A plant growth regulator-degrading strain DL-D2 and its application

Abstract

The invention discloses a plant growth regulator degradation strain DL-D2 and an application thereof. The degradation strain DL-D2 is identified as Cupriavidus agavae, has been deposited in the China Center for Type Culture Collection, and the deposit time is May 5, 2023, and the deposit number is CCTCC NO:M 2023677. The degradation strain DL-D2 obtained by separation and screening in the invention can effectively degrade the plant growth regulator 4-chlorophenoxyacetic acid sodium; the degradation rate of 50 mg / L plant growth regulator 4-chlorophenoxyacetic acid sodium can reach 100% within 24 hours, and the problem of harm of 4-chlorophenoxyacetic acid sodium residue to soil and water environment, crops and human health is effectively solved.

Description

Technical Field

[0001] The invention relates to microbial remediation of environmental pollution, and in particular to a plant growth regulator degrading strain DL-D2 and application thereof. Background Art

[0002] Sodium 4-chlorophenoxyacetate is also known as sodium p-chlorophenoxyacetate, with a molecular formula of C8H6ClNaO3, a molecular mass of 208.574, and a CAS number of 13730-98-8. The original drug of sodium 4-chlorophenoxyacetate is white needle-shaped or prismatic crystals with a slight phenolic smell, easily soluble in water, stable in nature, and will not deteriorate during long-term storage. The melting point is 156.5℃, and the boiling point is 315.2℃.

[0003] UV scanning revealed an absorption peak at 225nm and 280nm.

[0004]

[0005] Chemical structure of sodium 4-chlorophenoxyacetate

[0006] Sodium 4-chlorophenoxyacetic acid can be used as a plant growth regulator to improve the yield and quality of crops. In recent years, the research on the use of sodium 4-chlorophenoxyacetic acid to promote fruit enlargement has gradually increased. Some studies have shown that sodium 4-chlorophenoxyacetic acid can increase the fruit setting rate. Sodium 4-chlorophenoxyacetic acid can generate p-chlorophenoxyacetic acid after acidification, which can increase the average single fruit weight of tomatoes, promote early maturity of tomato fruits and accelerate fruit enlargement. Some research results show that 8% sodium 4-chlorophenoxyacetic acid is safe for tomatoes at lower doses, and no deformed fruits appear, but with the increase in dosage, the enlargement rate and deformed fruit rate increase. Sodium 4-chlorophenoxyacetic acid is a good growth regulator for increasing greenhouse tomato yield and improving tomato quality. 20mg / L p-chlorophenoxyacetic acid can make seedless watermelon grow strongest and have the best fruit quality in greenhouse cultivation, while high concentration treatment inhibits growth, causing leaves to shrink, curl, and premature aging. The subacute toxicity of medium and high doses of sodium 4-chlorophenoxyacetic acid can affect the growth of mice and damage the liver and kidneys. The wide application of sodium 4-chlorophenoxyacetate on crops will lead to the residue of sodium 4-chlorophenoxyacetate in crops, soil and water. There are relatively few literature reports on the degradation of plant growth regulators by microorganisms, especially the degradation of sodium 4-chlorophenoxyacetate by microorganisms, which has not been reported in the prior art. Summary of the invention

[0007] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a plant growth regulator degrading strain DL-D2, which can efficiently degrade the plant growth regulator 4-chlorophenoxyacetic acid sodium.

[0008] Another object of the present invention is to provide a degradation bacterial agent produced by the degradation strain and its application.

[0009] Technical solution: In order to achieve the above-mentioned purpose, the plant growth regulator degradation strain DL-D2 described in the present invention is identified as Cupriavidus agavae, which has been deposited in the China Center for Type Culture Collection on May 5, 2023, and the deposit number is CCTCC NO:M 2023677. The strain of the present invention was isolated by the inventor from the sewage collected from the sewage outlet of the production base of Sichuan Guoguang Agrochemical Co., Ltd., which has been producing 4-chlorophenoxyacetic acid sodium for a long time, and was named DL-D2. The strain can achieve a degradation rate of 100% for 50 mg / L of 4-chlorophenoxyacetic acid sodium within 24 hours. The colony DL-D2 has an overall circular shape on the LB solid plate, with a raised surface, neat edges, moist, smooth, light yellow, and Gram-negative staining.

