Priestia megaterium AS and application thereof in degrading methyl-parathion

Abstract

The present application relates to the technical field of microbial pesticide pollution remediation, and specifically discloses a strain capable of efficiently degrading MCPA and application thereof. The strain is Priestia megaterium AS, and the preservation number is CCTCC No. M 20242160. The present application provides a method for isolating and screening the strain, which grows well in a culture medium containing 500 μmol / L MCPA and can degrade MCPA as a carbon source, with a degradation rate of 500 μmol / L MCPA exceeding 50% within 7 days. It is proved by a potting test that inoculation of the strain or its preparation can significantly reduce the MCPA residue in the rhizosphere soil, effectively alleviate the toxic effect of MCPA on sensitive crops such as melons and pumpkins, and promote the growth recovery of plants. The strain and its bacterial agent provide an efficient and environmentally friendly biological technology solution for remediation of MCPA contaminated soil and guarantee of safe production of melon crops.

Description

Technical Field

[0001] This invention belongs to the field of microbial pesticide pollution remediation technology, and specifically discloses a strain that can efficiently degrade MCPA and its application. Background Technology

[0002] 2,4-methyl-4-chlorophenoxyacetic acid (MCPA), a hormone-based selective herbicide, is widely used for weed control in rice, wheat, and corn fields due to its high efficiency in controlling broadleaf weeds in gramineous crops. However, MCPA has a certain persistence in the environment, and its residues can cause soil and water pollution and may accumulate in organisms through the food chain, posing potential environmental and health risks. Studies have shown that MCPA exposure can have toxic effects on non-target organisms (such as mammals) and cause severe growth inhibition or even death in sensitive broadleaf crops (such as melons and cotton), which limits its safe use in crop rotation systems or areas adjacent to sensitive crops.

[0003] Currently, the main methods for controlling pesticide residues include physical, chemical, and biodegradation methods. Among these, utilizing microorganisms to degrade pesticide residues is considered a promising strategy due to its environmental friendliness, low cost, and low likelihood of secondary pollution. Existing technologies have reported on microorganisms (such as certain bacteria and fungi) capable of degrading phenoxycarboxylic acid herbicides; however, these microbial resources are still relatively limited, and in practical applications, they often suffer from low degradation efficiency, poor environmental adaptability, or unclear protective effects on specific crops. In particular, for the bioremediation of MCPA residues' toxic effects on high-value cucurbit crops (such as melons and pumpkins), there is a lack of efficient, specific microbial resources with clearly defined application effects.

[0004] Therefore, there is an urgent need in this field to screen for a new strain of microorganism that can efficiently degrade MCPA and effectively alleviate the toxic effects of MCPA on sensitive crops. This is of great significance for ensuring the safety of agricultural products, reducing environmental risks, and promoting sustainable agricultural development. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a strain of *Priestia megaterium* AS capable of efficiently degrading MCPA and its application in mitigating MCPA toxicity in sensitive crops. The aim of this approach is to obtain a patented strain with stable MCPA tolerance and degradation capabilities through targeted enrichment and high-concentration stress screening from long-term contaminated soil. This strain is then used to prepare microbial agents, which, through their colonization and degradation functions, specifically relieve the growth inhibition of sensitive crops such as cucurbits caused by MCPA, thereby achieving effective removal of pesticide residues, significant mitigation of crop phytotoxicity, and synergistic promotion of farmland soil ecological safety.

[0006] To achieve the above objectives, the present invention includes the following technical solutions.

[0007] A strain of *Priestia megaterium* AS, with accession number CCTCCNo.M20242160, has the ability to degrade MCPA.

[0008] Furthermore, the aforementioned *Priestella giantiformis* AS is characterized by having a 16S rRNA gene sequence as shown in SEQ ID No. 1.

[0009] The present invention also discloses a method for isolating the above-mentioned *Priscilla megaterium* AS, comprising the following steps:

[0010] S1. Collect soil samples containing 2,4-D residues;

[0011] S2. Dilute the soil sample and spread it on a solid culture medium containing 500 μmol / L dimethyltetrachloride;

[0012] S3. Incubate at 25℃ for 3-5 days, then pick single colonies for purification;

[0013] S4. The purified strain was continuously cultured in a medium containing 500 μmol / L dimethyltetrachloride to obtain a strain that is tolerant to and degrades dimethyltetrachloride.

