Kutta gibsonii B7 for degrading paclobutrazol and application of Kutta gibsonii B7

Abstract

The invention relates to the field of microorganisms, in particular to a Kutta gibsonii B7 for degrading paclobutrazol and application of the Kutta gibsonii B7. The Kutta gibsonii B7 is preserved in the China General Microbiological Culture Collection Center on October 23, 2024, and the preservation number is CGMCC No.32305. The Kutta gibsonii B7 can grow in a liquid culture medium containing the paclobutrazol, and the Kutta gibsonii B7 can be used for degrading the paclobutrazol. The content of paclobutrazol in the culture medium can be reduced, and the toxic effect of paclobutrazol on thalli is relieved; 95 mg / L paclobutrazol can be degraded in 9 days, and the degradation rate can reach 74%; a degradation product obtained after paclobutrazol is degraded has obviously weakened stress and phytotoxicity effects on plants, and is low in cost compared with physical and chemical cost and free of secondary pollution; the strain can be used for preparing a microbial agent for degrading paclobutrazol in soil, can obviously relieve the apical inhibition effect of paclobutrazol on crops, and has a relatively great application prospect.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a strain of Coxsackievirus B7 capable of degrading paclobutrazol and application thereof. Background Art

[0002] Paclobutrazol is a plant growth regulator, belonging to the triazole class of compounds. It is a highly effective, low-toxic plant growth retardant that can be absorbed by the roots, stems, and leaves of plants and transmitted to the site of action through the xylem. It mainly works by inhibiting the biosynthesis of gibberellins (GA) in plants. Gibberellins can promote cell elongation, while paclobutrazol inhibits key enzymes in the synthesis of gibberellins, such as kaurene oxidase, thereby reducing the amount of gibberellins. Plant cell elongation is inhibited, making the plants dwarfed and the internodes shortened. At the same time, it can also affect the balance of other hormones in plants, such as increasing the content of abscisic acid (ABA) and promoting plant stress resistance. Long-term or excessive use of paclobutrazol will lead to the accumulation of paclobutrazol residues in the soil, which is specifically manifested in reducing the types and number of soil microorganisms and affecting soil enzyme activity, which should be directed to the growth and development of subsequent crops. Residues may cause phytotoxicity to sensitive plants planted subsequently; it will also change the community structure of soil microorganisms, which may inhibit the growth and reproduction of some microorganisms, and may also promote the growth of other microorganisms. Since paclobutrazol affects plant growth, it indirectly affects soil fertility; in an aerobic environment, the half-life of paclobutrazol in soil is 43-618 days (average 182 days). Compared with soil without paclobutrazol, the bacteria, actinomycetes and fungi in the soil after paclobutrazol was applied were reduced by 58%, 28% and 28% respectively.

[0003] At present, the main treatment methods for paclobutrazol are to plow the farmland before planting the second crop, add nitrogen fertilizer or gibberellin to the soil for rescue; appropriately increase the effective iron ion concentration and pH value in the soil to reduce the content of paclobutrazol; use physical and chemical methods such as adsorption and advanced oxidation to remove residual paclobutrazol in the soil. The above methods have certain limitations in terms of degradation efficiency, safety, economic benefits and secondary pollution. Bioremediation has the characteristics of low cost, simple operation and short repair cycle, and has great advantages in pollution control. It is an emerging remediation technology. At present, there are not many reports on the microbial degradation of paclobutrazol.

[0004] Studies have shown that the genus Kutschibacterium can effectively degrade polycyclic aromatic hydrocarbons, cypermethrin, benzoconazole, dichlorodiphenyltrichloroethane (DDT), acetochlor, and sulfonylurea herbicides. In addition, it can also degrade triphenylmethane dyes, azo dyes, and aromatic dyes, as well as phenolic compounds and microcystins. In addition, the genus Kutschibacterium also exhibits stress resistance characteristics such as salt and alkali resistance, heat resistance, and resistance to organic solvents, showing great development and application potential in the field of microbial remediation. However, there is no report on the degradation of paclobutrazol by Kutschibacterium gibbsii. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the above-mentioned existing technologies, and provide a strain of Kurthia gibsonii B7 for degrading paclobutrazol. The Kurthia gibsonii B7 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on October 23, 2024, with a deposit number of CGMCC No.32305.

