A strain of *Pseudomonas oxidans* and its application

Abstract

This invention relates to a strain of *Pseudomonas oxidans* D-14 with accession number CGMCC No. 31959. Pseudarthrobacter oxydans This bacterium has several applications, including degrading carbendazim and promoting the growth of leafy greens. It can be prepared as a microbial agent, which, after colonizing leafy greens, effectively promotes the degradation of carbendazim in both the vegetables and the soil, increasing the biomass and nutritional indicators of the vegetables. As a rhizosphere bacterium, *Pseudomonas oxysporum* can colonize the roots of leafy greens, promoting vegetable growth in addition to its carbendazim degradation properties. The use of this microbial agent helps reduce carbendazim residues in the environment and crops, thus contributing to environmental and agricultural product safety.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and food safety, and in particular to a strain of Pseudarthrobacter oxydans D-14 and its application in degrading carbendazim and promoting crop growth. Technical Background

[0002] Carbendazim is a widely used, highly effective, low-toxicity, broad-spectrum systemic fungicide that effectively controls fungal diseases in crops such as Fusarium head blight, black spot, and anthracnose, making it widely used in agricultural production. However, due to its frequent use during plant growth and development, carbendazim residues are easily detected in common everyday foods such as vegetables, fruits, grains, oilseeds, and medicinal herbs. Carbendazim is chemically stable, has a long residual period in the natural environment, and is difficult to degrade. Continuous use over many years causes soil, water, and air pollution. Furthermore, carbendazim can be absorbed by plants, entering their bodies and affecting crop growth and quality, posing food safety risks, and contributing to the deterioration of the agricultural environment. Timely removal of pesticide residues from soil and crops is an urgent problem that needs to be solved in agricultural production.

[0003] The degradation of carbendazim in the environment includes physical, chemical, and microbial degradation. Microbial degradation is considered a promising method for degradation or detoxification due to its high efficiency, economy, and eco-friendliness. Currently, there are limited applications of *Pseudomonas oxysporum*, with some applications focusing on alleviating crop rotation obstacles and improving heavy metal tolerance. However, there are no literature reports on the degradation of the pesticide carbendazim by *Pseudomonas oxysporum*. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a strain of *Pseudomonas oxysporum* (D-14), which can degrade carbendazim in the Chinese cabbage planting system, reduce the accumulation of carbendazim in the above-ground and underground parts of Chinese cabbage, and at the same time promote the growth and improve the nutritional indicators of Chinese cabbage.

[0005] Specifically, this application is implemented through the following technical solution:

[0006] First, this application provides a strain of *Pseudarthrobacter oxydans* with accession number CGMCC No. 31959. This strain is a Gram-negative bacterium, appearing as short rods, arranged in clusters, and without spores. The applicant has named it D-14. This strain was isolated from the rhizosphere of vegetables grown in pesticide-contaminated soil and is harmless to plants and animals. The applicant deposited this strain on September 12, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Postcode: 100101; accession number: CGMCC No. 31959.

[0007] Secondly, this application also provides the application of *Pseudomonas oxidans* with accession number CGMCC No. 31959 in the degradation of carbendazim, including its application in the degradation of carbendazim in the environment (such as soil) and in crops (such as leafy greens).

[0008] Furthermore, the application of *Pseudomonas oxysporum* in degrading carbendazim in vegetables and the planting environment involves the following steps: Root irrigation is performed on crops with *Pseudomonas oxysporum* inoculant (D-14 inoculant) to degrade carbendazim residues in crops and soil; the above-mentioned root irrigation refers to two treatments: one before transplanting and one in the second week after transplanting, with 100 ml per plant each time.

[0009] Third, this application also provides the application of *Pseudomonas oxidans* (accession number CGMCC No. 31959) in promoting the growth of Chinese cabbage and improving its biomass and quality (soluble sugars and soluble proteins). Specifically, the application involves drenching the roots of Chinese cabbage with *Pseudomonas oxidans* inoculant (D-14 inoculant) to promote growth and improve biomass and quality. The root drenching is performed twice: once before transplanting and once in the second week after transplanting, with 100 ml per plant each time.

[0010] The above-mentioned *Pseudomonas oxidans* inoculum (D-14 inoculum) is composed of MSM medium and *Pseudomonas oxidans* with accession number CGMCC No. 31959. The composition of each liter of MSM medium is as follows: 0.4g MgSO4·7H2O, 0.2g FeSO4·7H2O, 0.2g K2HPO4, 0.2g (NH4)2SO4, 0.08g CaSO4, 1L deionized water, pH 7.0. The preferred final concentration of *Pseudomonas oxidans* in the above-mentioned D-14 inoculum is 10. 7 cfu / mL.