[0010] The invention discloses an application of the strain DL-D2 in degrading plant growth regulators.

[0011] Wherein, the plant growth regulator is sodium 4-chlorophenoxyacetate.

[0012] The strain is used in degrading the plant growth regulator sodium 4-chlorophenoxyacetate in soil or water environment.

[0013] The invention discloses an application of the strain DL-D2 in degrading 4-chlorocatechol.

[0014] The degrading bacterial agent produced by the plant growth regulator degrading strain DL-D2 of the present invention.

[0015] Wherein, the fermentation of the bacterial agent comprises the following steps:

[0016] 1) Inoculating the bacterial liquid of strain DL-D2 into a fermentation medium, and culturing with shaking until the logarithmic phase to obtain a fermentation strain;

[0017] 2) inoculating the cultured fermentation bacteria into the culture medium of the seed tank and culturing the culture medium until the logarithmic growth phase to obtain a seed solution;

[0018] 3) The seed liquid is connected to the culture medium of the production tank for cultivation; after the fermentation is completed, the culture liquid is taken out of the tank and packaged into liquid preparations.

[0019] The degrading bacterial agent of the present invention is used in degrading the plant growth regulator 4-chlorophenoxyacetic acid sodium.

[0020] Among them, the degrading bacterial agent is used in degrading the plant growth regulator sodium 4-chlorophenoxyacetate in the soil or water environment.

[0021] The degrading bacterial agent of the present invention is used in degrading 4-chlorocatechol.

[0022] The present invention screens and separates a 4-chlorophenoxyacetic acid sodium efficient degradation strain from sewage, named DL-D2. The strain DL-D2 is identified and analyzed by the 16s rRNA gene system evolutionary status, and it is found that the strain DL-D2 is a cupriavidus (Cupriavidus agavae), and the 16SrDNA sequence similarity with the strains Cupriavidus agavae strain ASC-992 and ASC-9912 is the highest, which is 99.86%. The growth characteristics, degradation characteristics and substrate spectrum of the strain DL-D2 are studied, and it is found that the strain DL-D2 can completely degrade 50 mg / L of 4-chlorophenoxyacetic acid sodium within 24 hours, and can use 4-chlorophenoxyacetic acid sodium as a carbon source for growth. The optimum growth temperature of the strain DL-D2 is 30°C. The optimum growth pH is 7.0. The intermediate product generated by the degradation of 4-chlorophenoxyacetic acid sodium by strain DL-D2 reached a peak at 20h, and the optimum temperature for the degradation of 4-chlorophenoxyacetic acid sodium by strain DL-D2 was 30℃. The optimum pH was 7.0. The degradation efficiency of 4-chlorophenoxyacetic acid sodium by strain DL-D2 was positively correlated with the inoculation amount of strain DL-D2, and the initial concentration of 4-chlorophenoxyacetic acid sodium was negatively correlated with the degradation rate. Cu 2+ It has an inhibitory effect on the degradation of sodium 4-chlorophenoxyacetate by strain DL-D2. Among the several chlorobenzene toxic reagents tested, strain DL-D2 can efficiently degrade sodium 4-chlorophenoxyacetate and 4-chlorocatechol, but has no degradation effect on 4-chlorophenol and 2,4-D.

[0023] The strain DL-D2 of the present invention is sequenced through the whole genome, and the genome size is 6.59M, and 6095 genes are predicted. According to the physiological and biochemical characteristics and the phylogenetic tree analysis based on the 16s rDNA sequence, it can be found that DL-D2 is more closely related to the strain Cupriavidus agavae, and DL-D2 is identified as Cupriavidus agavae.