[0006] The second object of the present invention is to provide a bacterial agent, comprising the Kutschibacterium gibbsii B7.

[0007] The third object of the present invention is to provide the use of the Kochbacterium gibbsii B7 or the bacterial agent in the preparation of a soil remediation agent.

[0008] The fourth object of the present invention is to provide the use of the aforementioned Gastrointestinalis B7 or the aforementioned bacterial agent in the preparation of a soil conditioner.

[0009] The fifth object of the present invention is to provide the use of the Kochbacterium gibbsii B7 or the bacterial agent in promoting plant growth.

[0010] The sixth object of the present invention is to provide the use of the Kochbacterium gibbsii B7 or the bacterial agent in degrading paclobutrazol.

[0011] The seventh object of the present invention is to provide a soil conditioner, which contains the Gibberellic acid B7 or the bacterial agent, and auxiliary materials.

[0012] The eighth object of the present invention is to provide a plant growth promoter for use in soil with paclobutrazol residues, wherein the plant growth promoter contains the Gibberellic acid B7 or the bacterial agent, and auxiliary materials.

[0013] Compared with the prior art, the present invention has the following advantages and effects:

[0014] (1) The present invention screened out a strain of Kurthia gibsonii B7, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on October 23, 2024, with a deposit number of CGMCC No.32305; the Kurthia gibsonii B7 can grow in a liquid culture medium containing paclobutrazol, can reduce the content of paclobutrazol in the culture medium, and eliminate the toxic effect of paclobutrazol on the bacteria.

[0015] (2) The bacterium Gibbsella B7 can degrade 95 mg / L of paclobutrazol in 9 days, with a degradation rate of up to 74%.

[0016] (3) The degradation products of paclobutrazol after being degraded by the bacterium Gibbsella B7 significantly reduce the stress and phytotoxicity to plants, have low cost compared to physical and chemical methods, and do not cause secondary pollution.

[0017] (4) The Gibberellic acid B7 can be used to prepare a bacterial agent for degrading paclobutrazol in soil, which can significantly alleviate the apical inhibitory effect of paclobutrazol on crops and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Paclobutrazol standard curve

[0019] Figure 2 Degradation rates of the 12 strains screened

[0020] Figure 3 Colony morphology of Koch's bacterium B7.

[0021] Figure 4 Phylogenetic tree of Kochnerella gibbsii B7.

[0022] Figure 5 Degradation characteristics of Kochnerella gibbsii B7 under different temperature conditions.

[0023] Figure 6 Degradation characteristics of Kochnerella gibbsii B7 under different pH conditions.

[0024] Figure 7 Degradation characteristics of Koch's bacterium B7 under different initial paclobutrazol concentrations.

[0025] Figure 8 Response surface and contour plots of temperature (A) and initial paclobutrazol concentration (C).

[0026] Fig. 9 Response surface and contour plots of pH (B) and initial paclobutrazol concentration (C).

[0027] Fig.10 This paper is to analyze the phytotoxicity of paclobutrazol and its degradation products. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0029] The methods described in the following examples are all conventional methods, which can be obtained from the literature, and the reagents used can be purchased from the market.

[0030] The formula of the enrichment medium described in the following examples is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and natural pH.

[0031] The formula of the LB solid medium described in the following examples is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar powder.

[0032] The formula of the basal salt medium MSM described in the following examples is: glucose 1.0 g / L, KH2PO4 1.5 g / L, K2HPO4 0.5 g / L, NaCl 1.0 g / L, MgSO4·7H2O 0.2 g / L, pH natural.

[0033] Example 1: Isolation, purification, screening and identification of strains

[0034] 1. Isolation and purification of strains

[0035] The soil samples contaminated with paclobutrazol were enriched and cultured, and the enriched culture solution was diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Different concentrations were applied to LB solid culture medium containing paclobutrazol, and cultured upside down in an incubator at 30°C. Single colonies were picked and streaked for purification to obtain 12 different strains for the next experiment.

