Method for degrading allelochemicals in tobacco-growing soils
By combining dye-loving bacteria with inorganic salt solutions, allelochemicals in tobacco-growing soil are efficiently degraded, solving the problem of continuous tobacco cropping and improving soil quality and tobacco yield.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WUHAN POLYTECHNIC UNIVERSITY
- Filing Date
- 2023-04-25
- Publication Date
- 2026-06-12
AI Technical Summary
The problem of tobacco continuous cropping obstacles is mainly caused by allelochemicals in the tobacco-growing soil, which leads to deterioration of soil physical and chemical properties, increase of tobacco diseases and pests, and decline in yield and quality. Existing technologies are unable to effectively degrade these toxic substances.
The dye-loving bacterium Pigmentiphaga sp. CHJ04 was mixed with tobacco-growing soil, and a specific inorganic salt solution was added under aerobic conditions to degrade allelochemicals in the tobacco-growing soil.
Under natural conditions, it can effectively degrade 11 allelopathic toxic substances within 2 to 6 days, solve the problem of continuous tobacco cropping, and improve soil quality and tobacco yield.
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Abstract
Description
Technical Field
[0001] This invention relates to the fields of agricultural production and microbial technology, and in particular to a method for degrading allelochemicals in tobacco-growing soil. Background Technology
[0002] Tobacco, as a crop susceptible to continuous cropping problems, often exhibits continuous cropping obstacles, such as deterioration of soil physical and chemical properties, increased pests and diseases affecting tobacco leaves, and decreased yield and quality. Numerous studies have shown that tobacco continuous cropping obstacles are closely related to allelopathic autotoxicity. Allelopathic substances secreted by the plant's roots can affect the plant's growth by influencing cell membrane permeability, enzyme activity, ion absorption, water absorption, photosynthesis, and protein and DNA synthesis. Therefore, these allelopathic substances are also known as allelopathic autotoxic substances. Continuous cropping of tobacco leads to their accumulation in the field, resulting in poor crop growth, reduced yield, and decreased quality, which has become one of the major problems hindering the sustainable development of agriculture. Summary of the Invention
[0003] The main objective of this invention is to propose a method for degrading allelochemicals in tobacco-growing soil, aiming to effectively degrade allelochemicals in tobacco-growing soil.
[0004] To achieve the above objectives, this invention proposes a method for degrading allelochemicals in tobacco-growing soil, comprising the following steps:
[0005] Dye-loving bacteria were used to degrade tobacco-growing soil.
[0006] Optionally, the step of using dyeophilic bacteria to degrade tobacco-growing soil includes:
[0007] S10. After inoculating the dye-loving bacteria onto the culture medium and culturing them into a bacterial solution, mix it with the tobacco-planting soil to obtain a mixture;
[0008] S20. Add an inorganic salt solution to the mixture and degrade it under aerobic conditions.
[0009] Optionally, step S10 includes:
[0010] S11. Inoculate the dye-loving bacteria onto the culture medium and culture them into a bacterial suspension;
[0011] S12. After drying the bacterial solution into powder, it is mixed with tobacco-growing soil to obtain a mixture.
[0012] Optionally, in step S10,
[0013] The culture medium includes LB medium; and / or,
[0014] The dyeophilic bacteria include dyeophiles ( Pigmentiphaga sp. CHJ04.
[0015] Optionally, in step S10, the inoculation amount of the dyeophilic bacteria is 0.1% to 0.5%.
[0016] Optionally, in step S20, the inorganic salt solution comprises components with the following concentrations:
[0017] Na2HPO4·12H2O 10.2~14.3g / L, KH2PO42~3g / L, FeSO4·7H2O 0.2~0.3mg / L, MnSO4·H2O 0.05~0.07mg / L, MgSO4·7H2O 0.015~0.02mg / L, CaCl20.25~0.3mg / L, CuSO40.0125~0.2mg / L, ZnSO40.0125~0.2mg / L and H3BO30.0125~0.2mg / L, (NH4)2SO42~3mM.
[0018] Optionally, in step S20, the pH value of the inorganic salt solution is 7.0 to 7.5.
[0019] Optionally, in step S20, the inorganic salt solution further includes agar powder.