[0024] The strain DL-D2 of the present invention is a copper-greedy bacteria (Cupriavidus agavae), which is specific for the degradation of sodium 4-chlorophenoxyacetate. DL-D2 cannot degrade 2,4-D, etc., indicating that the strain is a specific degradation strain with a completely new function, and its degradation mechanism and the gene function involved in the degradation are greatly different from the currently disclosed strain alkaliphilic copper-greedy bacteria.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0026] The strain DL-D2 obtained by separation and screening can effectively degrade plant growth regulators into sodium 4-chlorophenoxyacetate, and can completely degrade the sodium 4-chlorophenoxyacetate in an inorganic salt culture medium. Meanwhile, the degradation bacterial agent prepared by the strain can degrade high-concentration sodium 4-chlorophenoxyacetate in soil or water in a short time, thereby solving the problem of harm of residual sodium 4-chlorophenoxyacetate in the environment to soil and water environment, crops and human health.

[0027] The degradation bacterial agent prepared by the strain of the invention can be produced by using general fermentation equipment in the fermentation industry, has the advantages of low production cost, convenient use and good removal effect, and is suitable for the restoration of soil and water bodies polluted by 4-chlorophenoxyacetic acid sodium. The invention has important significance for protecting the ecological environment, reducing the phytotoxicity of 4-chlorophenoxyacetic acid sodium to crops and protecting human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the UV scanning spectrum of the degradation enrichment solution of sodium chlorophenoxyacetate;

[0029] Figure 2 This is the UV scanning spectrum of the purified strain DL-D2 degrading sodium 4-chlorophenoxyacetate;

[0030] Figure 3 is the colony image of the purified strain DL-D2;

[0031] Figure 4 The genome extraction and 16S rDNA amplification gel electrophoresis pattern of strain DL-D2;

[0032] Figure 5 is the phylogenetic tree of DL-D2 constructed based on the 16S rRNA gene;

[0033] Figure 6 is the growth curve of strain DL-D2;

[0034] Figure 7 is the effect of temperature on the growth of DL-D2;

[0035] Figure 8 is the effect of pH on the growth of DL-D2;

[0036] Fig. 9 is the standard curve of 4-chlorophenoxyacetic acid sodium concentration;

[0037] Fig.10 It is the UV scanning spectrum and HPLC spectrum of time-sharing sampling of 4-chlorophenoxyacetic acid sodium;

[0038] Fig.11 is the degradation curve of sodium 4-chlorophenoxyacetate;

[0039] Fig.12This is the curve of the change of DL-D2 bacterial volume during the degradation process;

[0040] Fig.13 The effect of temperature on the degradation of sodium 4-chlorophenoxyacetate by strain DL-D2;

[0041] Fig.14 The effect of pH on the degradation of sodium 4-chlorophenoxyacetate by strain DL-D2;

[0042] Fig.15 The effect of inoculum size on the degradation of sodium 4-chlorophenoxyacetate by strain DL-D2;

[0043] Fig.16 The effect of the initial concentration of sodium 4-chlorophenoxyacetate on the degradation of sodium 4-chlorophenoxyacetate by strain DL-D2;

[0044] Fig.17 Cu 2+ UV scanning spectrum of the effect on the degradation of sodium 4-chlorophenoxyacetate by strain DL-D2;

[0045] Fig.18 Cu 2+ HPLC spectrum of the effect on the degradation of sodium 4-chlorophenoxyacetate by strain DL-D2;

[0046] Fig.19 This is the UV scanning spectrum of the degradation of several other chlorobenzene-type toxic reagents by strain DL-D2. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0048] Unless otherwise specified, the raw materials and reagents in the present invention are commercially available.

[0049] Reagents: 4-chlorophenoxyacetic acid sodium (purity 98%) was purchased from Sichuan Guoguang Agrochemical Co., Ltd. Methanol (chromatographic grade), acetic acid, glycerol, 4-chlorophenol (purity 98%), 2,4-dichlorophenoxyacetic acid (purity 97%), 4-chlorocatechol (purity 98%) were purchased from Anaiji Chemical, bacterial genomic DNA extraction kit (DP302) was provided by Tiangen Biochemical Technology (Beijing) Co., Ltd., 2X Taqplus PCR MasterMix kit was purchased from Biosharp, and PCR primers were synthesized by Anhui General Biological Co., Ltd.