[0036] 2. Screening of strains

[0037] 2.1 Standard curve of paclobutrazol

[0038] Prepare 5000 mg / L paclobutrazol mother solution with chromatographic methanol, and then dilute it into a series of standard sample solutions (10, 25, 50, 100, 125, 250 mg / L) for high performance liquid chromatography determination. Draw a standard curve with the concentration of paclobutrazol as the horizontal axis and the peak area of ​​the target substance as the vertical axis.

[0039] The results are as follows Figure 1 As shown, it can be seen that the concentration of paclobutrazol has a good linear correlation with the peak area, and its linear regression equation is, the correlation coefficient R 2 =0.9993.

[0040] The 12 isolated strains were inoculated into MSM medium with 25 mg / L paclobutrazol as the sole nitrogen source, and cultured at 30°C with shaking (180 rpm) for 7 days. MSM medium containing an equal amount of paclobutrazol without inoculation was used as a control. The paclobutrazol content was determined by HPLC, and its degradation rate was calculated.

[0041] The results are as follows Figure 2 As shown, the strain numbered B7 had the highest degradation rate, reaching 41.9%.

[0042] 1.3 Identification of strains

[0043] The strain with good degradation effect was named B7. The colony was light yellow with irregular and raised edges. Figure 3 As shown. Through 16s rDNA molecular biological identification, the phylogenetic tree based on 16s rDNA sequence is shown Figure 4 As shown, the strain was determined to be Kurthiagibsonii B7, and the 16s rDNA sequence of Kurthiagibsonii B7 was shown in SEQ ID No.1.

[0044] The screened Kurthia gibsonii B7 was deposited in the China General Microbial Culture Collection Center, the deposit address of which is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No.32305, and the contact number is: 010-64807355.

[0045] In the following examples, Kurthia gibsonii B7 is abbreviated as Kurthia gibsonii B7.

[0046] Example 2: Degradation characteristics of Kochbacterium gibbsii B7 under different temperature conditions

[0047] A single colony of the purified and cultured Cootielia gissoni B7 was picked up, inoculated into 100 ml LB liquid culture medium, and cultured at 30°C and 180 rpm with sufficient shaking until the logarithmic growth phase to serve as a seed solution; the cultured seed solution was centrifuged to collect the bacteria, which were washed with sterile water and then resuspended to obtain a Cootielia gissoni B7 bacterial suspension with a concentration of 0.05 g / ml.

[0048] The suspension of B. gissoni B7 was inoculated into 30 ml MSM medium with a paclobutrazol concentration of 25 mg / L at 5% inoculum, and cultured in a shaking table at 180 rpm at different temperatures (20°C, 25°C, 30°C, 35°C and 40°C). Samples were taken on the 9th day, and the paclobutrazol content was determined by high performance liquid chromatography (HPLC) to analyze the degradation effect.

[0049] The results are as follows Figure 5 As shown in the figure, at a temperature of 25°C, the degradation rate of paclobutrazol by Gibberella B7 can reach 50%, and at 30°C, the degradation rate of paclobutrazol by Gibberella B7 can reach 35%. When the temperature is 20, 35 and 40°C, the degradation of paclobutrazol by Gibberella B7 is significantly inhibited, and the degradation rate is significantly reduced.

[0050] Example 3: Degradation characteristics of Kochbacterium gibbsii B7 under different pH conditions

[0051] The suspension of B. gissoni B7 was inoculated into 30 ml MSM medium with a paclobutrazol concentration of 25 mg / L at 5% inoculum, and cultured at different pH values ​​(5, 6, 7, 8, and 9) at 25°C and 180 rpm. Samples were taken on the 9th day, and the paclobutrazol content was determined by high performance liquid chromatography (HPLC) to analyze the degradation effect.

[0052] The results are as follows Figure 6 As shown in the figure, under weak acid conditions of pH 6, the degradation effect of GABA on paclobutrazol by GABA B7 was better, and the degradation rate could reach 60%. When the pH was at neutral and alkaline conditions such as 7, 8, and 9, the degradation of paclobutrazol by GABA B7 was inhibited, and the degradation rate decreased significantly.

