Pseudomonas oxal beijerinckii and application thereof in relieving continuous cropping obstacles of plants

Abstract

This invention belongs to the field of biotechnology and discloses a strain of *Pseudomonas oxysporum* and its application in alleviating continuous cropping obstacles in plants. The preservation number of *Pseudomonas oxysporum* is CCTCC NO: M2023054. This invention isolated a strain of *Pseudomonas oxysporum* RH60 from soil in a cucumber-pepper rotation system. This strain can effectively solve the problem of aggravated root-knot nematode disease under continuous cucumber cropping conditions and efficiently degrades the autotoxic substance p-hydroxybenzoic acid produced by continuous cucumber cropping, while also promoting plant growth. The *Pseudomonas oxysporum* provided by this invention has potential application prospects in the prevention and control of root-knot nematode disease and in solving continuous cropping obstacles caused by autotoxic substances.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a strain of *Pseudomonas oxysporum* and its application in alleviating continuous cropping obstacles in cucumbers. The *Pseudomonas oxysporum* strain provided by this invention can control plant pathogenic nematodes, degrade the autotoxic substance p-hydroxybenzoic acid, and promote plant growth. Background Technology

[0002] Continuous cropping obstacles refer to the abnormal growth and development of plants caused by continuously cultivating the same or closely related crops on the same land, ultimately leading to reduced crop yield and quality. In recent years, with the increasing planting area of ​​cucumbers in plastic greenhouses, continuous cropping obstacles have become a major factor restricting cucumber production. There are many causes of continuous cropping obstacles, including increased pests and diseases, accumulation of plant autotoxins, and excessive depletion of soil nutrients.

[0003] Studies have reported that root-knot nematodes are one of the important pathogens affecting vegetable production in my country. Cucumbers grown in continuous cropping are highly susceptible to root-knot nematode diseases caused by the southern root-knot nematode (Meloidogyne incognita) (see Jin Na, Research Progress on Occurrence, Damage and Green Control of Root-Knot Nematodes in Vegetables in my country, DOI: 10.13802 / j.cnki.zwbhxb.2022.2022828). Simultaneously, root-knot nematodes cause mechanical damage to plants, reducing their defense capabilities and making it easier for soil-borne pathogens such as Fusarium wilt and root-rot fungi to invade the root system, forming complex infections. This leads to widespread wilting and death of plants, resulting in reduced yields. Currently, chemical pesticides remain the main measure for nematode control; however, long-term pesticide use leads to pesticide resistance in pathogens and damages the environment, reducing the content of beneficial microorganisms in the soil and thus exacerbating continuous cropping obstacles. Using microorganisms to control root-knot nematode disease is a good approach, as it can kill harmful bacteria, insects, and eggs, and is also safe for the environment.

[0004] Another important reason for continuous cropping obstacles is the accumulation of autotoxic substances. Plants release allelochemicals through root exudates or plant residues, which, when accumulated in the soil to a certain concentration, inhibit the growth of crops in the same or subsequent crops. Phenolic acids, terpenes, and flavonoids are common autotoxic substances. Their mechanisms of action include altering cell membrane permeability, affecting protective enzyme systems, and influencing photosynthesis and respiration, thus reducing the plant's ability to respond to stress and causing continuous cropping obstacles. Studies have found significant accumulations of p-hydroxybenzoic acid, p-coumaric acid, ferulic acid, vanillic acid, vanillin, and syringic acid in cucumber-continuously cropped fields, and a decrease in the abundance of bacteria and fungi in the soil (Soil phenolics in a continuously mono-cropped cucumber (Cucumis sativus L.) system and their effects on cucumber seedling growth and soil microbial communities, DOI:10.1111 / j.1365-2389.2012.01442.x). The abundance of microorganisms that degrade autotoxins decreases in continuously cropped soils. Therefore, inoculating the soil with autotoxin-degrading bacteria can effectively promote plant growth and overcome the obstacles of continuous cropping.