[0050] Example 1

[0051] Isolation and identification of strain DL-D2:

[0052] Enrichment: Collect sewage from the sewage outlet of the production base of Sichuan Guoguang Agrochemical Co., Ltd., which has been producing 4-chlorophenoxyacetic acid sodium for a long time, and acclimate and enrich it. The specific method is as follows: Measure 1mL of sewage and inoculate it into a sterilized inorganic salt culture medium containing 50mg / L 4-chlorophenoxyacetic acid sodium. Cultivate in a rotary shaking incubator at 30℃ and 150rpm for 5 days, then take 5mL of the culture solution and transfer it to the same fresh inorganic salt for further cultivation. After 3 transfers, the target enrichment solution is obtained.

[0053] Screening: Take 1 mL of the enriched solution and dilute it to 10 with sterile water by volume. -7 , and then take 10 -4 , 10 -5 , 10 -6 , 10 -7 100 μL of each was spread on LB solid medium to make the colonies evenly distributed on the surface of the medium. Finally, the culture dish was placed in a 35°C constant temperature incubator for 1-2 days. When colonies grew on the surface of the medium, a single colony was randomly picked with a pipette tip and cultured in 3 mL LB overnight to make its OD 600 When the pH value reaches between 1.5 and 2.0, take 1 mL of bacterial solution, centrifuge at 12000 rpm for 1 min, discard the supernatant, wash twice with sterilized inorganic salts, add 20 mL of sterilized inorganic salts containing 50 mg / L 4-chlorophenoxyacetic acid sodium, and culture in a rotary shaking incubator at 30°C and 150 rpm for 5 days. Then take 1 mL of supernatant, centrifuge at 12000 rpm for 1 min, take the supernatant, scan the wavelength range of 200-350 nm with an ultraviolet spectrophotometer, and compare the shape of the absorption peaks of the standard control group and the treatment group to preliminarily determine whether 4-chlorophenoxyacetic acid sodium is degraded. The samples with changes in the shape of the absorption peaks are the degradation strains obtained in the initial screening.

[0054] The enriched solution after three subcultures was detected by UV-visible spectrophotometry, and it was found that 4-chlorophenoxyacetic acid sodium had been degraded. The enriched solution was able to completely degrade 4-chlorophenoxyacetic acid sodium within 5 days ( Figure 1 ). The scanning results in the wavelength range of 200-350nm showed that compared with the standard control group, the absorption peak intensity of 4-chlorophenoxyacetic acid sodium in the treatment group (enriched solution) was significantly reduced, and the peak shape changed, 4-chlorophenoxyacetic acid sodium was completely degraded, and the initial screening degradation strain was obtained.

[0055] Purification: streak the bacterial liquid of the degradation strain in solid LB medium, place the culture dish in a constant temperature incubator at 35°C and culture for 1-2 days, repeat the above test experiment to detect the effect of single bacteria in degrading 4-chlorophenoxyacetic acid sodium, until a single colony with stable properties of degrading 4-chlorophenoxyacetic acid sodium is isolated.

[0056] The overnight cultured bacteria were streaked in solid LB medium until a single colony grew out, then inoculated into 3 mL LB liquid medium for overnight culture. 1 mL of bacterial solution was taken, centrifuged at 5000 rpm for 3 min, washed twice with sterile MSM, resuspended with sterile MSM, and inoculated into 10 mL of inorganic salt medium containing 50 mg / L sodium 4-chlorophenoxyacetic acid for 5 days. The UV scan was consistent with the peak of the enrichment solution, and the degradation was complete ( Figure 2 ), the purified strain DL-D2 single bacterial morphology is shown in Figure 3 The whole body is round, with a raised surface, neat edges, moist, smooth, light yellow color, and Gram-negative staining. The strain was named DL-D2.