[0053] Example 4: Degradation characteristics of different concentrations of paclobutrazol by Kochbacterium gibbsii B7

[0054] The suspension of B. gissoni B7 was inoculated into 30 ml MSM medium at pH 6 with a 5% inoculum, and paclobutrazol was added to each treatment to a concentration of 10, 20, 30, 50, 100, 150 and 200 mg / L, and cultured at 25°C and 180 rpm in a shaking table. Samples were taken on the 9th day, and the paclobutrazol content was determined by high performance liquid chromatography (HPLC) to analyze the degradation effect.

[0055] The results are as follows Figure 7 As shown, when the initial concentration of paclobutrazol was 100 mg / L, the degradation rate of paclobutrazol by K. gibberellicus B7 could reach 70.23%.

[0056] Example 5: Optimization of degradation conditions of Kochbacterium gibbsii B7

[0057] RSM was used, with temperature (A), pH value (B) and initial paclobutrazol concentration (C) as independent variables and Y (paclobutrazol degradation rate) as the corresponding value, and the Box-Behnken design condition optimization experiment was carried out. The design of the experiment is shown in Table 1, and the response results are shown in Table 2. By performing polynomial regression analysis on the response value Y, that is, the degradation rate of paclobutrazol, a quadratic response regression model was established, and the quadratic polynomial response surface regression equation of the paclobutrazol degradation rate (Y) was fitted:

[0058] Y%=71.13+9.05A-10.33B-6.11C-0.47AB+6.64AC+5.30BC-16.78A 2 -16.64B 2 -

[0059] 9.08C 2

[0060] The variance analysis of the fitting model is shown in Table 3. As shown in Table 3, the effects of temperature (A), pH value (B), initial paclobutrazol concentration (C) and their quadratic terms as well as the interactions between the factors (AC, BC) on the degradation of paclobutrazol by G. gissoni B7 are all at a significant level. The multiple regression between the dependent variable degradation rate and the independent variables (temperature, pH and initial paclobutrazol concentration) is significant. The correlation coefficient of the model is 0,9803, but the results of the lack-of-fit experiment are not significant, indicating that the model has a good fitting effect and is suitable for the theoretical prediction of paclobutrazol degradation by G. gissoni B7.

[0061] The response surface and contour plots of the interactive effects of temperature and initial paclobutrazol concentration on the degradation of paclobutrazol by G. jirovecii B7 are shown in Figure 2. Figure 8 As shown in the figure. When the initial paclobutrazol concentration is constant, the degradation ability of G. gissori B7 on paclobutrazol increases rapidly with the increase of temperature, then gradually decreases after reaching the optimal temperature; when the temperature remains unchanged, with the increase of the initial paclobutrazol concentration, the degradation rate of G. gissori B7 shows a trend of first increasing and then decreasing. It can be seen from the contour map that the contour lines are elliptical, and the interaction between temperature and initial paclobutrazol concentration has a significant effect on the response value.

[0062] The response surface and contour plots of the interactive effects of pH and initial paclobutrazol concentration on the degradation of paclobutrazol by G. jirovecii B7 are shown in the figure. Fig. 9 As shown in the figure. When the pH value is constant, the degradation ability of Bacillus gissoni B7 on paclobutrazol first increases and then decreases with the increase of the initial paclobutrazol concentration. When the initial paclobutrazol concentration remains unchanged, the degradation ability of Bacillus gissoni B7 is significantly improved under weak acid conditions, and gradually becomes neutral when the pH is close to 7, and the degradation ability is significantly inhibited. It can be seen from the contour map that the contour lines are elliptical, and the interaction between pH value and initial paclobutrazol concentration has a significant effect on the response value.

[0063] According to the response value of the degradation rate, the quadratic polynomial equation was analyzed. Within the level range of 3 factors, the pH of the culture medium was 5.6, the culture temperature was 26°C, and the initial paclobutrazol concentration was 90 mg / L, which was the theoretical optimal degradation condition for Cootieella gissoni B7. At this time, the model predicted that the optimal degradation rate of Cootieella gissoni B7 was 74.03%.