[0014] The present invention also discloses a microbial inoculant containing the aforementioned Priestella giantiflora AS and an agriculturally acceptable carrier.

[0015] Furthermore, the aforementioned microbial agent can be a liquid agent, a solid agent, or a lyophilized powder.

[0016] The present invention also discloses the application of the above-mentioned Priestella giantiflora AS in the degradation of 2,4-D.

[0017] The present invention also discloses the application of the above-mentioned Priestella megaterium AS in mitigating the toxic effects of 2,4-D on plants.

[0018] Furthermore, in the above application, the plant is a cucurbit crop, preferably a melon or a pumpkin.

[0019] The present invention also discloses a method for degrading 2,4-D in soil or water, comprising applying the microbial agent described in the present invention to the soil or water.

[0020] Furthermore, in the above method, the application rate of the microbial agent is 1×10⁻⁶ per hectare. 8 -1×10 12 CFU.

[0021] Compared with the prior art, the present invention has the following outstanding advantages:

[0022] 1. Highly efficient and specific degradation ability: Strain AS can tolerate and efficiently degrade MCPA. Within 7 days, the degradation rate of 500 μmol / L MCPA can reach more than 50%, which is highly targeted and has a high repair efficiency.

[0023] 2. Significant crop detoxification and protection functions: Pot experiments have confirmed that inoculation with this strain can significantly reduce MCPA residues in the soil, effectively reverse the inhibitory effect of MCPA on the growth of melon and pumpkin seedlings, alleviate symptoms of pesticide damage such as stem shrinkage and leaf yellowing, and restore normal plant growth. This provides a direct and effective biological means to solve the harm of MCPA to sensitive crops.

[0024] 3. Environmentally friendly and highly safe: It adopts a microbial degradation method, which does not introduce secondary chemical pollution. Moreover, the strains are isolated and screened from nature, which have good environmental compatibility and are safe to use.

[0025] 4. Flexible application: The strains can be prepared into various forms of microbial agents, such as liquid, solid or dry powder, which are convenient for storage, transportation and field application, and can be adapted to different agronomic operation needs.

[0026] The preservation information for the strain is as follows:

[0027] Name of depositary institution: China Center for Type Culture Collection;

[0028] Address of the depository: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province;

[0029] Deposit date: October 11, 2024

[0030] Accession number CCTCC No. M20242160

[0031] Taxonomic nomenclature: Priestia megaterium AS. Attached Figure Description

[0032] Figure 1 Images of colonies and cell morphology of selected MCPA-tolerant strains: A, Left: Colony image on medium without MCPA; Right: Image of the strain on medium containing MCPA (500 μmol); B, Growth status of purified strains on medium containing MCPA; C, Single cell image on normal medium; D, Single cell image under 500 μmol MCPA; E, Single cell image under 900 μmol MCPA.

[0033] Figure 2 Schematic diagram of the developmental tree for identifying strains. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1

[0036] Isolation, purification and identification of strains

[0037] 1. Sample Source and Collection

[0038] The strain of this invention was isolated from soil in a greenhouse vegetable field that had been using MCPA herbicide for a long period of time. During collection, soil samples were collected from multiple points in the topsoil layer (0-20 cm) using sterile paper bags. After being mixed evenly, approximately 1.0 kg of the sample was placed in a 4°C low-temperature incubator and brought back to the laboratory as soon as possible.

[0039] 2. Strain Isolation

[0040] Weigh 1.0 g of the above soil sample and add it to a centrifuge tube containing 9 mL of sterile physiological saline (0.85% NaCl). Shake to mix well and prepare 10... -1 Soil suspension. Subsequently, it was serially diluted 10-fold to 10... -5 Take 10 samples respectively. -3 10 -4 10 -5Three gradient dilutions, 200 μL each, were spread onto MCPA screening agar plates. The screening medium consisted of (per liter): 10 g bacterial peptone, 1.0 g tryptone, 3.0 g sodium chloride, 15 g agar, with MCPA added to a final concentration of 500 μmol / L. The pH was adjusted to 6.8-7.0 with NaOH or HCl, and the plates were sterilized at 121°C for 20 min. The spread plates were then incubated upside down in a 25°C incubator for 3-5 days.