[0020] Optionally, the mass of the agar powder in each 100 mL of the inorganic salt solution is 1.5 to 2 g.
[0021] Optionally, in step S20, the degradation time is 2 to 6 days.
[0022] The technical solution provided by this invention proposes a method for degrading allelochemicals in tobacco-growing soil. Using the dyeophilic bacteria provided by this invention, 11 out of 13 allelochemicals in tobacco-growing soil can be degraded, including catechol, phthalic acid, p-hydroxybenzoic acid, butanol, benzoic acid, 3-methoxy-4-hydroxybenzoic acid, p-hydroxybenzaldehyde, 3-methoxy-4-hydroxybenzaldehyde, diisobutyl phthalate, dibutyl phthalate, and terephthalic acid. Under natural conditions, the above 11 substances can be degraded to a concentration of 0.5 mM within 2-6 days, achieving the degradation of allelochemicals in tobacco-growing soil and overcoming the obstacles of continuous tobacco cropping. Detailed Implementation
[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.
[0024] It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, solution B, or a solution that simultaneously satisfies A and B. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, such a combination should be considered non-existent and not within the scope of protection claimed by this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.
[0025] Tobacco, as a crop susceptible to continuous cropping problems, often exhibits continuous cropping obstacles, such as deterioration of soil physical and chemical properties, increased pests and diseases affecting tobacco leaves, and decreased yield and quality. Numerous studies have shown that tobacco continuous cropping obstacles are closely related to allelopathic autotoxicity. Allelopathic substances secreted by the plant's roots can affect the plant's growth by influencing cell membrane permeability, enzyme activity, ion absorption, water absorption, photosynthesis, and protein and DNA synthesis. Therefore, these allelopathic substances are also known as allelopathic autotoxic substances. Continuous cropping of tobacco leads to their accumulation in the field, resulting in poor crop growth, reduced yield, and decreased quality, which has become one of the major problems hindering the sustainable development of agriculture.
[0026] In view of this, the present invention proposes a method for degrading allelochemicals in tobacco-growing soil, which aims to effectively degrade allelochemicals in tobacco-growing soil.
[0027] To achieve the above objectives, this invention proposes a method for degrading allelochemicals in tobacco-growing soil, comprising the following steps:
[0028] Dye-loving bacteria were used to degrade tobacco-growing soil.
[0029] The technical solution provided by this invention proposes a method for degrading allelochemicals in tobacco-growing soil. Using the dyeophilic bacteria provided by this invention, 11 out of 13 allelochemicals in tobacco-growing soil can be degraded, including catechol, phthalic acid, p-hydroxybenzoic acid, butanol, benzoic acid, 3-methoxy-4-hydroxybenzoic acid, p-hydroxybenzaldehyde, 3-methoxy-4-hydroxybenzaldehyde, diisobutyl phthalate, dibutyl phthalate, and terephthalic acid. Under natural conditions, the above 11 substances can be degraded to a concentration of 0.5 mM within 2-6 days, achieving the degradation of allelochemicals in tobacco-growing soil and overcoming the obstacles of continuous tobacco cropping.
[0030] Thirteen allelochemicals in tobacco-growing soils include catechol, phthalic acid, p-hydroxybenzoic acid, butanol, benzoic acid, 3-methoxy-4-hydroxybenzoic acid, p-hydroxybenzaldehyde, 3-methoxy-4-hydroxybenzaldehyde, diisobutyl phthalate, o-, p-di-tert-butylphenol, dibutyl phthalate, diisooctyl phthalate, and terephthalic acid. These allelochemicals are contributing factors to continuous tobacco cropping obstacles. This invention utilizes dyeophilic bacteria to degrade allelochemicals in tobacco-growing soils, specifically degrading 11 of the thirteen allelochemicals. It is primarily applied to the treatment and comprehensive utilization of allelochemical accumulation in soils during continuous tobacco cropping, effectively addressing continuous tobacco cropping obstacles and promoting comprehensive application.
