteilii BDLZ-3 and application thereof in relieving continuous cropping obstacles of crops

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to pseudomonas monteilii BDLZ-3 and application thereof in relieving continuous cropping obstacles of crops. The invention discloses a strain of pseudomonas monteilii BDLZ-3, the preservation number of which is CGMCC (China General Microbiological Culture Collection Center) No.34349. The pseudomonas monteilii BDLZ-3 is separated and screened from the nature for the first time, phenolic acid autotoxic substances generated in the ginseng continuous cropping obstacle process can be efficiently degraded, the degradation rate of benzoic acid can reach 95.36%, various phenolic acids such as vanillic acid, cinnamic acid, palmitic acid and gallic acid can be effectively degraded, and the degradation spectrum is wide; when the pseudomonas monteilii BDLZ-3 is applied in a field, the content of benzoic acid in soil can be effectively reduced, meanwhile, the occurrence of ginseng root diseases can be controlled to a certain extent, the prevention and treatment effect reaches 41.23%, the growth indexes such as root length and root thickness are remarkably improved, and the yield increasing effect is achieved.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Pseudomonas montmorillonii BDLZ-3 and its application in alleviating crop continuous cropping obstacles. Background Technology

[0002] Ginseng ( Panax ginseng Ginseng, a perennial herbaceous plant belonging to the genus *Panax* of the family Araliaceae, is rich in polysaccharides and saponins, possessing various health benefits such as improving eyesight, regulating blood pressure, and enhancing intelligence. It is a valuable traditional Chinese medicine. However, as a perennial herb, ginseng has a long cultivation cycle and requires specific growing environments, which further exacerbates the problem of continuous cropping obstacles. Specifically, this manifests as low seedling survival rates, increased root diseases, and decreased yield and quality, severely hindering the sustainable development of the industry. Therefore, effectively alleviating the continuous cropping obstacles of ginseng has become a crucial issue that urgently needs to be addressed. Summary of the Invention

[0003] The purpose of this invention is to solve the aforementioned problems existing in the prior art and to find an effective solution to alleviate the continuous cropping obstacles of ginseng. To this end, this invention provides a strain of *Pseudomonas montmorillonii* BDLZ-3 and its application in alleviating continuous cropping obstacles.

[0004] This invention provides a strain of Pseudomonas montelukastii ( Pseudomonas monteilii BDLZ-3, with accession number CGMCC No.34349.

[0005] The present invention also provides a method for culturing the aforementioned Pseudomonas montelukastii BDLZ-3, comprising the following steps: inoculating Pseudomonas montelukastii BDLZ-3 into a culture medium for culturing to obtain a culture of Pseudomonas montelukastii BDLZ-3; The culture medium comprises the following components in weight percentage: 0.3% to 0.7% yeast extract, 1% to 1.5% soybean peptone, and anhydrous magnesium sulfate.

[0006] The present invention also provides a microbial agent, wherein the effective component of the microbial agent includes Pseudomonas montelukastiensis BDLZ-3 as described in the above technical solution or a culture of Pseudomonas montelukastiensis BDLZ-3 obtained by the culture method described in the above technical solution.

[0007] Preferably, the effective viable count of *Pseudomonas montelukastiensis* BDLZ-3 in the microbial agent is 4 × 10⁻⁶. 7 CFU / mL ~4×10 9 CFU / mL.

[0008] The present invention also provides the application of Pseudomonas montmorillonii BDLZ-3 as described in the above technical solution, the culture of Pseudomonas montmorillonii BDLZ-3 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.

[0009] Preferably, the mitigation of crop rotation obstacles includes degrading autotoxic substances produced by crops in the soil; The autotoxic substances include at least one of benzoic acid, vanillic acid, cinnamic acid, palmitic acid, and gallic acid.

[0010] The present invention also provides a method for alleviating crop rotation obstacles, comprising the following steps: The soil in continuously cropped fields is treated with Pseudomonas montmorillonii BDLZ-3 as described in the above technical solution, the culture of Pseudomonas montmorillonii BDLZ-3 obtained by the culture method described in the above technical solution, or the microbial agent described in the above technical solution.

[0011] Preferably, the treatment of the soil in the continuously cropped land includes mixing the above-described Pseudomonas montmorillonii BDLZ-3, the culture of Pseudomonas montmorillonii BDLZ-3 obtained by the above-described culture method, or the above-described microbial agent into the soil. The concentration of the strain in the culture or microbial agent is 4 × 10⁻⁶. 7 ~4×10 9 CFU / mL; The dosage of the culture or microbial agent added is 500~1000 mL / m 2 .