[0041] 3. Strain purification and preservation

[0042] After incubation, observe the colony growth on the plates. Select dominant colonies that grow vigorously and exhibit a uniform morphology on plates containing 500 μmol / L MCPA, and perform multiple streaking separations on fresh screening plates with the same composition until a pure culture is obtained. Name the purified single colony AS.

[0043] The pure bacterial strain was inoculated into LB liquid medium and cultured to the late logarithmic growth stage. One mL of the bacterial suspension was then mixed thoroughly with an equal volume of 40% (v / v) sterile glycerol and stored at -80°C for long-term preservation. This strain was also deposited at the China Center for Type Culture Collection (CCTCC, Wuhan University) on October 11, 2024, with accession number CCTCC No. M20242160.

[0044] 4. Strain identification

[0045] (1) Morphological observation: After culturing for 48 hours on LB plates, strain AS formed round, milky-white colonies with smooth, moist surfaces and regular edges. Gram staining showed it to be Gram-positive, and under a microscope, the cells appeared rod-shaped and could form spores. Figure 1 As shown in Figure A, the left side shows the typical colony morphology of strain AS on MCPA-free medium, while the right side shows the colonies grown on selection medium containing 500 μmol / L MCPA, indicating that this strain can grow normally under high concentration MCPA stress. Figure 1 B further demonstrates the individual cell morphology of the purified strain on MCPA-containing medium.

[0046] (2) Molecular biological identification: Total DNA was extracted from strain AS using a bacterial genomic DNA extraction kit. Using this DNA as a template, the 16S rRNA gene sequence was amplified using universal bacterial primers. The PCR product was purified and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequence is shown in SEQ ID No. 1, and its length is approximately 1500 bp.

[0047] The sequenced data were BLAST-aligned with the NCBI database, and the results showed that it had more than 99% similarity to multiple type strains of *Priestiamegaterium*. The phylogenetic tree is shown below. Figure 2 As shown, based on its morphological characteristics, it was finally identified as Priestia megaterium and named Priestia megateriumAS.

[0048] SEQ ID No.1=

[0049] CTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAACT

[0050] GATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGACGGGTGAGTAACACG

[0051] TGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAAT

[0052] ACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGG

[0053] CTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAAC

[0054] GGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCAC

[0055] ACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAA

[0056] TCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGA

[0057] AGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACGAGAG

[0058] TAACTGCTCGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTAC

[0059] GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTAT

[0060] TGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCA

[0061] CGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAGACTTGAGTGCAGAAG

[0062] AGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGG

[0063] AACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGC

[0064] GAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTA

[0065] AACGATGAGTGCTAAGTGTTAGAGGGTTTCGCCCTTTAGTGCTGCAGCT

[0066] AACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCA

[0067] AAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCG

[0068] AAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAG

[0069] AGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTG

[0070] TCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCCGCAACGAGCGCAA

[0071] CCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCC

[0072] GGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTT

[0073] ATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAG

[0074] ACCCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAG

[0075] GCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGC

[0076] ATGCCGCGGTGAATACGT.

[0077] Example 2

[0078] Determination of the strain's ability to degrade MCPA.

[0079] 1. Experimental Design

[0080] Prepare an inorganic salt liquid medium (MSM) with MCPA as the sole carbon source: (NH4)2SO4 1.0 g, Na2HPO4·12H2O 1.5 g, KH2PO4 0.5 g, MgSO4·7H2O 0.2 g, CaCl2 0.01 g, trace element solution 1 mL, and distilled water to a final volume of 1 L, pH 7.0. Add MCPA standard to the MSM to prepare culture media with final concentrations of 100 μmol / L, 500 μmol / L, and 900 μmol / L, respectively.