[0031] Specifically, the steps involving the degradation of tobacco-growing soil by dye-loving bacteria include:
[0032] S10. After inoculating the dye-loving bacteria onto the culture medium and culturing them into a bacterial solution, mix it with the tobacco-planting soil to obtain a mixture;
[0033] S20. Add an inorganic salt solution to the mixture and carry out degradation under aerobic conditions. When dyeophilic bacteria are used in tobacco-growing soil, the inorganic salt solution provides nutrients for the bacteria, leading to more efficient degradation of allelochemicals.
[0034] The technical solution provided by this invention proposes a method for degrading allelochemicals in tobacco-growing soil. Using the dyeophilic bacteria provided by this invention, 11 out of 13 allelochemicals in tobacco-growing soil can be degraded, including catechol, phthalic acid, p-hydroxybenzoic acid, butanol, benzoic acid, 3-methoxy-4-hydroxybenzoic acid, p-hydroxybenzaldehyde, 3-methoxy-4-hydroxybenzaldehyde, diisobutyl phthalate, dibutyl phthalate, and terephthalic acid. Under natural conditions, the above 11 substances can be degraded to a concentration of 0.5 mM within 2-6 days, achieving the degradation of allelochemicals in tobacco-growing soil and overcoming the obstacles of continuous tobacco cropping.
[0035] Preferably, step S10 includes:
[0036] S11. Inoculate the dye-loving bacteria onto the culture medium and culture them into a bacterial suspension;
[0037] S12. After drying the bacterial solution into powder, mix it with tobacco-growing soil to obtain a mixture. Mixing the dye-loving bacteria as powder with tobacco-growing soil makes the process easier.
[0038] Preferably, in step S10,
[0039] The culture medium mentioned includes LB medium, which is the name of a culture medium commonly used in biochemical molecular experiments to pre-culture bacterial strains, allowing the strains to multiply and reach the required levels for use. It can be divided into liquid and solid media. LB is generally interpreted as Luria-Bertani medium; however, according to its inventor, Giuseppe Bertani, the name comes from the English term "lysogeny broth." The formulation of LB medium is as follows: Tryptone 10 g / L, Yeast extract 5 g / L, and Sodium chloride (NaCl) 10 g / L. Using this medium allows for more efficient degradation of allelochemicals in the soil.
[0040] The dyeophilic bacteria include dyeophiles ( Pigmentiphaga sp. CHJ04. This strain has been deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 2023329. Deposit date: March 15, 2023. This chromophilic bacterium ( Pigmentiphaga sp. CHJ04 is filtered using the following method:
[0041] Sludge samples collected from Machiqiao, Wuhan City, were thoroughly mixed. Two mL of the sludge sample was added to a liquid inorganic salt culture medium containing 2 mM isophthalic acid and 0.5 M (NH4)2SO4. The medium was incubated at 28°C on a shaker at 200 rpm, with three replicates. After 10 days of incubation, the sample was allowed to stand for 30 minutes. Then, a 10% (v / v) inoculum was added to the same culture medium and incubated on a shaker at 200 rpm. After 7 days of incubation, the sample was allowed to stand for 30 minutes. Then, a 1% (v / v) inoculum was added to the same culture medium and incubated on a shaker at 200 rpm. This process was repeated three times to obtain a mixed microbial culture in which isophthalic acid-degrading bacteria were dominant. Further separation and purification experiments were then performed.
[0042] The enriched bacterial culture was streaked onto inorganic salt medium containing 2 mM isophthalic acid and incubated upside down at 28°C. After 24 h, different single colonies were picked and placed in 5 mL of liquid inorganic salt medium containing 2 mM isophthalic acid. The same concentration of (NH4)2SO4 was added, and the culture was incubated at 28°C with a shaker at 200 rpm. This streaking and single colony picking process was repeated three times to obtain morphologically consistent single colonies. The obtained single colonies were then used for further molecular biological identification of the strain.
[0043] Single colonies were picked and cultured in an inorganic salt medium (containing 2 mM isophthalic acid and (NH4)2SO4). The 16S rRNA sequence of the bacterial culture was amplified by PCR. The PCR product with a band of approximately 1500 bp was sent to a sequencing company for sequencing.
[0044] The sequence was analyzed and assembled using software and submitted to the NCBI database. BLAST alignment results showed that the sequence was consistent with... Pigmeutiphaga daegueusis The identity of strain K110 (GenBank: NR044082.1) was 99.65%, and it was named *Dystrophophila*. Pigmentiphaga sp. CHJ04. Hereinafter referred to as CHJ04, the CHJ04 selected in this application can more efficiently degrade allelochemicals in the soil.
