Bacterium degrading autotoxic substances and application thereof

Abstract

This invention discloses a bacterium that degrades autotoxic substances and its applications. The specific bacterium providing this invention is *Pseudomonas plecoglossicida*, strain number 56-21, with accession number CGMCC No. 26763 at the China General Microbiological Culture Collection Center. *Pseudomonas plecoglossicida* 56-21 (CGMCC No. 26763) provided by this invention can utilize p-hydroxybenzoic acid or vanillin as the sole carbon source for growth and can degrade autotoxic substances such as p-hydroxybenzoic acid or vanillin, which is of great significance for alleviating continuous cropping obstacles and improving the soil environment.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a bacterium that degrades autotoxic substances and its applications. Background Technology

[0002] Continuous cropping obstacles are prevalent in tobacco production, referring to the phenomenon of abnormal crop growth, reduced yield, lower quality, and increased plant diseases and pests caused by continuously planting the same or closely related crops on the same land. Autotoxicity is one of the important causes of continuous cropping obstacles, and phenolic acids are common autotoxic substances. Studies have shown that phenolic acids have an autotoxic effect on tobacco growth, which increases with increasing concentration (Cheng Yadong et al., 2020), and the amount of autotoxic substances in tobacco soil increases with the length of continuous cropping (Sun Jingguo et al., 2021). The accumulation of autotoxic substances in continuously cropped soil is caused by a reduction in functional bacteria. Inoculating continuously cropped soil with toxin-degrading bacteria can effectively reduce the content of autotoxic substances, promote plant growth, and thus improve the problem of continuous cropping obstacles (Dong et al., 2018).

[0003] Both p-hydroxybenzoic acid and vanillin belong to the phenolic acid class of compounds and are important autotoxic substances in crop soils. Studies have shown that exogenous addition of p-hydroxybenzoic acid strongly inhibits the growth of tobacco seedlings, manifested as a reduction in the number of lateral roots, slowed root elongation, decreased photosynthesis, disruption of protective enzyme systems, and potassium deficiency (Wu Wenxiang, 2010). Furthermore, phenolic acids also affect the microbial community structure, leading to an increase in pathogens, a decrease in beneficial bacteria, and soil deterioration. For example, vanillin can increase the number of soil-borne pathogenic Fusarium, exacerbating plant diseases (Zhou et al., 2018). Therefore, screening strains with the ability to degrade autotoxic substances and measuring their degradation capacity can provide a reference for tobacco continuous cropping obstacle control technology based on microecological regulation, and provide a scientific basis for improving the tobacco soil environment and effectively increasing tobacco yield and quality. Summary of the Invention

[0004] The purpose of this invention is to provide a bacterium that degrades autotoxic substances and its applications.

[0005] In a first aspect, the present invention claims protection for a strain of Pseudomonas splecoglossicida.

[0006] The strain number of *Pseudomonas plecoglossicida* claimed in this invention is 56-21, and its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 26763.

[0007] The single colonies of *Pseudomonas plecoglossicida* 56-21 were round, with regular, raised edges, a smooth and moist surface, and a white color. The physiological and biochemical characteristics of this strain are shown in Table 1.

[0008] Secondly, the present invention claims protection for cultures of *Pseudomonas plecoglossicida* as described in the first aspect above.

[0009] The culture of *Pseudomonas plecoglossicida* claimed in the first aspect above is a substance obtained by culturing *Pseudomonas plecoglossicida* in a bacterial culture medium.

[0010] In the above-mentioned cultures, the bacterial culture medium can be a solid culture medium or a liquid culture medium.

[0011] The term "culture" refers to any liquid or solid culture medium that has grown a microbial community after artificial inoculation and cultivation. It is the product obtained by growing and / or amplifying microorganisms; it can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process. The term "culture" also includes passaged cultures obtained by subculturing microorganisms; these can be cultures of a single generation or mixtures of several generations.