[0012] This invention also provides the application of *Pseudomonas montelukastiensis* BDLZ-3 as described in the above-described technical solutions, the culture of *Pseudomonas montelukastiensis* BDLZ-3 obtained by the culture method described in the above-described technical solutions, 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 crop root diseases; ③ Improve crop growth indicators; ④ Increase crop yield.

[0013] Preferably, the crop is ginseng.

[0014] Beneficial effects: This invention provides a strain of *Pseudomonas montmorillonii* BDLZ-3, with the preservation number CGMCC No. 34349. This *Pseudomonas montmorillonii* BDLZ-3, obtained for the first time from nature, can efficiently degrade phenolic acid autotoxic substances caused by continuous cropping obstacles in ginseng. Its degradation rate for benzoic acid reaches 95.36%, and it can also effectively degrade various phenolic acids such as vanillic acid, cinnamic acid, palmitic acid, and gallic acid, exhibiting a broad degradation spectrum. Furthermore, field application of this *Pseudomonas montmorillonii* BDLZ-3 can effectively reduce the benzoic acid content in the soil while also controlling the occurrence of ginseng root diseases to a certain extent, achieving a control effect of 41.23%. It significantly improves growth indicators such as root length and diameter and also has a yield-increasing effect.

[0015] Biological Preservation Information Pseudomonas montelukastii BDLZ-3, classified as Pseudomonas monteilii It was deposited on April 25, 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. 34349. Attached Figure Description

[0016] 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 A statistical chart showing the degradation effect of strain BDLZ-3 on different phenolic acids; Figure 2 The images show the colony morphology, Gram staining, and cell morphology of strain BDLZ-3; where A is the colony morphology of strain BDLZ-3, B is the Gram staining result of strain BDLZ-3, and B is the electron micrograph of strain BDLZ-3. Figure 3 This is a schematic diagram of the phylogenetic tree of strain BDLZ-3 constructed using the maximum likelihood method based on the 16S rRNA sequence. Figure 4 The figure shows the effect of initial pH on the growth of strain BDLZ-3 and its degradation effect on benzoic acid. Figure 5 The figure shows the effect of substrate concentration on the growth of strain BDLZ-3 and its degradation effect on benzoic acid. Figure 6 Figure 1 shows the effect of inoculum size on the growth of strain BDLZ-3 and its degradation of benzoic acid. Figure 7 Figure showing the effect of culture temperature on the growth of strain BDLZ-3 and its degradation of benzoic acid. Figure 8The figure shows the interaction effect of culture temperature and initial pH on the benzoic acid degradation efficiency of strain BDLZ-3; where a is the contour plot of the interaction effect and b is the response surface plot of the interaction effect. Figure 9 The figure shows the interaction effect of inoculum size and initial pH on the benzoic acid degradation efficiency of strain BDLZ-3; where a is the contour plot of the interaction effect and b is the response surface plot of the interaction effect. Figure 10 The figure shows the interaction effect of inoculum size and culture temperature on the benzoic acid degradation efficiency of strain BDLZ-3; where a is the contour plot of the interaction effect and b is the response surface plot of the interaction effect. Figure 11 The 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. Figure 12 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. Figure 13 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. Figure 14 The graph shows the changes in benzoic acid content in the soil after treatment with strain BDLZ-3. Detailed Implementation

[0018] This invention provides a strain of Pseudomonas montelukastii ( Pseudomonas monteiliiBDLZ-3, with preservation number CGMCC No. 34349, is the first *Pseudomonas montmorillonii* BDLZ-3 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 95.36% for benzoic acid. It can also effectively degrade various phenolic acids such as vanillic acid, cinnamic acid, palmitic acid, and gallic acid, exhibiting a broad degradation spectrum. Furthermore, field application of *Pseudomonas montmorillonii* BDLZ-3 can effectively reduce benzoic acid content in the soil while also controlling ginseng root diseases to a certain extent, achieving a control effect of 41.23%. It significantly improves growth indicators such as root length and diameter, and also has a yield-increasing effect. On NA medium, *Pseudomonas montmorillonii* BDLZ-3 colonies are white, opaque, round, with smooth and regular edges, a raised center, and are relatively moist. The bacteria are short rod-shaped or ellipsoidal, do not produce spores, and are Gram-negative. Transmission electron microscopy (TEM) showed that strain BDLZ-3 was rod-shaped, measuring (1.5~2.0) μm × (0.5~1.0) μm, and possessed flagella. The *Pseudomonas montelukastii* strain described in this invention showed positive results in physiological and biochemical tests for starch hydrolysis, indole, fluorescent dye, hanging drop kinetics, malonic acid, and citrate, while showing negative results in tests for oxidase, gelatin liquefaction, methyl red, and VP assay. The 16S rRNA sequence of *Pseudomonas montelukastii* BDLZ-3 described in this invention is shown in SEQ ID NO.1: Pseudomonas monteilii .