[0045] Preferably, in step S10, the inoculation amount of the dyeophilic bacteria is 0.1% to 0.5%. The inoculation amount refers to the ratio of the volume of the seed liquid to the volume of the culture medium after inoculation, that is, the ratio of the volume of the dyeophilic bacteria to the volume of the culture medium after inoculation. In the embodiments of the present invention, the inoculation amount of the dyeophilic bacteria can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, etc., which can achieve efficient degradation of allelochemicals in the soil with low strain dosage.
[0046] Furthermore, specifically, in step S20, the inorganic salt solution comprises components of the following concentrations:
[0047] The following inorganic salts provide energy for the growth, reproduction, and degradation of allelochemicals in CHJO4, enabling efficient degradation of allelochemicals in the soil. These include Na2HPO4·12H2O (10.2~14.3 g / L), KH2PO4 (2~3 g / L), FeSO4·7H2O (0.2~0.3 mg / L), MnSO4·H2O (0.05~0.07 mg / L), MgSO4·7H2O (0.015~0.02 mg / L), CaCl2 (0.25~0.3 mg / L), CuSO4 (0.0125~0.2 mg / L), ZnSO4 (0.0125~0.2 mg / L), H3BO3 (0.0125~0.2 mg / L), and (NH4)2SO4 (2~3 mM).
[0048] The present invention does not limit the pH value of the inorganic salt solution. Preferably, in step S20, the pH value of the inorganic salt solution is 7.0 to 7.5, for example, it can be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, etc. The above pH value is more conducive to the growth, reproduction and degradation of allelochemicals of CHJ04.
[0049] Preferably, in step S20, the inorganic salt solution further includes agar powder. This allows for the efficient degradation of allelochemicals in the soil.
[0050] The present invention does not limit the amount of agar powder added. Preferably, the mass of agar powder in each 100 mL of the inorganic salt solution is 1.5~2 g, for example, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2 g, etc., so as to degrade allelochemicals in the soil more efficiently.
[0051] Preferably, in step S20, the degradation time is 2 to 6 days. Experiments show that by using the above method, the degradation time is 2 to 6 days, which can basically achieve the degradation of the allelochemicals in the above 11.
[0052] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.
[0053] Example 1
[0054] (1) Dyephilic bacteria ( Pigmentiphaga sp. CHJ04 was inoculated onto LB medium and cultured into a bacterial suspension at an inoculum size of 0.3%.
[0055] (2) After drying the bacterial solution into powder, it is mixed with the root soil of tobacco plants in non-continuously cropped fields to obtain a mixture;
[0056] (3) Under aerobic conditions, degradation was carried out for 7 days.
[0057] Example 2
[0058] (1) Dyephilic bacteria ( Pigmentiphaga sp. CHJ04 was inoculated onto LB medium and cultured into a bacterial suspension at an inoculum size of 0.1%.
[0059] (2) After drying the bacterial solution into powder, it is mixed with the root soil of tobacco plants in a field that has been continuously cropped for 1 year to obtain a mixture;
[0060] (3) Under aerobic conditions, degradation was carried out for 7 days.
[0061] Example 3
[0062] (1) Dyephilic bacteria ( Pigmentiphaga sp. CHJ04 was inoculated onto LB medium and cultured into a bacterial suspension at an inoculum size of 0.5%.
[0063] (2) After drying the bacterial solution into powder, it is mixed with the root soil of tobacco plants in fields that have been continuously cropped for 2 years to obtain a mixture;
[0064] (3) Under aerobic conditions, degradation was carried out for 7 days.
[0065] Example 4
[0066] (1) Dyephilic bacteria ( Pigmentiphaga sp. CHJ04 was inoculated onto LB medium and cultured into a bacterial suspension at an inoculum size of 0.4%.
[0067] (2) After drying the bacterial solution into powder, it is mixed with the root soil of tobacco plants in fields that have been continuously cropped for 4 years to obtain a mixture;
[0068] (3) Under aerobic conditions, degradation was carried out for 7 days.