[0012] In a specific embodiment of the present invention, the bacterial culture medium is specifically an inorganic salt liquid culture medium containing p-hydroxybenzoic acid and / or vanillin.

[0013] Thirdly, this invention claims protection for a microbial agent.

[0014] The microbial agent claimed in this invention contains *Pseudomonas plecoglossicida* as described in the first aspect above and / or the culture described in the second aspect above.

[0015] The bacterial agent is used to degrade autotoxic substances.

[0016] In the aforementioned microbial agent, in addition to the active ingredient, a carrier is also included. The carrier can be a commonly used and biologically inert carrier in the pesticide field. The carrier can be a solid or liquid carrier; the solid carrier can be a mineral material, plant material, or polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material can be at least one of corn flour, soybean flour, and starch; the polymer compound can be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent can be decane and / or dodecane.

[0017] The above-mentioned microbial agents can be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0018] Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the bacterial agent.

[0019] Fourthly, the present invention claims protection for the use of *Pseudomonas plecoglossicida* as described in the first aspect above, or the culture as described in the second aspect above, or the bacterial agent as described in the third aspect above, in any of the following:

[0020] (A1) Degradation of autotoxic substances;

[0021] (A2) Prepare products for degrading autotoxic substances;

[0022] (A3) Degrades phenolic acid compounds;

[0023] (A4) Prepare products for the degradation of phenolic acid compounds;

[0024] (A5) Degrades p-hydroxybenzoic acid;

[0025] (A6) Prepare products for the degradation of p-hydroxybenzoic acid;

[0026] (A7) Degradation of vanillin;

[0027] (A8) Prepare products for the degradation of vanillin;

[0028] (A9) Alleviate continuous cropping obstacles;

[0029] (A10) Prepare products for alleviating continuous cropping obstacles;

[0030] (A11) Improve the soil environment;

[0031] (A12) Prepare products for improving the soil environment.

[0032] Fifthly, this invention claims protection for a product.

[0033] The active ingredient of the product claimed in this invention is *Pseudomonas plecoglossicida* as described in the first aspect above, or the culture as described in the second aspect above, or the bacterial agent as described in the third aspect above.

[0034] The product has any of the following functions:

[0035] (a1) Degradation of autotoxic substances;

[0036] (a2) Degradation of phenolic acid compounds;

[0037] (a3) Degradation of p-hydroxybenzoic acid;

[0038] (a4) Degradation of vanillin;

[0039] (a5) Alleviate continuous cropping obstacles;

[0040] (a6) Improve the soil environment.

[0041] Sixthly, the present invention claims protection for any of the following methods:

[0042] Method I: A method for degrading autotoxic substances, comprising the following steps: treating the autotoxic substance to be degraded with Pseudomonas plecoglossicida as described in the first aspect above, or the culture as described in the second aspect above, or the bacterial agent as described in the third aspect above.

[0043] Method II: A method for degrading phenolic acid compounds, comprising the following steps: treating the phenolic acid compounds to be degraded with *Pseudomonas plecoglossicida* as described in the first aspect above, or the culture as described in the second aspect above, or the bacterial agent as described in the third aspect above.

[0044] Method III: A method for degrading p-hydroxybenzoic acid, comprising the following steps: treating the p-hydroxybenzoic acid to be degraded with *Pseudomonas plecoglossicida* as described in the first aspect above, or the culture as described in the second aspect above, or the bacterial agent as described in the third aspect above.

[0045] Method IV: A method for degrading vanillin, comprising the following steps: treating the vanillin to be degraded with *Pseudomonas plecoglossicida* as described in the first aspect above, or the culture as described in the second aspect above, or the microbial agent as described in the third aspect above.

[0046] Method V: A method for alleviating continuous cropping obstacles, comprising the following steps: treating the soil causing the continuous cropping obstacles with Pseudomonas plecoglossicida as described in the first aspect above, or the culture as described in the second aspect above, or the inoculant as described in the third aspect above.