[0057] The genomic DNA of strain DL-D2 was extracted, and PCR amplification was performed using the genomic DNA of strain DL-D2 as a template and the universal primers of 16S rDNA gene as primers to obtain a gene sequence with a total length of 1443 bp (SEQ ID NO.1), as shown in FIG. Figure 4 As shown. The sequence was compared in the NCBI database, and the results showed that strain DL-D2 was most closely related to Cupriavidus. Among them, the 16S rDNA sequence similarity with Cupriavidus agavae strain ASC-992 and ASC-9912 was the highest, reaching 99.86%. According to physiological and biochemical characteristics, phylogenetic tree analysis based on 16s rDNA sequences, and genome analysis, the strain was identified as Cupriavidus agavae and named Cupriavidus agavae.DL-D2. The phylogenetic tree of strain DL-D2 and model strains in the genus Cupriavidus was constructed by the neighbor-joining method. Figure 5 The strain DL-D2 was sent to the China Center for Type Culture Collection (CCTCC) in Wuhan, China for preservation on May 5, 2023, with the preservation number CCTCC NO:M 2023677.

[0058] Example 2

[0059] Growth characteristics and optimal growth conditions of strain DL-D2

[0060] 1. Determination of growth curve

[0061] In the clean bench, a single colony of DL-D2 was picked up with a sterile pipette tip and inoculated into the sterilized LB liquid medium. The culture was placed in a rotary shaking incubator at 30°C and 150 rpm for overnight. The 0D 600 = 1.5, re-inoculate 30 μL of bacteria into 15 mL LB and adjust the initial OD 600The value is about 0.003. Starting from the time of inoculation, 1 mL of culture medium is drawn with a sterile pipette in the clean bench every 3 hours, and its OD600 is measured with a spectrophotometer. Each experiment is repeated 3 times. Figure 6 As shown, the strain entered the logarithmic growth phase after 10 hours of culture, and the growth of the strain reached saturation after 18 hours.

[0062] 2. Optimum growth temperature

[0063] In the clean bench, use a sterile pipette to pick up a single colony, inoculate it into the sterilized LB liquid medium, and place it in a rotary shaking incubator at 30°C and 150rpm to cultivate the OD 600 =1.5, take 10 μL of culture solution to 3 mL of LB, put it in a shaker at 20°C, 25°C, 30°C, 37°C, and 42°C, shake the bacteria at 150 rpm for 15 h, and then measure the OD with a spectrophotometer 600 Each experiment was repeated 3 times. The growth of strain DL-D2 under different temperature conditions is shown in Figure 7 As shown, DL-D2 grows better between 25-37℃, and temperatures above 42℃ are not conducive to the growth of the strain. The optimal growth temperature of strain DL-D2 is 30℃.

[0064] 3. Optimal growth pH

[0065] In the clean bench, use a sterile pipette to pick up a single colony, inoculate it into the sterilized LB liquid medium, and place it in a rotary shaking incubator at 30°C and 150rpm to culture until the OD 600 =1.5, take 10 μL of culture solution to 3 mL LB, set the pH gradient to 5.0, 6.0, 7.0, 8.0, 9.0. Shake the bacteria at 30°C at 150 rpm for 15 h, then measure the OD with a spectrophotometer 600 Each experiment was repeated 3 times. The growth of strain DL-D2 under different pH conditions is shown in Figure 8 As shown, DL-D2 grows better between pH 6-8, and pH below 5 is not conducive to the growth of the strain. The optimal growth pH of strain DL-D2 is 7.0.