[0019] This invention also provides a method for culturing *Pseudomonas montelukastii* BDLZ-3, comprising the following steps: inoculating *Pseudomonas montelukastii* BDLZ-3 into a culture medium for cultivation to obtain a culture of *Pseudomonas montelukastii* BDLZ-3; the culture medium comprises the following components by weight percentage: yeast extract 0.3%~0.7%, soybean peptone 1%~1.5%, and anhydrous magnesium sulfate 1%~1.5%. As one embodiment, the culture medium of this invention comprises the following components by weight percentage: yeast extract 0.6%, soybean peptone 1.2%, and anhydrous magnesium sulfate 1.4%. As another embodiment, the culture medium of this invention comprises the following components by weight percentage: yeast extract 5.9 g / L, soybean peptone 11.96 g / L, and anhydrous magnesium sulfate 13.99 g / L. As one embodiment, the culture temperature of strain BDLZ-3 of this invention is 25℃~31℃, the initial pH is 6~8, and the initial inoculum size is 1%~3%. In another embodiment, the strain BDLZ-3 of the present invention was cultured at a temperature of 27.87℃, an initial pH of 7.45, an initial inoculum size of 2.042%, a rotation speed of 180 r / min, and a bottling volume of 100 mL / 250 mL.

[0020] This invention also provides a microbial inoculant, the effective component of which includes *Pseudomonas montelukastiensis* BDLZ-3 as described in the above-described technical solution, or a culture of *Pseudomonas montelukastiensis* BDLZ-3 obtained by the culture method described in the above-described technical solution. As one embodiment, the effective viable count of *Pseudomonas montelukastiensis* BDLZ-3 in the microbial inoculant of this invention is 4 × 10⁻⁶. 7 CFU / mL ~ 4×10 9 CFU / mL. As another embodiment, the effective viable count of *Pseudomonas montelukastiensis* BDLZ-3 in the microbial agent of the present invention is 4 × 10⁻⁶. 8 CFU / mL.

[0021] This invention also provides the application of *Pseudomonas montmorillonii* BDLZ-3 as described in the above-described technical solutions, cultures of *Pseudomonas montmorillonii* BDLZ-3 obtained by the cultivation methods described in the above-described technical solutions, or microbial agents as 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, vanillic acid, cinnamic acid, palmitic acid, and gallic acid. As one embodiment, *Pseudomonas montmorillonii* BDLZ-3 of this invention, in MSM inorganic salt medium with benzoic acid as the sole carbon source, can achieve a degradation rate of 95.36% for benzoic acid at 50 µg / mL, and can also effectively degrade various phenolic acids such as vanillic acid, cinnamic acid, palmitic acid, and gallic acid, exhibiting a broad degradation spectrum.

[0022] The present invention also provides a method for alleviating crop rotation obstacles, comprising the following steps: The soil in continuously cropped fields is treated with Pseudomonas montmorillonii BDLZ-3 as described in the above technical solution, the culture of Pseudomonas montmorillonii BDLZ-3 obtained by the culture method described in the above technical solution, or the microbial agent described in the above technical solution.

[0023] As one embodiment, the treatment of soil in continuously cropped fields according to the present invention includes mixing *Pseudomonas montmorillonii* BDLZ-3 as described in the above-described technical solution, a culture of *Pseudomonas montmorillonii* BDLZ-3 obtained by the cultivation method described in the above-described technical solution, or a microbial agent as described in the above-described technical solution into the soil; the concentration of the strain in the culture or microbial agent is 4 × 10⁻⁶. 7 ~4×10 9 CFU / mL; the dosage of the culture or microbial agent added is 500~1000 mL / mL. 2 In another embodiment, the concentration of the bacterial strain in the culture or microbial agent of the present invention is 4 × 10⁻⁶. 8 CFU / mL; the dosage of the culture or microbial agent added is 750 mL / mL. 2 .

[0024] This invention also provides the application of *Pseudomonas montelukastiensis* BDLZ-3 as described in the above-described technical solutions, the culture of *Pseudomonas montelukastiensis* BDLZ-3 obtained by the culture method described in the above-described technical solutions, 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 crop root diseases; ③ Improve crop growth indicators; ④ Increase crop yield.