[0047] In a specific embodiment of the present invention, the mitigation of continuous cropping obstacles is specifically manifested in any of the following: mitigating the inhibitory effect of autotoxic substances (such as vanillin) on plant height; mitigating the inhibitory effect of autotoxic substances (such as vanillin) on the fresh weight of aboveground parts of plants; and reducing the content of autotoxic substances (such as vanillin) in the soil.

[0048] In a seventh aspect, the present invention claims a method for culturing the Pseudomonas plecoglossicida described in the first aspect above.

[0049] The method for culturing the *Pseudomonas plecoglossicida* claimed in this invention may include the following steps: culturing the *Pseudomonas plecoglossicida* in a culture medium using p-hydroxybenzoic acid as the sole carbon source.

[0050] Furthermore, the culture medium is an inorganic salt liquid culture medium.

[0051] Eighthly, the present invention claims a method for culturing the Pseudomonas plecoglossicida described in the first aspect above.

[0052] The method for culturing the *Pseudomonas plecoglossicida* claimed in this invention may include the following steps: culturing the *Pseudomonas plecoglossicida* in a culture medium using p-vanillin as the sole carbon source.

[0053] Furthermore, the culture medium is an inorganic salt liquid culture medium.

[0054] Ninthly, the present invention claims the use of *Pseudomonas splecoglossicida* as described in the first aspect above in the preparation of the culture described in the second aspect above or the bacterial agent described in the third aspect above.

[0055] Experiments have shown that the Pseudomonas plecoglossicida 56-21 (CGMCC No. 26763) provided by this invention can utilize p-hydroxybenzoic acid or vanillin as the sole carbon source for growth and can degrade autotoxic substances such as p-hydroxybenzoic acid or vanillin, which is of great significance for alleviating continuous cropping obstacles and improving the soil environment.

[0056] Preservation Instructions

[0057] Classification and naming: Pseudomonas plecoglossicida;

[0058] Biological materials from ginseng: 56-21;

[0059] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0060] The abbreviation for the depository institution is CGMCC.

[0061] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0062] Date of deposit: March 7, 2023;

[0063] Registration number at the Preservation Center: CGMCC No. 26763. Attached Figure Description

[0064] Figure 1 The colony morphology of strain 56-21 on inorganic salt solid medium.

[0065] Figure 2 Phylogenetic tree of strain 56-21 constructed based on 16S rDNA sequence.

[0066] Figure 3 The figure shows the growth curve of strain 56-21 using p-hydroxybenzoic acid as a substrate. In the figure, Pseudomonasplecoglossicida represents strain 56-21.

[0067] Figure 4 This is the standard curve for p-hydroxybenzoic acid.

[0068] Figure 5The figure shows the degradation curve of strain 56-21 using p-hydroxybenzoic acid as a substrate. In the figure, Pseudomonasplecoglossicida is strain 56-21.

[0069] Figure 6 The figure shows the growth curve of strain 56-21 using vanillin as a substrate. In the figure, Pseudomonasplecoglossicida is strain 56-21.

[0070] Figure 7 This is the standard curve for vanillin.

[0071] Figure 8 The figure shows the degradation curve of strain 56-21 using vanillin as a substrate. In the figure, Pseudomonasplecoglossicida is strain 56-21.

[0072] Figure 9 The effect of strain 56-21 on the growth of tobacco seedlings in continuously cropped soil treated with autotoxin. A represents plant height; B represents aboveground fresh weight; C represents belowground fresh weight. Different lowercase letters indicate significant differences at the P < 0.001 level.

[0073] Figure 10 The effect of strain 56-21 on vanillin content in continuously cropped soil treated with autotoxic substances. Different lowercase letters indicate significant differences at the P < 0.001 level. Detailed Implementation

[0074] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0075] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0076] The experimental materials involved in the following embodiments:

[0077] (1) Inorganic salt culture medium from Beijing Coolbo Technology Co., Ltd.