[0064] Table 1 Response surface experimental design

[0065]

[0066] Table 2 Response results of response surface

[0067]

[0068]

[0069] Table 3 Analysis of variance in response surface experiments of Cootieella gibberelli B7

[0070]

[0071] Example 6. Phytotoxicity of Paclobutrazol and Its Degradation Products

[0072] Wheat seeds were used as the research object to evaluate the phytotoxicity of paclobutrazol and its degradation products. The inoculum of 5% was inoculated into MSM medium with a paclobutrazol concentration of 100 mg / L. The degradation products after 5 days and 9 days of degradation were selected, centrifuged at 10000×g for 10 minutes to remove bacterial cells, and the supernatant was collected and filtered with a 0.22μm sterile filter membrane. The solution of undegraded paclobutrazol with the same concentration was used as a positive control. The surface of wheat seeds was disinfected in 2% H2O2 for 10 minutes and washed with sterile water. Then 25 wheat seeds were placed on a culture dish covered with double-layer filter paper. The culture dish was placed in a light incubator for culture (12h light and 12h dark). 20mL of undegraded paclobutrazol solution (PBZ) and the product of paclobutrazol degradation for 5 days (5d) and the product of paclobutrazol degradation for 9 days (9d) were added to each treatment every day. MSM medium without paclobutrazol (CK) was used as a control. Three parallels were set for each group of experiments. After 5 days, the seed germination rate (%), root length (cm), and stem length (cm) were recorded.

[0073] Table 4 Phytotoxicity studies of paclobutrazol and its degradation products

[0074]

[0075] Note: CK: inorganic salt medium without PBZ added; 9d: PBZ degradation product after 9 days of degradation; 5d: PBZ degradation product after 5 days of degradation; PBZ: undegraded PBZ; Different letters indicate significant differences among treatments at the P<0.05 level.

[0076] The results are as follows Fig.10 It can be seen that undegraded paclobutrazol (PBZ) has a great impact on the germination and growth of wheat seeds, resulting in short plants, small tubers, deformities, curled leaves, twisted young leaves, and shrinkage. The toxicity of the product (9d) of paclobutrazol degradation for 9 days was significantly reduced, and the germination rate and growth of wheat seeds were significantly better than those of the undegraded treatment group. The toxicity of the product (9d) of paclobutrazol degradation for 9 days was also lower than that of the product (5d) of paclobutrazol degradation for 5 days. The data of plant growth were statistically analyzed using the software Image J, and the statistical data are shown in Table 4.

[0077] In summary, the present invention screened out a strain of Kurthia gibsonii B7, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 23, 2024, with a deposit number of CGMCC No.32305; the Kurthia gibsonii B7 can grow in a liquid culture medium containing paclobutrazol, can reduce the content of paclobutrazol in the culture medium, and eliminate the toxic effect of paclobutrazol on the bacteria; 95 mg / L of paclobutrazol can be degraded in 9 days, and the degradation rate can reach 74%. The degradation products after degrading paclobutrazol have significantly weakened stress and phytotoxic effects on plants, and the cost is low compared to physical and chemical methods, and there is no secondary pollution.

Claims

1. A strain of Kurthia gibsonii B7 for degrading paclobutrazol, characterized in that: The described Kochbacterium gibbsii B7 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on October 23, 2024, with the deposit number CGMCC No.32305.

2. A bacterial agent, characterized in that Including the Kochbacterium gibbsii B7 described in claim 1.

3. Use of the Gibberellic acid B7 as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of a soil remediation agent.

4. Use of the Gibberellic acid B7 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a soil conditioner.

5. Use of the Gibberellic acid B7 according to claim 1 or the bacterial agent according to claim 2 in promoting plant growth.

6. Use of the Kochbacterium gibbsii B7 as claimed in claim 1 or the bacterial agent as claimed in claim 2 in degrading paclobutrazol.

7. A soil conditioner, characterized in that: The soil conditioner contains the Gibberellic acid B7 described in claim 1 or the bacterial agent described in claim 2, and auxiliary materials.

8. A plant growth promoter used in soil with paclobutrazol residues, characterized in that: The plant growth promoter contains the Gibberellic acid B7 described in claim 1 or the bacterial agent described in claim 2, and auxiliary materials.

Images