[0081] The activated Priestia megaterium AS strain was inoculated into LB liquid medium and cultured at 30°C with shaking at 180 rpm until the mid-log phase (OD2). 600 ≈ 0.8). Collect bacterial cells by centrifugation, wash twice with sterile physiological saline, and resuspend at OD0.05. 600 = 1.0. The bacterial suspension was inoculated into MSM media containing different concentrations of MCPA at an inoculum rate of 5% (v / v). Simultaneously, a drug-containing medium without inoculation was set up as a blank control. Each treatment was performed in triplicate.

[0082] The inoculated and control Erlenmeyer flasks were placed in a shaker at 30°C and 180 rpm and incubated in the dark.

[0083] 2. Sample Collection and Testing

[0084] At 0, 1, 3, 5, and 7 days of culture, 5 mL of culture medium was aseptically aspirated from each treatment. The culture medium was centrifuged at 12,000 rpm for 10 min, and the supernatant was collected, filtered through a 0.22 μm microporous membrane, and analyzed by high-performance liquid chromatography (HPLC).

[0085] HPLC detection conditions: C18 reversed-phase column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile: 0.1% phosphoric acid aqueous solution = 60:40 (v / v); flow rate: 1.0 mL / min; column temperature: 30℃; detection wavelength: 280 nm; injection volume: 20 μL. The residual amount of MCPA in the sample was calculated using the external standard method based on the MCPA standard curve.

[0086] 3. Results Analysis

[0087] The formula for calculating the degradation rate is: Degradation rate (%) = [(C0 - C] t [) / C0] × 100%, where C0 is the initial concentration of MCPA on day 0, C t The residual concentration is shown in Table 1 after t days of cultivation. The results are shown in Table 1.

[0088] Table 1: Degradation effect of strain AS on different concentrations of MCPA

[0089]

[0090] Experimental results showed that strain AS exhibited the ability to degrade MCPA at concentrations of 100, 500, and 900 μmol / L, and the degradation rate was correlated with the initial concentration. At an initial concentration of 500 μmol / L, the degradation rate reached over 50% after 7 days of cultivation, while the MCPA concentration in the blank control group showed no significant change. This indicates that *Priestia megaterium* AS can utilize MCPA as both a carbon and energy source for growth and metabolism, and effectively degrade it.

[0091] Example 3

[0092] Verification of the detoxification effect of the strain on cucurbit crops under MCPA stress (pot experiment)

[0093] 1. Experimental Materials and Design

[0094] The tested crops were: melon ('Jintian' variety) and pumpkin ('Zhenmu No. 6' variety).

[0095] Test strain: Priestia megaterium AS, prepared at a concentration of 1×10⁻⁶. 8 CFU / mL bacterial suspension.

[0096] Experimental substrate: peat nutrient soil sterilized at high temperature.

[0097] The experiment was set up as follows:

[0098] ① CK (blank control): Watered with sterile water, without adding MCPA, and without inoculating with bacterial strains.

[0099] ② MCPA (stress control): Irrigate with an aqueous solution containing 100 μmol / L MCPA to simulate residual stress in the field, without inoculating with bacterial strains.

[0100] ③ MCPA+AS (treatment group): Irrigate with an aqueous solution containing 100 μmol / L MCPA, and on the same day, irrigate the roots of each seedling with 10 mL of the above AS bacterial suspension (approximately 1×10⁻⁶). 9 CFU / strain).

[0101] Each treatment was repeated at least 3 times, with 6 seedlings per replicate, arranged in a randomized block design.

[0102] 2. Test Methods

[0103] Select plump and uniform melon and pumpkin seeds, sterilize them, and germinate them. When the radicle has grown to about 1 cm, sow them in seedling trays filled with sterilized substrate. Cultivate the seedlings in a greenhouse (day / night temperature 28 / 20℃, light 12 h / d) until they have one true leaf. Transplant uniformly growing seedlings into plastic flowerpots (15 cm in diameter), one seedling per pot. After a 3-day recovery period following transplanting, begin the above treatment. MCPA solution and bacterial suspension are applied via root drenching. All treatments maintain the same routine water and fertilizer management during the experiment.