[0078] (2) R2A culture medium from Beijing Xinkailong Biotechnology Co., Ltd.

[0079] (3) p-hydroxybenzoic acid and vanillin (analytical grade 99.5%) from Shanghai Yuanye Biotechnology Co., Ltd.

[0080] (4) Amplification primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGCTACCTTGTTACGACTT-3') synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0081] (5) Generic PCR Mix from Novizan Biotechnology Co., Ltd.

[0082] Example 1: Isolation, purification and identification of strain 56-21

[0083] I. Isolation and purification of strain 56-21

[0084] 10g of soil (collected from Niulanjiang Town, Songming County, Kunming City, Yunnan Province (25°17'N; 103°13'E)) was added to 100mL of sterile water and incubated at 28℃ and 180rpm with shaking for 30min. The mixture was then aliquoted into centrifuge tubes and centrifuged at 8000×g for 10min. 5mL of the soil suspension was transferred to inorganic salt liquid medium (containing 0.5mg / mL p-hydroxybenzoic acid) and incubated at 28℃ and 180rpm for 3 days. Similarly, 5mL of the culture was added to inorganic salt liquid medium, and incubation was repeated for two more rounds. Finally, the strain suspension was diluted with sterile water and plated onto inorganic salt solid medium. After incubation at 28℃ for 3 days, single colonies were streaked three times for further purification. The purified strain was named 56-21 and stored long-term in an ultra-low temperature freezer at -80℃.

[0085] II. Identification of strain 56-21

[0086] 1. Morphological identification of strain 56-21

[0087] The strain 56-21 isolated in step one was inoculated onto an inorganic salt solid medium using the streak plating method. Simultaneously, it was diluted with sterile water to form a bacterial suspension, which was then spread onto the inorganic salt solid medium. The culture was carried out at 28°C for 3-5 days. After single colonies appeared, their morphology, including colony size, shape, color, and transparency, was observed.

[0088] The results showed that the tested strain 56-21 had single, round colonies with neat, raised edges, a smooth and moist surface, and a white color. Figure 1 ).

[0089] 2. Physiological and biochemical characteristics of strain 56-21

[0090] The test strain was inoculated onto R2A solid medium and cultured at 28°C for 1 day. A single colony was collected with a cotton swab and inserted into the bottom of an inoculation tube containing IF-A inoculation solution; the mixture was stirred thoroughly to obtain a bacterial suspension. The bacterial suspension was then added to GEN III microplates and cultured at 28°C for 1 day. The results were read using a BIOLOG automated microbial identification system, and the identification results were evaluated based on similarity values.

[0091] The results showed that strain 56-21 was Pseudomonas putida (Table 1), with a SIM value of 0.724.

[0092] Table 1. Physiological and biochemical characteristics of strain 56-21

[0093]

[0094]

[0095] Note: "+" indicates a positive result; "w" indicates a weak positive result.

[0096] III. Molecular biological identification of strain 56-21

[0097] DNA from strain 56-21 was extracted using a thermal lysis method. A small amount of single colony was scraped from a tube and added to 100 μL of ddH2O. After vortexing and mixing, the mixture was incubated at 100°C for 10 min, then centrifuged at maximum speed for 2 min at room temperature. The supernatant was transferred to a new centrifuge tube, and the extracted DNA was stored at -20°C. The target fragment was amplified using the universal 16S rDNA primers 27F / 1492R. The PCR amplification reaction system consisted of 20 μL: 10 μL of 2×Taq PCR Mix, 0.5 μL each of forward and reverse primers, 1 μL of template, and ddH2O to make up the volume. The reaction conditions were: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 57°C annealing for 15 s, 72°C extension for 15 s, 35 cycles; and 72°C final extension for 5 min. Bidirectional sequencing was performed after detection by 1% agarose gel electrophoresis. The sequencing results were analyzed using BLAST on NCBI, homologous sequences were downloaded, and a phylogenetic tree was constructed using the neighbor-joining method with MEGA software to clarify its taxonomic position. Cellvibrio japonicus was selected as the outgroup.