[0025] In one embodiment, the crop described in this invention is ginseng. In another embodiment, the *Pseudomonas montmorillonii* BDLZ-3 described in this invention can increase the survival rate of ginseng at harvest to 73.08%, significantly higher than the 48.08% in the control group without *Pseudomonas montmorillonii* BDLZ-3 treatment. In yet another embodiment, the *Pseudomonas montmorillonii* BDLZ-3 described in this invention 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 a yield increase rate of up to 34.10%. In yet another embodiment, the *Pseudomonas montmorillonii* BDLZ-3 described in this invention can effectively control the occurrence of ginseng root diseases, achieving a control effect of 34.42%~41.23% on root diseases (including red skin disease, rust rot, and root rot), demonstrating a certain degree of control effect on root diseases.

[0026] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a strain of Pseudomonas montmorillonii BDLZ-3 provided by the present invention 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.

[0027] Example 1 Isolation and screening of strain BDLZ-3 1. Isolation and initial screening of bacteria in soil samples Microorganisms were isolated from 60 soil samples collected from ginseng-continuous cropping sites using an enrichment-accumulation-re-separation method. A total of 213 bacterial strains were obtained. The degradation ability of the 213 bacterial strains to benzoic acid was determined by ultraviolet spectrophotometry, and 6 strains with a benzoic acid degradation rate of more than 80% were obtained.

[0028] 2. Bacterial rescreening of soil samples Six bacterial strains obtained from the initial screening were re-screened using high-performance liquid chromatography (HPLC) to further determine their degradation effects and degradation spectra on benzoic acid and other phenolic acid autotoxic substances (vanillic acid, cinnamic acid, palmitic acid, and gallic acid) in soil. Chromatographic analysis was performed using an Aichrom Bond-AQ C18 column (4.6 mm × 250 mm, 5 µm) with an acetonitrile-formic acid aqueous solution (80:20 v / v) as the mobile phase. The formic acid aqueous solution was 0.1% (v / v) formic acid. The flow rate was 0.8 mL / min, the UV detection wavelength was 230 nm, the column temperature was 30 °C, the injection volume was 10 µL, and isocratic elution was used.

[0029] The test strain was cultured in LB liquid medium, and a 2% (volume fraction) bacterial suspension was taken ( OD 600=1.0) was inoculated into MSM-BA medium containing 50 mg / L, with MSM-BA medium without added bacteria as a control. Samples were taken after 3 days, 10 mL each time, extracted three times with an equal volume of ethyl acetate, evaporated to dryness using a rotary evaporator, and then dissolved in 10 mL of methanol. The solution was filtered through a 0.22 µm filter. The degradation rate ( D The calculation formula for is shown in Equation I.

[0030] , Formula I; In the formula, Co The initial concentration of the sample is (mg / L). Ct The concentration of the sample is measured in mg / L.

[0031] After secondary screening, strain BDLZ-3 was the only one among the six initially screened strains to exhibit degradation activity against vanillic acid, cinnamic acid, palmitic acid, and gallic acid. The degradation rates of these five phenolic acids by strain BDLZ-3 are shown below. Figure 1 As shown in Table 1, in addition to exhibiting high degradation activity for benzoic acid, BDLZ-3 strain also showed significant degradation effects on vanillic acid and gallic acid, with degradation rates reaching 90.42% and 61.75%, respectively.

[0032] Table 1. Degradation effect of strain BDLZ-3 on phenolic acids

[0033] Example 2 Identification of strain BDLZ-3 1. Morphological identification The colony morphology of the degrading strain BDLZ-3 on NA plates was observed, and the strain was subjected to Gram staining and transmission electron microscopy. The colony morphology of strain BDLZ-3 was obtained as follows: Figure 2 As shown in A, the Gram staining results of strain BDLZ-3 are as follows: Figure 2 As shown in B, the electron microscopic observation results of strain BDLZ-3 are as follows: Figure 2 As shown in C.

[0034] As can be seen, strain BDLZ-3 colonies on NA medium are white, opaque, round, with smooth and regular edges, a raised center, and are relatively moist. The cells are short rod-shaped or ellipsoidal, do not produce spores, and are Gram-negative. TEM transmission electron microscopy shows that strain BDLZ-3 is rod-shaped, measuring (1.5~2.0) μm × (0.5~1.0) μm, and has flagella.

[0035] 2. Physiological and biochemical identification 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 BDLZ-3 are shown in Table 2, where "+" indicates a positive reaction and "−" indicates a negative reaction. The physiological and biochemical results showed that strain BDLZ-3 was positive for starch hydrolysis, indole, fluorescent dye, hanging drop kinetics, malonic acid, and citrate tests (Table 2). Based on the morphological characteristics and physiological and biochemical test results, and referring to Bergey's Manual of Bacterial Identification, strain BDLZ-3 was preliminarily identified as… Pseudomonas sp.