[0104] 3. Observational Indicators and Statistics

[0105] After 21 days of treatment, the following indicators were measured:

[0106] (1) Growth indicators: Measure plant height and stem base diameter; separate the above-ground parts of the plant from the root system, weigh the fresh weight of each, then kill the green at 105℃, dry at 80℃ to constant weight, and weigh the dry weight.

[0107] (2) Morphological observation: Record the color and shape of the leaves, and whether there are symptoms of pesticide damage such as wilting, curling, and deformity.

[0108] (3) MCPA residue: Rhizosphere soil samples from each treatment were collected, air-dried, sieved, extracted by accelerated solvent extraction, and the MCPA residue in the soil was detected by HPLC.

[0109] (4) Data analysis: All data were analyzed using SPSS 26.0 software for one-way ANOVA, with the significance level set at P < 0.05.

[0110] 4. Test Results

[0111] As shown in Table 2.

[0112] Table 2: Effects of inoculation with AS strain under MCPA stress on growth indicators of melon and pumpkin

[0113]

[0114] Note: All data are mean ± standard deviation (n=3). There was a significant difference between the MCPA+AS group and the MCPA group (P < 0.05).

[0115] The results showed that, compared with the control group (CK), the growth of melon and pumpkin seedlings in the MCPA treatment group was significantly inhibited (P < 0.05), manifested as dwarfing, weak stems, and significantly reduced biomass (fresh and dry weight). Leaves exhibited varying degrees of yellowing and wrinkling, typical symptoms of herbicide damage. MCPA stress led to severe dwarfing and stem shrinkage in melon and pumpkin seedlings, with growth almost completely halted. Compared with the MCPA-stressed control group, the seedlings in the Priestia megaterium AS treatment group (MCPA+AS) showed significant improvement in all growth indicators. Specifically, plant height, stem diameter, and aboveground dry weight significantly increased (P < 0.05), herbicide damage symptoms were significantly reduced, and plant growth approached that of the CK group. After inoculation with the AS strain, the growth status of melon and pumpkin seedlings significantly recovered, with increased plant height and morphology approaching normal. Simultaneously, the residual MCPA in the rhizosphere soil of the MCPA+AS group was significantly lower than that of the MCPA group. This demonstrates that the inoculated strain AS can effectively degrade MCPA in the rhizosphere, thereby significantly alleviating the toxic effects of MCPA on melon and pumpkin seedlings and promoting their normal growth.

[0116] Example 4

[0117] Fermentation of bacterial strains and preparation and application of inoculants

[0118] 1. Optimization of fermentation conditions

[0119] Priestia megaterium AS was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of seed culture medium (LB) and cultured at 30 °C and 200 rpm for 12 h with shaking to obtain the seed culture.

[0120] Seed culture was inoculated at a 5% inoculum into a 1 L fermenter containing different fermentation media for batch fermentation. The optimized conditions are as follows:

[0121] Culture media: Comparison with basal MSM (containing 500 μmol / L MCPA), LB, and optimized culture media (5 g tryptone, 3 g yeast extract, 5 g NaCl, 5 g / L glucose, pH 7.0).

[0122] Temperature: 28℃, 30℃, and 33℃ were tested.

[0123] pH: The pH is controlled at 6.5, 7.0, and 7.5 by adding acid / base.

[0124] Dissolved oxygen: The dissolved oxygen level is controlled between 20% and 40% by adjusting the stirring speed.

[0125] By measuring the bacterial cell concentration (OD) at different time points 600 The optimal fermentation conditions were determined by measuring the cell count and viable cell count (CFU / mL). Results showed that under optimized culture medium conditions, 30℃, pH 7.0, and dissolved oxygen levels of approximately 30%, the viable cell count could reach 5 × 10⁻⁶ after 24-36 h of fermentation. 9 CFU / mL or higher is suitable for large-scale culture.

[0126] 2. Preparation of microbial agents

[0127] (1) Liquid bacterial agent: The above fermentation broth is directly dispensed or concentrated by centrifugation and then resuspended with a preservative (such as 5% trehalose or 1% skim milk) to prepare a high-concentration liquid bacterial agent (≥1×10⁻⁶). 10 (CFU / mL).