[0098] The results showed that different species of Pseudomonas sp. could be completely separated by 16S rDNA. The 16S rDNA sequence of strain 56-21 (SEQ ID No. 1) had the highest similarity (99.79%) to Pseudomonas plecoglossicida (NR114226). Figure 2 ).

[0099] Based on the comprehensive morphological, physiological, biochemical, and molecular biological identification results, strain 56-21 was classified as *Pseudomonas plecoglossicida*, with the corresponding Chinese name *Pseudomonas plecoglossicida*. This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 7, 2023, with the accession number CGMCC No. 26763.

[0100] Example 2: Application of Pseudomonas plecoglossicida 56-21 in the degradation of autotoxic substances

[0101] I. Determination of the growth of strain 56-21 using p-hydroxybenzoic acid as the sole carbon source

[0102] *Pseudomonas plecoglossicida* 56-21 was cultured in inorganic salt liquid medium (containing 1 mg / mL p-hydroxybenzoic acid) at 28°C with shaking at 180 rpm for 24 h to prepare a bacterial suspension. 2 mL of the bacterial suspension was transferred to inorganic salt liquid medium and cultured at 28°C with shaking at 180 rpm for 2 days. Inorganic salt liquid medium (containing 1 mg / mL p-hydroxybenzoic acid) without strain 56-21 was used as a control. The culture was repeated four times. During the liquid culture period, samples were taken every 6 h, and the OD value of the bacterial suspension was measured at 600 nm using a spectrophotometer. A growth curve of strain 56-21 was plotted with time on the x-axis and OD value on the y-axis. An additional 1 mL of the bacterial suspension was used in the subsequent step two to determine the residual amount of p-hydroxybenzoic acid in the sample.

[0103] The results showed that strain 56-21 entered the exponential growth phase at around 6 hours, reached its maximum growth at around 24 hours, and entered the decline phase after 30 hours, which basically conforms to the growth pattern of bacteria, indicating that strain 56-21 can utilize p-hydroxybenzoic acid as the sole carbon source for growth. Figure 3 ).

[0104] II. Degradation effect of strain 56-21 on p-hydroxybenzoic acid in culture medium

[0105] 1. Construction of the standard curve for p-hydroxybenzoic acid

[0106] Inorganic salt liquid culture media containing different concentrations of p-hydroxybenzoic acid were prepared, placed in sterile centrifuge tubes, and stored at 4°C. After complete sampling, the OD values ​​corresponding to different p-hydroxybenzoic acid concentrations were measured together with the test samples at a wavelength of 250 nm. A standard curve for p-hydroxybenzoic acid was plotted with p-hydroxybenzoic acid concentration on the x-axis and OD values ​​on the y-axis, as shown below. Figure 4As shown. The results show that the standard curve formula for p-hydroxybenzoic acid is y = 41.957x + 0.1039, and the correlation coefficient R is... 2 = 0.9984, where x is the concentration of p-hydroxybenzoic acid and y is the corresponding OD. 250 value.

[0107] 2. Determination of the degradation curve of p-hydroxybenzoic acid

[0108] The OD value of p-hydroxybenzoic acid in the bacterial culture was measured at a wavelength of 250 nm, with sterile water serving as a blank control. A degradation curve of p-hydroxybenzoic acid was plotted with time on the x-axis and OD value on the y-axis. The degradation rate was calculated using the formula: Degradation rate (%) = 1 - (Initial p-hydroxybenzoic acid concentration - Residual p-hydroxybenzoic acid concentration) / Initial p-hydroxybenzoic acid concentration × 100%. The degradation rates of p-hydroxybenzoic acid at different sampling times are shown in Table 2.