[0036] Table 2. Physiological and biochemical assay results of strain BDLZ-3

[0037] 3. Molecular biological identification Bacterial DNA was extracted using a molecular biology identification kit (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.2 and SEQ ID NO.3. SEQ ID NO.2: 5'-AGAGTTTGATCCTGGCTCAG-3'; SEQ ID NO. 3: 5'-TACGGTTACCTTGTTACGACTT-3'.

[0038] 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. Escherichia coli strain U 5 / 41 NR 024570.1 is an outgroup. A phylogenetic tree was constructed using the maximum likelihood method, resulting in the identification phylogenetic tree of strain BDLZ-3 as follows: Figure 3 As shown, the measured 16S rRNA sequence was submitted to GenBank.

[0039] The 16S rRNA sequence of strain BDLZ-3 is shown in SEQ ID NO.1.

[0040] Using DNA from the degrading strain BDLZ-3 as a template, PCR amplification was performed using universal primers for the bacterial 16S rRNA gene. The sequence was compared using EzBioCloud and NCBI, and the results showed that the degrading strain BDLZ-3 was similar to *Pseudomonas montelukastii* (…). Pseudomonas monteiliiDB13 MN889014.1) belong to the same branch. Finally, based on morphological observation, physiological and biochemical index determination, and 16S rRNA sequence analysis, strain BDLZ-3 was identified as a strain of Pseudomonas montelukastii (…). Pseudomonas monteilii It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 25, 2025, with accession number CGMCC NO.34349.

[0041] Example 3 Optimization of benzoic acid degradation conditions and culture medium formulation for strain BDLZ-3 The tested strain BDLZ-3 was inoculated into LB liquid medium and cultured with shaking for 12 h (30℃, 180 r·min⁻¹) to obtain a bacterial suspension. The bacterial suspension was then inoculated into an inorganic salt medium containing 50 mg·L⁻¹ benzoic acid for single-factor experiments. The pH (5.0, 6.0, 7.0, 8.0, 9.0), temperature (25℃, 28℃, 31℃, 34℃, 37℃), inoculum size (1%, 2%, 3%, 4%, 5%), and substrate concentration (10 mg·L⁻¹) were measured. -1 30 mg·L -1 50 mg·L -1 70 mg·L -1 90 mg·L -1 The effects of each single factor on the degradation efficiency of the strain were investigated. After 3 days of cultivation, the growth rate and benzoic acid degradation efficiency of the strain were measured to clarify the influence of each factor on the degradation efficiency. The effects of initial pH, substrate concentration, culture temperature, and inoculum size on the growth and benzoic acid degradation ability of strain BDLZ-3 were obtained as follows: Figures 4-7 As shown, where, Figure 4 The figure shows the effect of initial pH on the growth of strain BDLZ-3 and its degradation effect on benzoic acid. Figure 5 The figure shows the effect of substrate concentration on the growth of strain BDLZ-3 and its degradation effect on benzoic acid. Figure 6 Figure 1 shows the effect of inoculum size on the growth of strain BDLZ-3 and its degradation of benzoic acid. Figure 7 The figure shows the effect of culture temperature on the growth of strain BDLZ-3 and its degradation effect on benzoic acid. Figures 4-7 The results show that pH 6–8 and substrate concentration 50–90 mg·L are suitable for optimal substrate conditions. -1 When the temperature was 28℃ and the inoculum size was 2%, strain BDLZ-3 showed the best degradation effect on benzoic acid, with a degradation rate of more than 80.00%, and the bacterial cells grew well.

[0042] 1. Degradation condition optimization experiment Based on 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 (6, 7, 8), and initial inoculum size (1%, 2%, 3%). Box-Behnken design was used to optimize the response surface methodology for these three key factors. A three-factor, three-level experiment was designed, and the levels of each factor were coded according to Box-Behnken design requirements. The pH (6, 7, 8), temperature (25℃, 28℃, 31℃), and inoculum size (1%, 2%, 3%) corresponded to the coded values ​​-1, 0, and 1, respectively. The specific experimental design and grouping are shown in Table 3.

[0043] The strain BDLZ-3 was added to MSM inorganic salt medium (benzoic acid content 50 µg / mL) with benzoic acid as the sole carbon source, and the same MSM medium without the strain BDLZ-3 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 3.

[0044] Table 3. Box-Benhnken experimental design based on 3 factors and 3 levels.