[0066] Example 3

[0067] Study on the Degradation Characteristics of 4-Chlorophenoxyacetic Acid Sodium by Strain DL-D2

[0068] 1. Degradation curve of 4-chlorophenoxyacetic acid sodium by strain DL-D2

[0069] (1) Drawing of the standard curve of sodium 4-chlorophenoxyacetate

[0070] Dilute the sodium 4-chlorophenoxyacetic acid solution to 400 mg / L, 200 mg / L, 100 mg / L, 50 mg / L, and 25 mg / L with methanol (chromatographic grade) according to the gradient dilution method, filter through a 0.22 μm organic filter membrane, and then measure HPLC. Chromatographic conditions:

[0071] Chromatographic column: C-18 reverse phase column; column temperature: 40°C; the ratio of mobile phase A and B is methanol: 0.25% acetic acid solution (60:40); detection wavelength is 280nm; flow rate: 0.8mL / min; injection volume: 20μL, column pressure is about 9.5MPa.

[0072] According to the relationship between the detection concentration and area, a standard curve is prepared, such as Fig. 9 As shown, after fitting, the linear equation is: y = 0.0001x + 0.1974, R 2 =0.9999.

[0073] 2. Degradation curve

[0074] A single colony of strain DL-D2 cultured overnight was picked and inoculated into 100 mL LB liquid culture. After culturing at 30°C and 150 rpm for 16 h, the bacteria were collected by centrifugation at 5000 rpm for 5 min, washed three times with sterilized MSM, and then resuspended with sterilized MSM to obtain the DL-D2 seed solution.

[0075] The DL-D2 seed solution was inoculated into inorganic salt (pH = 7.0) and the initial bacterial concentration OD was adjusted. 600 To 0.1, add 4-chlorophenoxyacetic acid sodium with a final concentration of 50 mg / L as the only carbon source in the culture medium, culture at 30°C, 150rpm constant temperature shaker, sample every 6 hours, monitor the residual concentration of 4-chlorophenoxyacetic acid sodium and bacterial growth. The UV scanning spectrum and HPLC spectrum of the time-sharing sampling degradation of 4-chlorophenoxyacetic acid sodium are shown in Fig.10 According to the peak area change of 4-chlorophenoxyacetic acid sodium degradation, the residual concentration in the solution was calculated and the degradation curve was made as follows: Fig.11 As shown in the figure, 4-chlorophenoxyacetic acid sodium was completely degraded (100%) within 24 hours. During the degradation of 4-chlorophenoxyacetic acid sodium, intermediate products could be detected at 6 hours, reaching a peak between 18 and 24 hours, and then the concentration decreased. According to the OD of strain DL-D2 600 The value of the test is changed, and the bacterial change curve is made, such as Fig.12 As shown in the figure, the bacterial concentration tended to increase during the whole degradation process, indicating that the strain DL-D2 can use sodium 4-chlorophenoxyacetic acid as the sole carbon source for growth.

[0076] 3. Effects of temperature and pH on the degradation of sodium 4-chlorophenoxyacetic acid by strain DL-D2

[0077] The DL-D2 seed solution was inoculated into sterilized inorganic salt (pH = 7.0) and the bacterial concentration OD was adjusted. 600 The concentration of sodium 4-chlorophenoxyacetate in the degradation solution was determined after culturing at 20, 25, 30, 37, and 42°C and 150 rpm for 18 h.

[0078] The results of the degradation experiment of 4-chlorophenoxyacetic acid sodium after culturing for 18h under different temperature conditions showed that ( Fig.13 ), strain DL-D2 can effectively degrade sodium 4-chlorophenoxyacetic acid (degradation rate>49%) in the range of 20-30℃, with the optimum temperature at 30℃, and the degradation rate of sodium 4-chlorophenoxyacetic acid reaching 82.77%. When the temperature rises to 37℃, the degradation ability is significantly weakened, with a degradation rate of only 2.37%. When the temperature rises to 42℃, the degradation rate is only 0.31%. This shows that the strain has a certain degree of thermal sensitivity to the degradation of sodium 4-chlorophenoxyacetic acid, which may be related to the growth conditions of the strain and the temperature tolerance of the relevant degradation enzymes.

[0079] The DL-D2 seed solution was inoculated into inorganic salts with different pH values ​​(5.0, 6.0, 7.0, 8.0, and 9.0) and the OD 600 The concentration of sodium 4-chlorophenoxyacetate was 0.1, 4-chlorophenoxyacetate sodium was increased to a final concentration of 50 mg / L, cultured at 30°C, 150 rpm for 18 h, and the concentration of sodium 4-chlorophenoxyacetate in the degradation solution was detected.