[0005] This invention provides for the first time a strain of *Pseudomonas oxidans* that can control root-knot nematode disease, degrade the autotoxic substance p-hydroxybenzoic acid, and promote plant growth. It can provide a scientific basis for reducing soil-borne diseases in cucumber continuous cropping and effectively improving cucumber yield and quality. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of Pseudarthrobacter oxydans RH60 with strong biocontrol capabilities, the preservation number of which is CCTCC NO: M2023054.

[0007] Another object of the present invention is to provide the application of Pseudarthrobacter oxydans RH60 in alleviating continuous cropping obstacles in plants. The strain of the present invention alleviates continuous cropping obstacles by controlling southern root-knot nematode disease, degrading the autotoxic substance p-hydroxybenzoic acid, and promoting plant growth.

[0008] To achieve the above objectives, the present invention adopts the following technical measures:

[0009] Obtaining *Pseudomonas oxidans* RH60:

[0010] A strain of *Pseudarthrobacter oxydans* was isolated from the rhizosphere soil of cucumbers after pepper rotation at the experimental base of Huazhong Agricultural University. The 16S sequence fragment of this strain was amplified using universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-ACGGCTACCTTGTTACGACTT-3'). The sequenced sequences were then subjected to BLAST homology alignment on NCBI. A phylogenetic tree was constructed using sequences with high similarity to the aligned sequences, identifying *Pseudarthrobacter oxydans* as the closest genus to this strain. RH60 in the following examples refers to *Pseudarthrobacter oxydans*.

[0011] This strain was deposited at the China Center for Type Culture Collection on January 9, 2023, with the classification name: Pseudarthrobacter oxydans RH60, accession number: CCTCC NO: M2023054, and deposit address: Wuhan University, Wuhan, China.

[0012] Strawberry strain RH60 was streaked in Luria-Bertani (LB) solid medium and incubated at 30°C for 24 hours. The colonies were round, milky white, opaque, smooth, with regular edges, and 1 mm in diameter.

[0013] The scope of protection of this invention also includes:

[0014] The application of Pseudarthrobacter oxydans RH60 in the control of southern root-knot nematodes can be achieved by directly drenching the roots of plants with its fermentation supernatant or fermentation broth.

[0015] Pseudarthrobacter oxydans RH60 is used to prepare an insecticide for southern root-knot nematodes.

[0016] The preferred application of Pseudarthrobacter oxydans RH60 in the preparation of plant growth promoters is to directly drench the roots of plants with its fermentation supernatant or fermentation broth.

[0017] Application of Pseudarthrobacter oxydans RH60 in the degradation of p-hydroxybenzoic acid.

[0018] Application of Pseudarthrobacter oxydans RH60 in the preparation of p-hydroxybenzoic acid degrading agents.

[0019] Application of Pseudarthrobacter oxydans RH60 in alleviating plant continuous cropping obstacles.

[0020] In the above applications, the preferred plant is cucumber.

[0021] In the above-described applications, the *Pseudarthrobacter oxydans* RH60 refers to the *Pseudarthrobacter oxydans* RH60 strain, its fermentation broth, or its fermentation supernatant.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The *Pseudomonas oxysporum* provided by this invention has a highly effective control effect on root-knot nematode disease and can also promote plant growth. It can be used to prepare root irrigation agents, seed coating agents or plant growth regulators.

[0024] (2) The *Pseudomonas oxidans* provided by the present invention can degrade p-hydroxybenzoic acid, and includes the degradation method of the bacteria and its application to p-hydroxybenzoic acid in continuously cropped soil, which has application value in improving cucumber continuous cropping obstacles.

[0025] (3) The strains used in this invention are environmentally safe, harmless to the human body, simple to ferment, and low in cost. Attached Figure Description

[0026] Figure 1 RH60 was identified as *Pseudomonas oxidans*.