[0045] The quadratic polynomial regression model established through fitting is shown in Equation II: Y BDLZ-3 =95.3-0.8775* A -0.1675* B +0.0925* C -0.0250* AB +0.2650* AC -0.045* BC -3.48* A 2 -2.06* B 2 -2.545* C 2 Formula II.

[0046] In Equation II, 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 =99.54%, P=(0.0001)<0.05 is highly significant, and the lack-of-fit term =0.0787>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 interaction effects of inoculum size, culture temperature, and initial pH on the benzoic acid degradation efficiency of strain BDLZ-3, as shown below. Figures 8-10 As shown, where Figure 8 The results of the interaction between culture temperature and initial pH on the benzoic acid degradation efficiency of strain BDLZ-3 are shown in the figure, including the interaction contour plot as shown in a and the response surface plot as shown in b. Figure 9 The results of the interaction between inoculum amount and initial pH on the benzoic acid degradation efficiency of strain BDLZ-3 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 10 The interaction effects of inoculum size and culture temperature on the benzoic acid degradation efficiency of strain BDLZ-3 are shown in Figure 4, including the interaction contour plot (a) and the response surface plot (b). The optimal values ​​for the three main factors were obtained as follows: pH 7.454, temperature 27.866℃, and inoculum size 2.042%. Under these conditions, the predicted degradation rate was 94.193%, and the average of the three actual test results was 95.36%, higher than the predicted value. The experimental values ​​and predicted values ​​showed a high degree of fit, indicating that the established model is effective (Table 4).

[0047] Table 4 Results of Response Surface Methodology Experiment

[0048] 2. Culture medium formulation optimization experiment The tested strain BDLZ-3 was inoculated into LB liquid medium and cultured with shaking for 12 h (30℃, 180 r·min⁻¹) to obtain a bacterial suspension. The bacterial suspension was then inoculated into a medium containing 50 mg·L⁻¹ of LB liquid medium. -1In an inorganic salt culture medium containing benzoic acid, the effects of carbon source (0.1%, 0.3%, 0.5%, 0.7%, 0.9%), nitrogen source (0.5%, 0.75%, 1%, 1.25%, 1.5%), and inorganic salt (0.5%, 0.75%, 1%, 1.25%, 1.5%) on the growth of the bacterial strain were determined. After 3 days of culture, the effects of each single factor on the cell production were finally clarified, and the optimal culture medium formula was determined to be yeast extract, peptone, and anhydrous magnesium sulfate. Continuing with yeast powder additions of 0.3%, 0.5%, and 0.7%, soybean peptone additions of 1%, 1.25%, and 1.5%, and anhydrous magnesium sulfate additions of 1%, 1.25%, and 1.5%, the Box-Behnken design was used to optimize the response surface methodology for the coding levels of the three factors: yeast powder, soybean peptone, and anhydrous magnesium sulfate. A three-factor, three-level experiment was designed. The coding levels of the experimental factors and their corresponding actual concentrations are shown in Table 5, and the experimental design is shown in Table 6.

[0049] Table 5. Box-Behnken Design Experimental Factors and Coding Levels

[0050] Strain BDLZ-3 was added to MSM-BA medium, with the same MSM medium without strain BDLZ-3 as a control. After 1 day of culture, the OD600 of strain BDLZ-3 was measured (Table 6).

[0051] Table 6 Box-Benhnken Experimental Design

[0052] 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: Y BDLZ-3 =1.95+0.0574* A -0.0235* B +0.0356* C +0.0087* AB +0.0564* AC +0.0010* BC -0.0997* A 2 -0.0438* B 2 -0.0424* C 2 Formula III.

[0053] In Formula III, A, B, and C represent the coding values ​​for the amount of yeast powder, soybean peptone, and anhydrous magnesium sulfate added, respectively, and Y is the response value of the growth of strain BDLZ-3, i.e., OD600.

[0054] Table 7 Results of Response Surface Design Variance Analysis

[0055] The results of the response surface methodology variance analysis are shown in Table 7. Table 7 shows that the model… R 2 =95.85%; P =0.0005 < 0.01, highly significant; the lack-of-fit term 0.2529 > 0.05, not significant. This indicates 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 interaction effects of carbon source, nitrogen source, and inorganic salts, as shown below. Figures 11-13 As shown, where Figure 11 The response surface plot (b) and contour plot (a) show the interaction between carbon and nitrogen sources. Figure 12 The response surface plot (b) and contour plot (a) show the interaction between carbon source and inorganic salt. Figure 13 The 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 5.9 g / L, soybean peptone 11.96 g / L, and magnesium sulfate 13.99 g / L. Under these conditions, the predicted... OD 600 The maximum value was 1.953, based on three actual tests. OD 600 The average value was 2.099, 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.