[0011] This application marks the first discovery of *Pseudomonas oxidans*, a bacterium capable of degrading carbendazim, in cabbage plants grown in soil contaminated with carbendazim. The strain was then studied, identified, and preserved. Experiments have demonstrated that this strain can colonize the rhizosphere and roots of cabbage, reducing carbendazim residues in the soil and both above-ground and below-ground parts of the cabbage, thereby decreasing carbendazim residues in crops and the environment. Simultaneously, it promotes cabbage growth and improves quality indicators. Attached Figure Description

[0012] Figure 1 This is a scanning electron microscope image of *Pseudomonas oxidans* D-14.

[0013] Figure 2 This is a schematic diagram illustrating the in vitro degradation function of carbendazim by Pseudomonas oxidans D-14.

[0014] Figure 3 This is a schematic diagram showing the degradation results of carbendazim residues in soil and vegetables by D-14 inoculant.

[0015] Figure 4 This is a schematic diagram of growth-promoting index data for Bacillus oxidans D-14. Detailed Implementation

[0016] The culture medium involved in the examples:

[0017] LB medium / LB solid plates: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15-20 g / L, adjust pH to 7.0 with NaOH;

[0018] Inorganic salt liquid culture medium (MSM): MgSO4·7H2O (0.4g), FeSO4·7H2O (0.2g), K2HPO4 (0.2g), (NH4)2SO4 (0.2g), CaSO4 (0.08g), 1L deionized water, pH 7.0.

[0019] MSM solid medium: Add agar to an inorganic salt liquid medium to a final concentration of 20 g / L.

[0020] King B medium: 1000 mL deionized water, 32 g tryptone, 1.15 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate heptahydrate, 10 mL glycerol, plus 1 g L-tryptophan.

[0021] Nitrogen-fixing medium was purchased from Coolaber, and siderogenic medium (CAS) was purchased from Xianghai Biotechnology Co., Ltd. The preparation methods of the above media were in accordance with the instructions for use of the media.

[0022] All culture media must be autoclaved at 121°C for 20 minutes before use.

[0023] Unless otherwise specified, all reagents used in the following examples were purchased commercially.

[0024] Example 1: Isolation, Identification, and Degradation Function Verification of *Pseudomonas oxysporum* D-14

[0025] 1. Isolation and identification of strain D-14 (Pseudarthrobacter oxydans D-14)

[0026] In June 2023, the applicant collected samples of Chinese cabbage grown in pesticide-contaminated soil from an experimental field (E118°86′, N32°03′) of the Jiangsu Academy of Agricultural Sciences to isolate and screen rhizosphere bacteria with carbendazim degradation capabilities. The rhizosphere bacteria isolation and screening method involved adding sterilized LB medium containing 50 ppm carbendazim to the rhizosphere soil samples and incubating them at 30°C and 150 rpm for 1 day on a shaker; then, the bacterial suspension was used for 10... 4 10 5 10 6 The solution was diluted several times, and 100 μl of the diluted solution was evenly spread onto LB agar plates containing 50 ppm carbendazim and incubated at 30°C in the dark for 1–3 days. Single colonies with good growth from the LB plates were selected and streaked onto MSM agar plates containing 20 mg / kg carbendazim. The plates were incubated at 30°C for 1 day. The colonies on the MSM plates were continuously purified until a single clone was obtained. A rhizosphere bacterium with carbendazim degradation characteristics was obtained through screening. Its electron micrograph is shown below. Figure 1 As shown, the strain is short rod-shaped, arranged in clusters, and has no spores. The applicant named the strain D-14.

[0027] Based on physiological and biochemical characteristics and 16S rDNA conserved sequence comparison, strain D-14 was identified as *Pseudarthrobacter oxydans*. The applicant deposited this strain with the China General Microbiological Culture Collection Center (CGMCC) on September 10, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 22887, and classified it as *Pseudarthrobacter oxydans*.