[0027] Phylogenetic trees were constructed using neighbor-joining (1000 bootstrap) based on the 16S rRNA sequences of strain RH60 and other reference strains. The 16S rRNA sequences of *Pseudarthrobacter* spp. and *Micrococcus* spp. were downloaded from the NCBI database; the accession numbers are in parentheses. The lower left corner shows the colony morphology (Bar = 2 mm) and Gram staining (Bar = 100 μm) results for strain RH60.

[0028] Figure 2 A schematic diagram illustrating the lethal effect of RH60 fermentation broth on southern root-knot nematodes.

[0029] In this study, A shows the lethal effect of the RH60 fermentation broth on second-instar larvae of the southern root-knot nematode, and B shows the statistical analysis of the corrected mortality rates of the nematodes at 24 h and 48 h; the experiment was conducted in three biological replicates. Bar = 200 μm.

[0030] Figure 3A schematic diagram illustrating how RH60 promotes cucumber growth;

[0031] The Mock group was a control group with cucumbers added with sterile water, and the RH60 group was an experimental group with only RH60 bacteria inoculated.

[0032] Where A is a schematic diagram of sampling the above-ground parts and roots of cucumbers 14 days after inoculation, Bar = 5cm.

[0033] B represents the plant height of different groups; C represents the aboveground fresh weight of different groups; D represents the root length of different groups; E represents the root fresh weight of different groups; *, * and *** represent P<0.05, 0.01 and 0.001 respectively, and the test method is Student's t test.

[0034] Figure 4 RH60 can alleviate root-knot nematode disease in southern China and promote plant growth;

[0035] The Mock group was a control group consisting of cucumbers inoculated only with Southern Root-Knot Nematode, while the RH60 group was an experimental group consisting of cucumbers inoculated with RH60 bacteria before being inoculated with second-instar larvae of Southern Root-Knot Nematode 4 days after bacterial inoculation.

[0036] Figure A shows a diagram illustrating sampling of the above-ground parts and roots of cucumbers 30 days after inoculation, with the yellow arrow indicating the root knot. Bar = 5cm;

[0037] B represents the number of nematodes per 100 mL of soil; C represents the root knot index of different groups; D represents the plant height of different groups; E represents the aboveground fresh weight of different groups; F represents the root length of different groups; G represents the root fresh weight of different groups; * and *** represent P<0.05, 0.01 and 0.001 respectively, and the test method is Student's t test.

[0038] Figure 5 This is a schematic diagram illustrating the degradation effect of p-hydroxybenzoic acid by RH60.

[0039] A represents a schematic diagram of the degradation of p-hydroxybenzoic acid by RH60 at different culture times; DMSO was used to replace p-hydroxybenzoic acid and p-hydroxybenzoic acid was added only to the culture medium as controls, and the experiment was set up with 3 biological replicates;

[0040] B is a schematic diagram of the degradation effect of RH60 on p-hydroxybenzoic acid in continuously cropped soil. The experiment was set up with 4 biological replicates.

[0041] C represents the standard curve for p-hydroxybenzoic acid. Detailed Implementation

[0042] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0043] Example 1:

[0044] Isolation and identification of *Pseudomonas oxidans* RH60:

[0045] 1. The isolation method of *Pseudomonas oxidans* is as follows:

[0046] The bacterial strain of *Pseudomonas oxidans* was isolated from the rhizosphere soil of cucumbers after a cucumber-pepper-cucumber rotation at the experimental base of Huazhong Agricultural University.

[0047] Shake off the non-rhizosphere soil adhering to the surface of the cucumber roots, place the root sample in a 50mL test tube containing PBS buffer, and shake in a shaker at 30℃ and 120rpm for 20min. Remove the roots with sterile tweezers, centrifuge the remaining suspension, and obtain the rhizosphere soil.