[0109] Table 2. Degradation rate of p-hydroxybenzoic acid at different sampling times

[0110] Sampling time (hour) Degradation rate (%) 0 0 6 20.07 12 34.80 18 52.09 24 81.55 30 93.04 36 97.10 42 97.45 48 98.03

[0111] Based on the above values, a degradation curve for p-hydroxybenzoic acid was prepared as follows: Figure 5 As shown in the figure, the concentration of p-hydroxybenzoic acid (p-hydroxybenzoic acid) decreased continuously with the growth of strain 56-21. The p-hydroxybenzoic acid concentration began to decrease sharply after strain 56-21 entered the exponential growth phase, and the degradation rate of p-hydroxybenzoic acid also increased accordingly. Furthermore, the growth rate of strain 56-21 reached its maximum at 24 hours, at which point the p-hydroxybenzoic acid content was also close to its minimum, with a final degradation rate of 98.03%. This demonstrates that strain 56-21 isolated in Example 1 of this invention can effectively degrade p-hydroxybenzoic acid.

[0112] III. Growth of strain 56-21 with vanillin as the sole carbon source

[0113] Strain 56-21 was cultured in inorganic salt liquid medium (containing 1 mg / mL vanillin) at 28°C with shaking at 180 rpm for 24 h to prepare a bacterial suspension. 2 mL of the bacterial suspension was transferred to inorganic salt liquid medium and cultured at 28°C with shaking at 180 rpm for 2 days. Inorganic salt liquid medium (containing 1 mg / mL vanillin) without strain 56-21 was used as a control. The culture was repeated four times. During the liquid culture period, samples were taken every 6 h, and the OD value of the bacterial suspension was measured at 600 nm using a spectrophotometer. A growth curve of strain 56-21 was plotted with time on the x-axis and OD value on the y-axis. An additional 1 mL of the bacterial suspension was used in step four to determine the residual vanillin in the sample.

[0114] The results showed that strain 56-21 entered the exponential growth phase at around 6 hours, reached its maximum growth at around 18 hours, and entered the decline phase after 24 hours, which basically conforms to the growth pattern of bacteria, indicating that strain 56-21 can utilize vanillin as the sole carbon source for growth. Figure 6 ).

[0115] IV. Degradation of vanillin in culture medium by strain 56-21

[0116] 1. Drawing the vanillin standard curve

[0117] Prepare inorganic salt liquid culture media containing different concentrations of vanillin, place them in sterile centrifuge tubes, and store them at 4°C. After complete sampling, measure the OD values ​​corresponding to different vanillin concentrations together with the test samples at a wavelength of 250 nm. Plot a vanillin standard curve with vanillin concentration on the x-axis and OD values ​​on the y-axis, as shown below. Figure 7 As shown. The results show that the standard curve formula for vanillin is y = 12.043x + 0.3645, and the correlation coefficient R is... 2 = 0.9923, where x is the vanillin concentration and y is the corresponding OD. 250 value.

[0118] 2. Determination of vanillin degradation curve

[0119] The OD value of vanillin in the bacterial culture was measured at a wavelength of 250 nm, with ddH2O used as a blank control. A vanillin degradation curve was plotted with time on the x-axis and OD value on the y-axis. The degradation rate was calculated using the formula: degradation rate (%) = 1 - (initial vanillin concentration - residual vanillin concentration) / initial vanillin concentration × 100%. The degradation rates of vanillin at different sampling times are shown in Table 3.

[0120] Table 3. Concentration and degradation rate of vanillin at different sampling times

[0121] Sampling time (hour) Degradation rate (%) 0 0 6 11.08 12 72.15 18 91.77 24 95.78 30 97.05 36 97.78

[0122] Based on the above values, a vanillin degradation curve was constructed as follows: Figure 8 As shown in the figure, the vanillin concentration decreased continuously with the growth of strain 56-21. The vanillin concentration began to decrease sharply after strain 56-21 entered the exponential growth phase, and the degradation rate of vanillin increased accordingly. Furthermore, the growth rate of strain 56-21 reached its maximum at 18 hours, at which point the vanillin content was also close to its minimum, with a final degradation rate of 97.78%. This indicates that the isolated strain 56-21 can effectively degrade vanillin.