[0028] 2. Verification of the function of strain D-14 (Pseudarthrobacter oxydans D-14) in degrading carbendazim

[0029] First, culture the D-14 strain obtained in step 1 on LB medium for 12 hours. After centrifugation (6000 rpm, 5 min, 20℃), the bacterial suspension was resuspended three times in sterile inorganic salt solution (pH 7.0) and washed. The OD value was then adjusted. 600=1.0); then, at a volume ratio of 2%, the D-14 bacterial suspension was added to (10 μg / mL) inorganic salt liquid medium with carbendazim as the sole carbon source and cultured for 3, 7, and 14 days respectively; the residual amount of carbendazim in the inorganic salt liquid medium at different culture times was detected by liquid chromatography-mass spectrometry (LC-MS), and compared with the blank group (CK), the degradation rate of strain D-14 was calculated:

[0030] C = (AB) / A * 100%

[0031] A = Carbendazim concentration in CK, mg / L;

[0032] B = Carbendazim concentration in the experimental group, mg / L;

[0033] C = Carbendazim degradation rate

[0034] The carbendazim in vitro degradation ability of D-14 was calculated using this formula. The LC-MS parameters are as follows:

[0035] Analysis was performed using a Shimadzu high-performance liquid chromatography-tandem AB Sciex QTRAP 5500+ mass spectrometry system. The chromatographic column was a Kinetex C18 column (3.0 × 100 mm, 2.6 μm). The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B). The chromatographic gradient program started at 90% A, held for 1 min, then decreased to 10% A over 3 min and held for 1 min, then recovered to 90% A over 5.01 min and held for 0.9 min. The flow rate was 0.3 mL / min, the column temperature was 30 °C, and the injection volume was 2 μL. The scanning mode was positive ion mode. The electrospray voltage was +5.50 kV. The auxiliary gas (N2) and nebulizer gas (N2) were 55 psi, the ion source temperature was 550 °C, and the assay method was multiple reaction monitoring (MRM).

[0036] Test results as follows Figure 2 As shown, within 14 days, the D-14 bacterial culture (OD) 600 =1.0) The degradation rate of D-14 at a concentration of 10 mg / L carbendazim was 28.6%. This verifies that D-14 has the degradation properties of carbendazim.

[0037] Example 2: Preparation of D-14 bacterial agent

[0038] The specific preparation steps of D-14 inoculant in this embodiment are as follows:

[0039] 1) The D-14 strain isolated in Example 1 was inoculated into LB medium containing 10 mg / L (final concentration) carbendazim for enrichment. It was streaked successively on MSM solid plates containing 10 mg / L (final concentration) carbendazim and LB solid plates containing 10 mg / L (final concentration) carbendazim. Single colonies were then picked from the plates and placed in LB liquid medium. The culture was carried out at 30°C and 150-220 rpm for 12 h. This activation process was repeated twice to obtain activated bacterial solution.

[0040] 2) In a clean bench, place the activated bacterial solution into a 50ml sterile centrifuge tube and centrifuge at 20℃ and 6000rpm for 5 minutes to obtain precipitated bacterial cells. Autoclave the precipitate with MSM liquid medium for 20 minutes. Rinse the precipitate three times with MSM liquid medium and then add more MSM liquid medium to adjust the OD of the bacterial solution. 600 =1 (bacterial concentration is approximately 10) 10 (cfu / mL) is used to obtain D-14 bacterial agent.

[0041] Example 3D-14: Experiment on promoting the degradation of carbendazim in leafy greens and soil environment, and promoting the growth of leafy greens.

[0042] Soil samples were collected from the field at the Jiangsu Academy of Agricultural Sciences. After drying, the soil was crushed and sieved through a 20-mesh screen to remove impurities before use. Composition: Organic matter 4%; Silt 49%; Clay 22%; Sand 25%. Original soil nutrients: Total organic carbon 14.86 mg / kg; Available potassium 0.14 g / kg; Available phosphorus 0.05 g / kg; Total nitrogen 1.65 g / kg; pH 6.7.

[0043] Carbendazim treatment: Carbendazim technical grade was diluted and mixed into the soil. After the soil had aged for one week, a vegetable planting experiment was conducted (the concentration of carbendazim in the soil was about 5 ppm at the time of planting). 1.5 kg of soil was placed in each pot. There were inoculant treatment group and control group. Each treatment was replicated 3 times.

[0044] Shanghai bok choy seedlings were transplanted at the three true leaf stage. The specific experimental steps for the inoculum treatment group were as follows: The bacterial strain was inoculated onto LB medium and cultured in a shaker at 30℃ and 180 rpm for 24 hours. The culture was then centrifuged and washed three times with MSM medium. Bacterial cells were collected and diluted with MSM medium to adjust the bacterial concentration to OD0.05. 600 It reaches around 0.2 (the density of bacteria in a plate count is approximately 1 × 10⁻⁶). 7 The bacterial solution (cfu / mL) is the bacterial agent. The bacterial agent was applied as irrigation one day before transplanting and two weeks after transplanting, with each irrigation volume being 100 mL. The control group was treated with the same volume of sterile MSM medium. All other management practices were the same for both groups.