[0048] Weigh 1g of rhizosphere soil, add sterile water to a final volume of 10mL, vortex for 5min, let stand for 5min until stratification, then perform serial dilution to 10. 4 10 5 and 10 6 The culture was plated on LB agar (the agar formulation consisted of 5g yeast extract, 10g tryptone, 10g sodium chloride, and 15g agar added to ddH2O, pH adjusted to 7.0, and volume brought to 1L). The culture was incubated upside down at 30°C for 24 hours. Single colonies were then picked and inoculated into LB liquid medium, incubated at 30°C and 180 rpm for 24 hours to obtain a pure culture. Glycerol was added, and the culture was stored at -80°C.

[0049] The strain was streaked in LB solid medium and incubated at 30°C for 24 hours. The colonies were round, milky white, opaque, smooth, with regular edges, and 1 mm in diameter.

[0050] The strain was stained using a Gram staining kit, and the bacteria appeared purple under an optical microscope, confirming that it was a Gram-positive bacterium.

[0051] The 16S sequence fragments of the above strains were amplified using universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-ACGGCTACCTTGTTACGACTT-3'). The sequenced sequences were then subjected to BLAST homology alignment on NCBI. Sequences with high similarity to the aligned sequences were selected to construct a phylogenetic tree. The results are shown in […]. Figure 1The closest species to the above strain was identified as *Pseudarthrobacter oxydans*, and named *Pseudarthrobacter oxydans* RH60.

[0052] This strain was deposited at the China Center for Type Culture Collection on January 9, 2023, with the classification name: Pseudarthrobacter oxydans RH60, accession number: CCTCC NO: M2023054, and deposit address: Wuhan University, Wuhan, China.

[0053] Example 2:

[0054] Lethal effect of Bacillus oxidans RH60 fermentation broth on J2s second instar larvae of Southern root-knot nematode.

[0055] 1. Activation of *Pseudomonas oxidans* RH60 and preparation of fermentation broth:

[0056] The RH60 bacterial culture stored at -80℃ in Example 1 was activated, streaked on LB agar plates, and incubated at 30℃ for 24 hours. Single colonies were picked and added to 1 mL of LB liquid medium, and incubated overnight at 30℃ and 180 rpm to obtain the RH60 seed culture. 10 μL of the bacterial culture was inoculated into 10 mL of LB liquid medium and incubated at 30℃ and 180 rpm for 48 hours. The bacterial culture was centrifuged at 12000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the bacterial fermentation broth.

[0057] 2. Hatching of J2s, second instar larvae of the southern root-knot nematode

[0058] Southern root-knot nematodes reproduce using cucumber as a host plant. Cucumber roots with root knots were collected, and a 0.8% sodium hypochlorite solution was added. The mixture was shaken at 180 rpm for 10 minutes. Then, 40% sucrose was added, and the mixture was centrifuged at 3500 rpm for 5 minutes. The supernatant suspension of root-knot nematode eggs was collected and washed three times with sterile water to obtain a sterile suspension. This suspension was then placed in sterile water and incubated at 28°C. The hatched J2s were collected for experimental use.

[0059] 3. Lethal effect of Bacillus oxidans RH60 fermentation broth on J2s second instar larvae of Southern root-knot nematode;

[0060] The lethal effect of bacterial fermentation broth on nematodes was investigated in 96-well plates. 100 μL of RH60 fermentation broth and 100 μL of nematode suspension (containing 100 nematodes) were added to each well. The fermentation broth was replaced with 100 μL of LB medium for correction (i.e., control group). Three biological replicates were set up for each treatment. The 96-well plates were incubated statically at 28°C for 24 h and 48 h, and the mortality rate of nematodes in each well was calculated under a stereomicroscope. Results are as follows: Figure 2 As shown, the corrected mortality rates of nematodes after 24 h and 48 h of treatment with RH60 fermentation broth were 82.3% and 97.3%, respectively.