[0123] V. Degradation of vanillin in continuously cropped soil by strain 56-21

[0124] The role of strain 56-21 in degrading soil autotoxins was further verified through greenhouse experiments. Soil samples were collected from tobacco fields with significant continuous cropping obstacles after three years of continuous cropping. After sieving, the samples were sterilized at high temperature for use in pot experiments. Strain 56-21 was cultured in LB liquid medium for 24 hours, centrifuged, the supernatant was discarded, and the suspension was resuspended in sterile water at a concentration of 1×10⁻⁶. 9 CFU / mL. A pot experiment was conducted by mixing a suspension of strain 56-21 with sterilized continuously cropped soil. Three treatments were used: 1) X: vanillin; 2) X56: vanillin + strain 56-21; 3) W: sterilized water, with six replicates for each treatment (the mean was taken). The amount of vanillin added to the soil was 0.05 mg / g soil, and the amount of bacterial suspension added was 5% (v / v). The treated soil was added to flowerpots. Seedlings were raised in seedling trays and transplanted after the tobacco seedlings had two true leaves. The plants were cultivated in a greenhouse at 25℃ with a photoperiod of 16 h light / 8 h dark. The experiment ended after 8 weeks. The plant height, aboveground and underground fresh weight of the tobacco were recorded, and soil samples were collected for vanillin content determination using high-performance liquid chromatography (HPLC).

[0125] The results showed that adding vanillin to the soil inhibited tobacco growth. Figure 9 Adding strain 56-21 can alleviate this inhibitory effect; at the same time, strain 56-21 can also reduce the vanillin content in the soil. Figure 10 This helps alleviate the obstacles of continuous cropping.

[0126] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Kills *Pseudomonas aeruginosa* ( Pseudomonas plecoglossicida 56-21, characterized in that: Its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 26763.

2. The culture of *Pseudomonas aeruginosa* according to claim 1 is a substance obtained by culturing *Pseudomonas aeruginosa* according to claim 1 in a bacterial culture medium.

3. A microbial agent, characterized in that: The bacterial agent contains *Pseudomonas aeruginosa* as described in claim 1 and / or the culture as described in claim 2.

4. The microbial agent according to claim 3, characterized in that: The microbial agent is capable of degrading p-hydroxybenzoic acid and vanillin and can alleviate the obstacle of continuous tobacco cropping.

5. The use of the *Pseudomonas aeruginosa* of claim 1, the culture of claim 2, or the bacterial agent of claim 3 or 4 in any of the following: (A1) Degrades p-hydroxybenzoic acid and vanillin and alleviates tobacco continuous cropping obstacles; (A2) Prepare products for degrading p-hydroxybenzoic acid and vanillin and for alleviating tobacco continuous cropping obstacles.

6. A product capable of degrading p-hydroxybenzoic acid and vanillin and alleviating tobacco continuous cropping obstacles, wherein the active ingredient is *Pseudomonas aeruginosa* as described in claim 1, or the culture as described in claim 2, or the bacterial agent as described in claim 3 or 4.

7. A method for degrading p-hydroxybenzoic acid and vanillin and alleviating tobacco continuous cropping obstacles, comprising the following steps: treating soil containing p-hydroxybenzoic acid and vanillin that causes tobacco continuous cropping obstacles with the *Pseudomonas aeruginosa* of claim 1, the culture of claim 2, or the inoculant of claim 3 or 4.

8. The use of *Pseudomonas aeruginosa* according to claim 1 in the preparation of the culture according to claim 2 or the bacterial agent according to claim 3 or 4.

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