[0045] Harvested 30 days after transplanting, soil and vegetable samples were collected, with three parallel treatments (n=3). Test results are as follows: Figure 3As shown.

[0046] Carbendazim Accumulation and Degradation: At harvest, the carbendazim concentration in the soil of the inoculant-treated group was 4.15 mg / kg, while that in the control group was 5.95 mg / kg. The removal rate of carbendazim in the soil by the inoculant-treated group was 30.25%. The carbendazim content in the cabbage plants was tested. The carbendazim concentrations in the aboveground and underground parts of the cabbage in the inoculant-treated group were 2.34 and 3.41 mg / kg, respectively, while those in the control group were 4.17 and 5.36 mg / kg, respectively. The carbendazim concentrations in the aboveground and underground parts of the cabbage in the inoculant-treated group decreased by 43.88% and 36.38%, respectively. It can be seen that this strain has a good degradation effect on carbendazim residues in the soil and carbendazim in crop plants.

[0047] D-14 improves the biomass and quality of leafy greens, as shown in Table 1 below:

[0048] Table 1 Results of biomass and quality testing of Chinese cabbage

[0049]

[0050] The results of the D-14 inoculant treatment on improving the quality indicators of Chinese cabbage are shown in Table 1. Under carbendazim stress, the fresh weight of Chinese cabbage treated with D-14 at harvest on day 30 was significantly higher than that of the control group (CK group) and 30.97% higher than that of the uncontrolled group. Under carbendazim stress, the soluble sugar and soluble protein content of Chinese cabbage treated with D-14 at harvest on day 30 were increased by 10.94% and 14.29% respectively compared to the CK group.

[0051] Example 4: Experiment on the growth-promoting properties of D-14 and its effects on promoting growth and improving the quality of Chinese cabbage under carbendazim stress.

[0052] 1. IAA: The D-14 bacterial agent prepared in Example 2 was inoculated into King B medium containing L-tryptophan (1g) (the precursor for IAA synthesis). Uninoculated King B medium was used as the control group (CK). The culture was carried out at 32℃ and 180 rpm for 12 h on a shaker. After centrifugation at 8000 rpm for 5 min, the supernatant was mixed thoroughly with Salkowski's reagent (50 mL of 35% perchloric acid plus 1 mL of 0.5 mol / L ferric chloride) at a ratio of 1:2. The mixture was then placed in the dark for 30 min. The presence of a dark red substance in the D-14 group indicated that the bacteria could produce IAA (see...). Figure 4 (as shown in a).

[0053] 2. Nitrogen fixation ability: When D-14 bacterial culture is inoculated into nitrogen-fixing medium and cultured, a clear zone can be observed around D-14, indicating that D-14 has a certain nitrogen-fixing ability (see...). Figure 4 (As shown in b).

[0054] 3. CAS Production Capacity: When D-14 bacterial culture was inoculated into CAS medium and cultured, the strain grew normally and formed a yellow transparent zone around it, indicating that D-14 has the ability to produce siderophores (see...). Figure 4 (as shown in c).

[0055] The above experimental results show that D-14 inoculant has a good growth-promoting effect and can improve the biomass and quality of leafy greens.

Claims

1. A strain of *Pseudomonas oxidans* ( Pseudarthrobacter oxydans D-14, with accession number CGMCC No.31959.

2. The application of *Pseudomonas oxidans* as described in claim 1 in degrading carbendazim and promoting the growth of leafy greens; the promotion of leafy green growth refers to increasing the biomass, soluble sugar content, and soluble protein content of leafy greens.

3. The application as described in claim 2, characterized in that, The carbendazim mentioned refers to the carbendazim that degrades the environment and crop plants.

4. The application as described in claim 2, characterized in that, The application refers to the root irrigation treatment of leafy greens with an agent containing the *Pseudomonas oxidans* as described in claim 1.

5. The application according to claim 4, characterized in that, The bacterial agent containing *Pseudomonas oxidans* as described in claim 1 includes MSM culture medium and *Pseudomonas oxidans*.

6. A bacterial agent containing *Pseudomonas oxidans* with accession number CGMCC No. 31959.

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