[0128] (2) Solid microbial agent: The fermentation broth is mixed with a sterilized adsorbent carrier (such as peat moss, vermiculite, and diatomaceous earth mixed in a 1:1:1 ratio) at a ratio of 1:2 (v / w), and dried in a cool and ventilated place until the moisture content is 20%-30% to make a solid microbial agent with a viable count ≥1×10⁻⁶. 8 CFU / g.

[0129] (3) Dry powder inoculant: Add freeze-drying protectant to the fermentation broth, freeze-dry and grind into powder to make dry powder preparation, which is convenient for transportation and storage.

[0130] 3. Verification of the effectiveness of microbial agents

[0131] Referring to the pot experiment method in Example 3, the fresh bacterial suspension in the “MCPA+AS” treatment was replaced with the above-mentioned solid bacterial agent (applied to the rhizosphere at a rate of 10 g / pot) or liquid bacterial agent (diluted and applied to the roots) with the same number of live bacteria.

[0132] The results are shown in Table 3.

[0133] Table 3: Comparison of the effects of different formulations of AS inoculants on melon growth and MCPA degradation

[0134]

[0135] There were no significant differences between all formulations of inoculum and fresh bacterial suspension in promoting growth and degrading MCPA (P > 0.05), indicating good formulation stability.

[0136] The experimental results showed that the effects of applying the two formulations of microbial agents on promoting the growth of melon / pumpkin seedlings, alleviating pesticide damage symptoms, and reducing soil MCPA residues were not significantly different from those of using fresh bacterial suspension in Example 3 (P > 0.05). This indicates that the strains of the present invention can be used to prepare various forms of microbial remediation agents, and their application effects are stable.

[0137] In summary, the present invention fully illustrates the implementation process and effects of the technical solution through the following four embodiments:

[0138] Example 1 details the isolation source of strain AS (MCPA-contaminated soil), the targeted screening method using high-concentration MCPA stress, the purification process, and the systematic identification process (including colony morphology such as...). Figure 1 (As shown in the figure, Gram staining, 16S rRNA gene sequence analysis and phylogenetic identification) finally identified it as Priestiamegaterium and completed patent preservation.

[0139] Example 2: By setting different concentration gradients of MCPA as the sole carbon source, the degradation performance of strain AS was quantitatively determined. HPLC detection confirmed its efficient ability to degrade MCPA, and the specific degradation rate was calculated.

[0140] Example 3 used pot experiments on melons and pumpkins, designing three treatment groups: a blank control, MCPA stress, and MCPA stress plus bacterial inoculation. The detoxification effect of the bacterial strain was systematically evaluated. The results showed that inoculation with strain AS significantly improved the growth indicators of seedlings under MCPA stress, alleviated visible phytotoxicity symptoms, and effectively reduced the residual MCPA in the rhizosphere soil. Statistical analysis confirmed the significant effect.

[0141] Example 4 further provides the direction for optimizing the fermentation process of strain AS, the preparation methods of liquid and solid inoculants, and verifies that the prepared inoculant has a detoxification effect comparable to that of fresh inoculant in pot experiments, proving the industrialization and practical feasibility of its technical solution.

[0142] The above are merely a few preferred embodiments of the present invention, described in a relatively specific and detailed manner, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A strain of Priestella megaterium AS ( Priestia megaterium AS), with accession number CCTCC No: M20242160, has the ability to degrade MCPA.

2. The *Priestella giantiformis* AS according to claim 1, characterized in that, Its 16S rRNA gene sequence is shown in SEQ ID No.

1.

3. A microbial inoculant, characterized in that, Contains *Priestella giantiformis* AS as described in claim 1 or 2 and an agriculturally acceptable carrier.

4. The microbial agent according to claim 3, characterized in that, The bacterial agent can be a liquid bacterial agent or a solid bacterial agent.

5. The use of *Priestella giantiflora* AS as described in claim 1 or 2 in mitigating the toxicity of 2,4-D to melons or squash.

6. A method for degrading 2,4-D in soil, characterized in that, This includes applying the microbial agent of claim 3 or 4 to the soil.

7. The method according to claim 6, characterized in that, The application rate of the microbial agent is 1×10⁻⁶ per hectare. 8 -1×10 12 CFU.

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