[0080] The results of the degradation experiment of 4-chlorophenoxyacetic acid sodium after 18h of incubation under different pH conditions showed that ( Fig.14 ), the degradation efficiency of strain DL-D2 on sodium 4-chlorophenoxyacetate was quite different. Strain DL-D2 showed very good degradation ability in the pH range of 7.0-9.0. When the pH was acidic, the ability of strain DL-D2 to degrade sodium 4-chlorophenoxyacetate was significantly weakened. When the pH was 5, the degradation rate was 53.52%. The degradation rate was the highest at pH 7, reaching 86.96%, so the optimal pH for the strain to degrade sodium 4-chlorophenoxyacetate was 7.0.

[0081] 4. Effect of inoculation amount on the degradation of sodium 4-chlorophenoxyacetic acid by strain DL-D2

[0082] The DL-D2 seed solution was inoculated into freshly sterilized inorganic salt (pH = 7.0), the bacterial concentration OD600 was adjusted to 0.05, 0.1, 0.2, 0.4 and 0.6, and 4-chlorophenoxyacetic acid sodium was added to make the final concentration 50 mg / L. The culture was carried out at 30°C and 150 rpm for 14 hours, and the supernatant of the degradation solution was taken at 9h and 14h, and the concentration of 4-chlorophenoxyacetic acid sodium in the degradation solution was detected by HPLC.

[0083] The degradation results of 4-chlorophenoxyacetic acid sodium by different initial concentrations of strain DL-D2 are shown in Figure 2. Fig.15 As shown in the figure, with the increase of the inoculum amount of DL-D2, the degradation rate of sodium 4-chlorophenoxyacetate gradually increased. When the inoculum amount reached OD 600 =0.4, strain DL-D2 was able to completely degrade sodium 4-chlorophenoxyacetate within 14 h. The degradation efficiency of sodium 4-chlorophenoxyacetate was positively correlated with the inoculation amount of strain DL-D2. 600 When the concentration was as low as 0.05, strain DL-D2 could still degrade 77.50% of sodium 4-chlorophenoxyacetate within 14 hours, indicating that the strain had a high degradation activity.

[0084] 5. Effect of initial substrate concentration on the degradation of sodium 4-chlorophenoxyacetic acid by strain DL-D2

[0085] The DL-D2 seed solution was inoculated into freshly sterilized inorganic salt (pH = 7.0) and the OD 600 To 0.1, add sodium 4-chlorophenoxyacetate to make the final concentrations of 50mg / L, 100mg / L, 150mg / L, 200mg / L, 300mg / L, 400mg / L, 600mg / L, 800mg / L, 1000mg / L, 1200mg / L. After culturing at 30℃, 150rpm for 14h, the concentration of sodium 4-chlorophenoxyacetate in the degradation solution was detected by HPLC.

[0086] The results of the degradation experiment of 4-chlorophenoxyacetic acid sodium after culturing for 14h under different initial concentrations of 4-chlorophenoxyacetic acid sodium showed that ( Fig.16 )The initial concentration of sodium 4-chlorophenoxyacetate was negatively correlated with the degradation efficiency of sodium 4-chlorophenoxyacetate by strain DL-D2.

[0087] 6. Cu 2+ Effects on the degradation of sodium 4-chlorophenoxyacetate by strain DL-D2

[0088] The DL-D2 seed solution was inoculated into freshly sterilized inorganic salt (pH = 7.0) and the OD 600 to 0.1, then add 4-chlorophenoxyacetic acid sodium to the culture medium to a final concentration of 50 mg / L, and add 1 mM Cu 2+ , 30°C, 150 rpm, and then cultured for 12 h. The wavelength range of 200-350 nm was scanned by UV-Vis spectrophotometer. 2+ The reaction solution was used as the control, and the degradation of 4-chlorophenoxyacetic acid sodium in the control group and the treatment group was compared to determine the Cu 2+ Effect of sodium 4-chlorophenoxyacetate on the degradation of DL-D2.