[0069] Example 5
[0070] (1) Dyephilic bacteria ( Pigmentiphaga sp. CHJ04 was inoculated onto LB medium and cultured into a bacterial suspension at an inoculum size of 0.2%.
[0071] (2) After drying the bacterial solution into powder, it is mixed with the root soil of tobacco plants in a field that has been continuously cropped for 10 years to obtain a mixture;
[0072] (3) Under aerobic conditions, degradation was carried out for 7 days.
[0073] The contents of 13 allelochemicals in tobacco-grown soil were determined before and after degradation using the methods described in Examples 1 to 5, respectively. Table 1 shows the results. The contents before degradation and the detection methods were based on the literature report "Extraction and detection methods of allelochemicals in tobacco-grown soil (Invention Patent, 2019, Publication No.: CN109781872A)".
[0074] Table 1. Determination results (μg / kg) of Examples 1 to 5
[0075]
[0076] As shown in Table 1, the degradation methods for allelochemicals in tobacco-growing soil in Examples 1-5 of the present invention showed that the content of the first 11 of the 13 allelochemicals decreased significantly after degradation, indicating that the degradation was highly efficient. It can be seen that the use of dyeophilic bacteria to degrade allelochemicals in tobacco-growing soil in the embodiments of the present invention has obvious effects, can achieve the degradation of allelochemical toxic substances in tobacco-growing soil, and solve the obstacles of continuous tobacco cropping.
[0077] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.
Claims
1. A method for degrading allelochemicals in tobacco-growing soil, characterized in that, Includes the following steps: Dystrophic bacteria were used to degrade allelochemicals in tobacco-growing soil. The chromophilic bacterium includes Pigmentiphaga sp. CHJ04, which is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M2023329 and deposit date of March 15, 2023.
2. The method for degrading allelochemicals in tobacco-growing soil as described in claim 1, characterized in that, The steps involving the degradation of allelochemicals in tobacco-growing soil by dyeophilic bacteria include: S10. After inoculating the dye-loving bacteria onto the culture medium and culturing them into a bacterial solution, mix it with the tobacco-planting soil to obtain a mixture; S20. Add an inorganic salt solution to the mixture and degrade it under aerobic conditions.
3. The method for degrading allelochemicals in tobacco-growing soil as described in claim 2, characterized in that, Step S10 includes: S11. Inoculate the dye-loving bacteria onto the culture medium and culture them into a bacterial suspension; S12. After drying the bacterial solution into powder, it is mixed with tobacco-growing soil to obtain a mixture.
4. The method for degrading allelochemicals in tobacco-growing soil as described in claim 2, characterized in that, In step S10, the culture medium includes LB medium.
5. The method for degrading allelochemicals in tobacco-growing soil as described in claim 2, characterized in that, In step S10, the inoculum amount of the dye-loving bacteria is 0.1% to 0.5%.
6. The method for degrading allelochemicals in tobacco-growing soil as described in claim 2, characterized in that, In step S20, the inorganic salt solution comprises components of the following concentrations: Na2HPO4·12H2O 10.2~14.3 g / L, KH2PO4 2~3 g / L, FeSO4·7H2O 0.2~0.3 mg / L, MnSO4·H2O 0.05~0.07 mg / L, MgSO4·7H2O 0.015~0.02 mg / L, CaCl2 0.25~0.3 mg / L, CuSO4 0.0125~0.2 mg / L, ZnSO4 0.0125~0.2 mg / L and H3BO3 0.0125~0.2 mg / L, (NH 4)2 SO4 2~3 mM.
7. The method for degrading allelochemicals in tobacco-growing soil as described in claim 2, characterized in that, In step S20, the pH value of the inorganic salt solution is 7.0 to 7.
5.
8. The method for degrading allelochemicals in tobacco-growing soil as described in claim 2, characterized in that, In step S20, the inorganic salt solution also includes agar powder.
9. The method for degrading allelochemicals in tobacco-growing soil as described in claim 8, characterized in that, The mass of agar powder in every 100 mL of the inorganic salt solution is 1.5–2 g.
10. The method for degrading allelochemicals in tobacco-growing soil as described in claim 2, characterized in that, In step S20, the degradation time is 2 to 6 days.