[0061] Corrected mortality rate = (mortality rate of nematodes in the treatment group - mortality rate of nematodes in the control group) / (1 - mortality rate of nematodes in the control group) × 100.

[0062] Example 3:

[0063] Pot experiment on the promotion of cucumber growth by Bacillus oxidans RH60.

[0064] 1. Preparation of *Pseudomonas oxidans* RH60 suspension:

[0065] The activation method for strain RH60 was as described in Example 2. The RH60 seed culture was inoculated into LB liquid medium at a ratio of 1:1000 and cultured at 30°C with shaking at 180 rpm for 8-10 hours. After centrifugation at 12000 rpm for 5 minutes, the culture medium was removed, and the bacterial cells were resuspended in sterile water. Then, based on OD... 600 Adjust the value of the bacterial solution to 10. 7 CFU mL -1 A bacterial suspension with RH60 was prepared.

[0066] 2. Treatment of cucumber seedlings:

[0067] Select plump cucumber seeds of uniform size and soak them in sterile water for 6 hours. Disinfect the seeds with 75% ethanol, rinse three times with sterile water, and place them in petri dishes lined with germination paper to germinate. After germination, sow the cucumber seeds in sterilized nutrient soil, planting one seedling per seedling pot (10×10×8cm). When the cucumbers have developed one true leaf, inoculate them with bacterial solution (root drenching), using 10mL of a 10% concentration per seedling. 7 CFU mL -1 RH60 bacterial suspension. Cucumbers with added sterile water were used as a control, and 15 cucumber seedlings were planted in each treatment.

[0068] 3. Biomass survey of cucumbers:

[0069] Fourteen days after inoculation with the bacterial solution, the biomass of cucumbers was measured, including aboveground fresh weight, plant height, root fresh weight, and root length. Results from potted plants are as follows: Figure 3As shown, compared with the control group which only received sterile water, inoculation with RH60 bacterial solution significantly increased the aboveground fresh weight, plant height, root fresh weight, and root length of cucumbers. After RH60 treatment, the aboveground fresh weight of cucumbers increased from 2.4g in the control group to 3.8g; plant height increased from 12.1g to 12.6g; root fresh weight increased from 365.9g to 534.0g; and root length increased from 17.2g to 22.5g. These results indicate that RH60 can promote cucumber growth.

[0070] Example 4:

[0071] Pot experiment on the control of cucumber root-knot nematode disease using Bacillus oxysporum RH60.

[0072] 1. Preparation of *Pseudomonas oxidans* RH60 suspension:

[0073] The method for preparing RH60 bacterial suspension is the same as in Example 3, where the concentration of the bacterial suspension is adjusted to 10. 7 CFU mL - 1 2. Treatment of cucumber seedlings:

[0074] Select plump cucumber seeds of uniform size and soak them in sterile water for 6 hours. Disinfect the seeds with 75% ethanol, rinse three times with sterile water, and place them in petri dishes lined with germination paper to germinate. After germination, sow the cucumber seeds in sterilized nutrient soil, planting one seedling per seedling pot (10×10×8cm). When the cucumbers have developed one true leaf, inoculate them with bacterial solution (root drenching), using 10mL of a 10% concentration per seedling. 7 CFU mL -1 RH60 bacterial suspension was used. Four days after inoculation, 800 second-instar larvae (J2s) of the southern root-knot nematode were inoculated per plant. Cucumbers inoculated only with the southern root-knot nematode served as a control, and 15 cucumber seedlings were planted for each treatment.

[0075] 3. Survey of cucumber biomass and root-knot nematode disease

[0076] Thirty days after nematode inoculation, cucumber biomass was measured, including aboveground fresh weight, plant height, root fresh weight, and root length. Simultaneously, the number of root-knot nematodes and the root-knot index in the soil were recorded. 100 mL of soil was measured in a beaker and isolated using the shallow dish method (Whitehead and Hemming 1965). The disease severity grading criteria for root-knot nematodes are as follows: Grade 0: No root knots; Grade 1: A small number of root knots, accounting for 1%-25% of the total root system; Grade 2: Moderate number of root knots, accounting for 26%-50% of the total root system; Grade 3: A relatively large number of root knots, accounting for 51%-75% of the total root system; Grade 4: A very large number of root knots, accounting for 76%-100% of the total root system (Benjamn et al. 1987).