[0089] Cu was added 2+and without Cu addition 2+ The culture medium containing 50 mg / L of inorganic salt was cultured at the same initial bacterial volume, and samples were taken at 12h, 16h, 20h, and 24h, respectively, and scanned in the wavelength range of 200-350nm by UV-visible spectrophotometer. The results are shown in Fig.17 As shown, the supernatant after 12 hours of culture was tested by HPLC. The results are shown in Fig.18 The test results show that: Cu 2+ After that, the degradation rate of 4-chlorophenoxyacetic acid sodium by strain DL-D2 slowed down. 2+ It has a certain inhibitory effect on the degradation of sodium 4-chlorophenoxyacetate by strain DL-D2.

[0090] 7. Degradation of several other chlorobenzene-based toxic reagents by strain DL-D2

[0091] The DL-D2 seed solution was inoculated into freshly sterilized inorganic salt (pH = 7.0) and the OD 600 to 0.1, 4-chlorophenol, 2,4-D, 4-chlorocatechol and sodium 4-chlorophenoxyacetate were added respectively to make the final concentration of 100 mg / L, and cultured at 30°C and 150 rpm for 5 days. 1 mL of the solution was taken and centrifuged at 12000 rpm for 1 min. The supernatant was taken and scanned in the wavelength range of 200-350 nm by UV-visible spectrophotometer. The shape of the absorption peak of the reaction solution after treatment with the standard control group and the strain DL-D2 was compared to determine whether the substrate was degraded.

[0092] After testing, under the condition of substrate initial concentration of 100 mg / L, samples were taken after culturing for 24h, 36h, 48h, and 96h, and scanned in the wavelength range of 200-350nm by UV-visible spectrophotometer. The results are shown in Fig.19 As shown, the results show that the strain DL-D2 can degrade sodium 4-chlorophenoxyacetate and 4-chlorocatechol, but has no degradation effect on 4-chlorophenol and 2,4-D. At the same time, when the concentration of 4-chlorophenol and 2,4-D is adjusted to 50 mg / L, there is still no degradation effect (the result is consistent with 100 mg / L), which further illustrates the degradation specificity of the strain of the present invention.

Claims

1. A plant growth regulator-degrading strain DL-D2 was identified as Copper-Grubbing Bacteria ( Cupriavidus agavae ), has been deposited in the China Center for Type Culture Collection on May 5, 2023, with the deposit number CCTCC NO:M2023677.

2. Use of the strain DL-D2 according to claim 1 in degrading the plant growth regulator sodium 4-chlorophenoxyacetate.

3. The use according to claim 2, characterized in that: The strain is used in degrading the plant growth regulator sodium 4-chlorophenoxyacetate in soil or water environment.

4. Use of the strain DL-D2 according to claim 1 in degrading 4-chlorocatechol.

5. A degradation bacterial agent produced using the plant growth regulator-degrading strain DL-D2 according to claim 1.

6. The degradation bacterial agent according to claim 5, characterized in that The fermentation of the bacterial agent comprises the following steps: 1) Inoculate the bacterial liquid of strain DL-D2 into the fermentation medium, and culture with shaking until the logarithmic phase to obtain the fermentation strain; 2) inoculating the cultured fermentation bacteria into the culture medium of the seed tank and culturing until the logarithmic growth phase to obtain a seed solution; 3) The seed liquid is connected to the culture medium of the production tank for cultivation; after fermentation is completed, the culture liquid is taken out of the tank and packaged into liquid preparations.

7. Use of the degrading bacterial agent according to claim 5 in degrading the plant growth regulator sodium 4-chlorophenoxyacetate.

8. The use according to claim 7, characterized in that: The degrading bacterial agent is used in degrading the plant growth regulator sodium 4-chlorophenoxyacetate in soil or water environment.

9. Use of the degrading bacterial agent according to claim 5 in degrading 4-chlorocatechol.

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