[0056] In summary, the optimal fermentation medium formulation for strain BDLZ-3 was determined to be: yeast extract (5.90 g / L), soybean peptone (11.96 g / L), and anhydrous magnesium sulfate (13.99 g / L). Under the following conditions: pH 7.45, temperature 27.87℃, inoculum size 2.04%, rotation speed 180 r / min, and flask volume 100 mL / 250 mL, the degradation rate of benzoic acid at 50 µg / mL was 95.36%, and the optimized cell biomass reached 6.5 × 10⁻⁶. 10 The cfu / mL was increased by 13.0 times.

[0057] Example 4 Field application trial of benzoic acid degrading bacteria BDLZ-3 The degradation capacity of the bacterium BDLZ-3 on benzoic acid in ginseng-continuously cropped soil was evaluated under field conditions. The experiment was conducted at the ginseng experimental base of Jilin Agricultural University in Jingyue District, Changchun City. The bacterial cell count was 4 × 10⁻⁶. 9 4×10 8 and 4×10 7 CFU·mL -1 The fermentation broth of strain BDLZ-3 was fermented at 750 mL / m 2 The dosage was evenly mixed into the soil. Five treatments were included: a 2% biochar treatment and a control with an equal volume of fermentation medium. There were three replicates. The plot area was 2m². 2 Healthy ginseng plants with uniform growth were selected for transplanting. Samples were taken 120 days after treatment, and the change in benzoic acid content in the soil was detected using the Martens method. At harvest, agronomic traits and disease incidence of the ginseng were investigated and measured. The grading method for root diseases is shown in Table 8, and the calculation of the disease index and control efficacy is shown in Equations IV and V.

[0058] Table 8 Grading Standards for Ginseng Root Diseases

[0059] Disease index = Σ (number of disease roots at each level × representative value at each level) / (total number of disease roots surveyed × highest representative value) × 100, Equation IV; Prevention and control effect (%) = (disease index of control area - disease index of treatment area) / disease index of control area × 100, formula V.

[0060] 1. Detection of benzoic acid content in soil The changes in benzoic acid content in soil were detected using the Martens method, and the results of changes in benzoic acid content in soil after different treatments were obtained as follows: Figure 14 As shown in the figure. After measuring the benzoic acid content in the soil, it was found that the benzoic acid content in the soil treated with the control culture medium but not inoculated with strain BDLZ-3 remained almost unchanged at 7.30 mg·kg⁻¹. -1 The concentration of benzoic acid decreased to some extent in the uninoculated biochar treatment, with a benzoic acid content of 5.35 mg·kg⁻¹. -1 The degradation rate was 17.43%, but the effect was not significant. In contrast, the benzoic acid in the soil of the three treatments inoculated with BDLZ-3 showed varying degrees of degradation, with levels of 3.63 mg / kg. -1 3.01 mg·kg -1 3.90 mg·kg -1 The degradation rates of benzoic acid were 44.17%, 57.52%, and 49.18%, respectively, with the highest rate being 4.0 × 10⁻⁶. 8 CFU·mL -1 BDLZ-3 showed the best degradation effect.

[0061] 2. Effects of strain BDLZ-3 on ginseng growth At harvest, agronomic traits and disease incidence of ginseng were investigated and measured. The statistical results of the agronomic traits of ginseng treated with strain BDLZ-3 are shown in Table 9. It can be seen that adding different concentrations of fermentation broth from strain BDLZ-3 promoted the growth of ginseng in all indicators. The concentration of BDLZ-3 added was 4 × 10⁻⁶. 8 The bacterial suspension with CFU / mL showed the best effect, with significant differences compared to the control treatment at the 5% level. Simultaneously, the survival rate of ginseng at harvest was 65.38%–73.08%, significantly higher than all control treatments, and the difference was significant at the 5% level. This indicates that treating ginseng in soil containing benzoic acid with BDLZ-3 can alleviate the inhibition of ginseng growth, and all growth indicators are improved.

[0062] Table 9. Agronomic traits of ginseng after treatment with strain BDLZ-3

[0063] Note: CK1 and CK2 represent 2% biochar and culture medium treatments, respectively; BBA1, BBA2, and BBA3 represent inoculation with BDLZ-3 at 4×10⁻⁶ cm⁻¹. 9 4×10 8 4×10 8 CFU·mL -1 750 mL of bacterial culture was used for each treatment, with CK2 as the control, and the yield increase rate was calculated. Different lowercase letters after the data in the same column indicate the application of Duncan's new multiple range test. P The difference was statistically significant at the level of <0.05 (⁻x±s, n=3).