[0077]

[0078] The survey results of potted plants are as follows Figure 4 As shown, compared with the control group inoculated only with southern root-knot nematodes, inoculation with RH60 significantly increased the aboveground fresh weight, plant height, root weight, and root length of cucumbers; while the number of nematodes in the soil and the root knot index significantly decreased. After RH60 treatment, the aboveground fresh weight of cucumbers increased from 3.0g in the control group to 4.0g; plant height increased from 16.7g in the control group to 22.2g; root fresh weight increased from 1.7g in the control group to 2.4g; and root length increased from 18.4g in the control group to 23.1g. The number of nematodes in the soil decreased from 2626 in the control group to 1913; and the root knot index of cucumbers decreased from 80.0 in the control group to 61.7. These results indicate that RH60 can be used as a beneficial biocontrol agent to control southern root-knot nematodes and promote cucumber growth.

[0079] Example 5:

[0080] Determination of the degradation ability of *Pseudomonas oxidans* RH60 on p-hydroxybenzoic acid, an autotoxic substance in cucumber-continuously cropped soil:

[0081] 1. Growth of *Pseudomonas oxidans* RH60 on a medium with p-hydroxybenzoic acid as the sole carbon source.

[0082] The culture medium used in this experiment was M9 liquid medium (formulation: 1.0 g / L). -1 NH4Cl, 0.13 g L -1 MgSO4, 3.0 g / L -1 KH2PO4, 6.0g L -1 (Na2HPO4). The concentration of the p-hydroxybenzoic acid stock solution was 200 mg / mL. -1 It is dissolved in dimethyl sulfoxide (DMSO).

[0083] The activation method for the strain is as described in Example 2. The RH60 seed culture was inoculated into LB liquid medium at a ratio of 1:1000 and cultured at 30°C and 180 rpm for 8-10 hours until OD (digestion). 600 =0.6. Take 500μL OD 600 =0.6% bacterial suspension was inoculated into 50 mL of a solution containing 200 μg / mL. -1 In M9 medium containing p-hydroxybenzoic acid, an equal volume of DMSO was added instead of p-hydroxybenzoic acid as a control, with each treatment in triplicate. The culture was incubated at 30°C and 180 rpm, and the OD was measured at 12, 24, 36, and 48 hours. 600 And record the data.

[0084] OD of RH60 at various time periods 600 like Figure 5 As shown in Figure A, the results indicate that strain RH60 exhibits an exponential growth trend in the medium supplemented with p-hydroxybenzoic acid, suggesting that p-hydroxybenzoic acid can be used as the sole carbon source for growth.

[0085] 2. Degradation of *Pseudomonas oxidans* RH60 on a culture medium with p-hydroxybenzoic acid as the sole carbon source.

[0086] The bacterial degradation assay for p-hydroxybenzoic acid followed the same steps as described in section 1 above, with the control being the addition of only 200 μg / mL to M9 medium. -1 p-hydroxybenzoic acid was detected in three replicates for each treatment. At 12, 24, 36, and 48 hours, 1 mL of bacterial culture was centrifuged at 12000 rpm for 10 min. The supernatant was collected, filtered through a 0.22 μm filter, and the p-hydroxybenzoic acid content in the culture medium was determined by high-performance liquid chromatography (HPLC). The mobile phase for HPLC was methanol:water = 25:75 (v / v), the column was an Agilent C18, 250 × 4.6 mm, and the flow rate was 1 mL / min. -1 The injection volume was 20 μL, the detection wavelength was 280 nm, and the column temperature was 30 °C.