[0064] 3. Effects of strain BDLZ-3 on the occurrence of root diseases in ginseng Based on the grading method in Table 8, and Formulas IV and V, the disease incidence of ginseng after treatment with strain BDLZ-3 is shown in Table 10. It can be seen that the severity of ginseng root diseases (including red skin disease, rust rot, and root rot) varies. The culture medium control showed the most severe disease. Inoculation with strain BDLZ-3 alleviated the root disease, with control effects of 34.42%, 41.23%, and 35.88% respectively compared to the culture medium control. Although biochar can reduce the benzoic acid content in the soil, it cannot effectively control the occurrence of root diseases.

[0065] Table 10 Disease incidence in ginseng after treatment with strain BDLZ-3

[0066] Note: CK1 and CK2 represent 2% biochar and culture medium treatments, respectively; BBA1, BBA2, and BBA3 represent inoculation with BDLZ-3 at 4×10⁻⁶ cm⁻¹.9 4×10 8 4×10 7 CFU·mL -1 Treatment with 750 mL of bacterial suspension, with CK2 as the control, was used to calculate the control efficacy; different lowercase letters represent the efficacy tested using Duncan's multiple range test. P The difference was statistically significant at the level of <0.05 (⁻x±s, n=3).

[0067] In summary, the fermentation broth of strain BDLZ-3 at 4.0 × 10⁻⁶ is effective. 8 At a dosage of CFU / mL, the degradation rate of benzoic acid reached a maximum of 57.52%, which was significantly higher than that of the biochar control. Furthermore, the application of the degrading bacteria BDLZ-3 in the field could control the occurrence of root diseases to a certain extent, with a control effect of 34.42%~41.23% on root diseases. It could also significantly improve growth indicators such as root length and root diameter of ginseng, and had a certain yield-increasing effect.

[0068] 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 Pseudomonas montelukastii ( Pseudomonas monteilii BDLZ-3, characterized in that, The accession number is CGMCC No. 34349.

2. The method for culturing Pseudomonas montelukastii BDLZ-3 according to claim 1, characterized in that, Includes the following steps: Pseudomonas montmorillonii BDLZ-3 was inoculated into a culture medium and cultured to obtain a culture of Pseudomonas montmorillonii BDLZ-3. The culture medium comprises the following components in weight percentage: 0.3% to 0.7% yeast extract, 1% to 1.5% soybean peptone, and 1% to 1.5% anhydrous magnesium sulfate.

3. A microbial inoculant, characterized in that, The effective components of the microbial agent include the *Pseudomonas montelukastiensis* BDLZ-3 as described in claim 1 or a culture of *Pseudomonas montelukastiensis* BDLZ-3 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 *Pseudomonas montmorillonii* BDLZ-3 in the microbial agent was 4 × 10⁻⁶. 7 CFU / mL ~4×10 9 CFU / mL.

5. The application of the *Pseudomonas montmorillonii* BDLZ-3 as described in claim 1, the culture of *Pseudomonas montmorillonii* BDLZ-3 obtained by the culture method described in claim 2, or the microbial agent as described in claim 3 or 4 in alleviating crop continuous cropping obstacles.

6. The application according to claim 5, characterized in that, The measures to alleviate crop rotation obstacles include degrading crop autotoxic substances produced by crops in the soil; The autotoxic substances include at least one of benzoic acid, vanillic acid, cinnamic acid, palmitic acid, and gallic acid.

7. A method for alleviating crop rotation obstacles, characterized in that, Includes the following steps: Soil in continuously cropped fields can be treated with the *Pseudomonas montmorillonii* BDLZ-3 as described in claim 1, the culture of *Pseudomonas montmorillonii* BDLZ-3 obtained by the culture method described in claim 2, or the microbial agent as described in claim 3 or 4.

8. The method according to claim 7, characterized in that, The treatment of soil in continuously cropped fields includes mixing the culture of Pseudomonas montmorillonii BDLZ-3 as described in claim 1, the culture of Pseudomonas montmorillonii BDLZ-3 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 strain in the culture or microbial agent is 4 × 10⁻⁶. 7 ~4×10 9 CFU / mL; The dosage of the culture or microbial agent added is 500~1000 mL / m 2 .

9. The application of *Pseudomonas montelukastiensis* BDLZ-3 as described in claim 1, the culture of *Pseudomonas montelukastiensis* BDLZ-3 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 crop root diseases; ③ Improve crop growth indicators; ④ Increase crop yield.

10. The application according to any one of claims 5, 6, and 9, characterized in that, The crop in question is ginseng.

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