[0087] A standard curve was plotted based on the concentration and peak area of ​​the p-hydroxybenzoic acid standard sample. The curve equation is as follows: Figure 5 As shown in C, y = 11.158x - 19.35, R 2 =0.9989, where x represents the concentration of p-hydroxybenzoic acid and y represents the peak area at that concentration.

[0088] Based on the standard curve, the remaining content of p-hydroxybenzoic acid in the culture medium under RH60 treatment was calculated, and the results are as follows: Figure 5 As shown in Figure A, the content of p-hydroxybenzoic acid in the culture medium decreased with the growth of strain RH60. At 48 hours of culture, the OD of the strain... 600 The value reached its maximum, and the content of p-hydroxybenzoic acid also reached its minimum. At this time point, the degradation rate of RH60 reached 98.8%. The above results indicate that the isolated *Pseudomonas oxysporum* RH60 has the ability to degrade p-hydroxybenzoic acid.

[0089] 3. Degradation of p-hydroxybenzoic acid in cucumber-continuously cropped soil by *Pseudomonas oxidans* RH60.

[0090] To determine the degradation ability of strain RH60 on p-hydroxybenzoic acid produced by continuous cropping, soil was collected from a greenhouse where cucumbers were grown for three consecutive seasons. 50 mL of a 10% concentration was added to every 1 kg of soil. 7 CFU mL -1A bacterial suspension at RH60 was thoroughly mixed, and an equal volume of water was added to the soil sample as a control. Phenolic acids were extracted from the soil at 12, 24, 36, and 48 hours, and the content of p-hydroxybenzoic acid in the soil was determined. The extraction method for phenolic acids in the soil was as follows: 20g of soil sample was weighed and 100mL of 2mol / L solution was added. -1 The sample was incubated in NaOH solution at 28℃ and 120 rpm with shaking for 24 h. The incubator was aliquoted into 50 mL centrifuge tubes and centrifuged at 6000 rpm for 15 min. 45 mL of the supernatant was collected, and the pH was adjusted to 2.5 with hydrochloric acid. An equal volume of ethyl acetate was added for extraction, and the supernatant was collected. This extraction was repeated three times, and the supernatants were combined, evaporated to dryness by rotary evaporation, and then dissolved in methanol. Finally, the sample was filtered through a 0.22 μm filter membrane, and the p-hydroxybenzoic acid content was determined by liquid chromatography.

[0091] The results are as follows Figure 5 As shown in Figure B, the degradation rate of p-hydroxybenzoic acid in the soil was significantly higher after the addition of RH60 than that without addition. At 48 hours, the p-hydroxybenzoic acid content in the soil was 26.5 μg / g compared to the uninoculated soil. -1 Decreased to 3.8 μg g -1 The above results further validate the ability of strain RH60 to degrade p-hydroxybenzoic acid in soil.

Claims

1. A strain of *Pseudomonas oxidans* ( Pseudarthrobacter oxydans The preservation number of *Pseudomonas oxidans* RH60 is CCTCC NO: M 2023054.

2. The application of the *Pseudomonas oxidans* RH60 as described in claim 1 in the control of southern root-knot nematodes.

3. The application of the *Pseudomonas oxidans* RH60 as described in claim 1 in the preparation of an insecticide for southern root-knot nematodes.

4. The application of the *Pseudomonas oxidans* RH60 according to claim 1 in the preparation of cucumber growth promoter.

5. The application of the *Pseudomonas oxidans* RH60 as described in claim 1 in the degradation of p-hydroxybenzoic acid.

6. The use of the *Pseudomonas oxidans* RH60 according to claim 1 in the preparation of a p-hydroxybenzoic acid degrading agent.

7. The application of the *Pseudomonas oxidans* RH60 as described in claim 1 in alleviating cucumber continuous cropping obstacles.

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