Method for remedying acetochlor-polluted soil by compounding growth-promoting bacteria and soybeans

Abstract

The invention belongs to the technical field of soil remediation, and particularly relates to a method for remedying acetochlor-contaminated soil by compounding growth-promoting bacteria and soybeans. The efficient growth promoting function of the Klebsiella sp.D5A strain is utilized, the Klebsiella sp.D5A strain secretes growth hormone to promote soybean growth, and the adaptive capacity of soybeans to the acetochlor polluted environment is enhanced; after being combined with an acetochlor-tolerant soybean variety, the soybean repairing agent can realize synchronous improvement of the repairing efficiency and the soybean yield through a'growth promotion-stress resistance 'dual synergistic mechanism and by means of the acetochlor degradation capability of the tolerant soybean variety, and solves the problems of single microbial function and disjunction of repairing and production in the prior art.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of soil remediation, in particular to a method for remediation of acetochlor contaminated soil by using growth-promoting bacteria and soybeans. BACKGROUND

[0002] The rotation of corn and soybeans is a widely used ecological planting mode, which can effectively maintain the balance of soil fertility and ecological function. However, in actual planting, due to the long-term continuous planting of corn, combined with the overuse of herbicides, the cumulative amount of acetochlor in the soil continues to rise, causing increasingly serious harm to the soybeans planted in the following season. Acetochlor has potential carcinogenicity and poses risks to organisms and crops. Its widespread and frequent use has caused serious environmental harm and may cause genetic diseases, reproductive disorders and deformities in humans, aquatic organisms and herbivorous animals. International organizations have listed it as a B2 carcinogen. As a crop that is relatively sensitive to acetochlor stress, soybeans often exhibit significant growth inhibition in contaminated soil, thereby affecting yield and quality.

[0003] The yield reduction of soybeans caused by acetochlor pollution is a prominent problem in agricultural production. In existing remediation technologies, microbial remediation has become a research hotspot due to its environmental friendliness. However, single microbial agents often have functional limitations, and the resistance differences of soybean varieties also restrict the remediation efficiency. CN112931123A discloses a technology for rhizobium to alleviate atrazine phytotoxicity, which confirms that microorganisms can enhance plant stress resistance through growth promotion. However, this technology does not form a synergistic remediation system with resistant varieties. SUMMARY

[0004] Based on the above problems, the present application provides a method for remediation of acetochlor contaminated soil by using growth-promoting bacteria and soybeans. The core innovation lies in the use of the high-efficiency growth-promoting function of Klebsiella sp. D5A strain, which secretes growth hormones to promote soybean growth and enhance the adaptability of soybeans to acetochlor contaminated environment. After being combined with acetochlor-resistant soybean varieties, it can realize the synchronous improvement of remediation efficiency and soybean yield through the "growth promotion-stress resistance" dual synergistic mechanism, and the degradation ability of resistant soybean varieties to acetochlor, solving the problem of single microbial function and the disconnection between remediation and production in existing technologies.

[0005] Specifically, the method comprises the following steps:

[0006] Step 1: Transfer Klebsiella sp. D5A strain to LB solid plate culture medium, cultivate at 28-30℃, pick single colonies after growth, inoculate in LB liquid culture medium, ferment at 28-30℃, 180r / min for 48h to obtain fermentation liquor, mix the fermentation liquor with bran at a mass ratio of (10-30):(70-90) to obtain growth-promoting bacteria agent.

[0007] The Klebsiella sp. D5A is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO. 7248, the preservation address is No. 3 Datun Road, Haidian District, Beijing, and the preservation date is February 1, 2013.

[0008] In step two, the growth promoting bacteria agent is mixed with the seeds of the acetochlor tolerant soybean variety at a mass ratio of (2-3):100, and is sowed in the acetochlor contaminated soil.

[0009] Preferably, the acetochlor tolerant soybean variety is Hei Nong 54 or Ji Yu 100.

[0010] Preferably, the sowing is performed by hole sowing or strip sowing, and the sowing is performed by hole sowing or strip sowing, in the hole sowing, 3-4 seeds are sowed in each hole with a hole depth of 3-4 cm and a hole distance of 20-25 cm, in the strip sowing, the row distance is 30-40 cm, and the sowing depth is 3-4 cm. After sowing, 2-3 cm of soil is covered, and the seeds and the soil are lightly pressed to make them closely contact, which is beneficial to the seed to absorb water and nutrients.

[0011] Preferably, during the growth period of the soybean, the soil humidity is maintained at 60-70% of the field water holding capacity.

[0012] The present application has the following beneficial effects:

[0013] (1) The growth promoting function of the growth promoting bacteria assists in the repair: the Klebsiella sp. D5A significantly increases the biomass of the soybean through the growth promotion, enhances the growth ability of the soybean in the acetochlor contaminated environment, and assists the tolerant soybean variety to better play the repair role, so that the limitation of the single function microbial agent is solved.

[0014] (2) The production and repair are realized synchronously: in the acetochlor contaminated soil with ≤15 mg / kg (about 15 times of the normal field application amount), the yield of the soybean is recovered and exceeds the non-contaminated level by 28-45% during the repair period, relying on the degradation capacity of the tolerant soybean variety and the growth promoting capacity of the bacteria agent, so that the influence of the traditional repair on the agricultural production is overcome. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0016] Figure 1Figure for the growth state of soybean plants under the treatment of Klebsiella sp. D5A.

[0017] Figure 2 Figure for the effect of the addition of the microbial agent on the occurrence rate of soybean phytotoxicity in acetochlor-contaminated soil.

[0018] Figure 3 Figure for the relative abundance of Klebsiella sp. D5A in the rhizosphere of soybean.

[0019] Figure 4 Figure for the reduction effect of acetochlor after the planting of soybean under the treatment of Klebsiella sp. D5A.

[0020] Figure 5 Figure for the fresh weight of soybean under the treatment of Klebsiella sp. D5A.

[0021] Figure 6 Figure for the yield of soybean under the treatment of Klebsiella sp. D5A. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the application.

[0023] Embodiment 1

[0024] Step one, the Klebsiella sp. D5A strain was transferred to LB solid plate culture medium, and cultured at 29±1℃. After single colonies grew, a single colony was picked and inoculated in LB liquid culture medium. After fermentation at 29±1℃ and 180r / min for 48h, a fermentation liquor was obtained. The fermentation liquor was mixed with bran at a mass ratio of 20:80 to obtain a growth-promoting microbial agent.

[0025] Step two, the growth-promoting microbial agent was mixed with Hei 54 soybean seeds at a mass ratio of 2:100 for seed dressing, and then sowed in acetochlor-contaminated soil (the initial concentration of acetochlor in the soil was ≤15mg / kg).

[0026] Hole sowing or strip sowing was adopted. When hole sowing was adopted, the hole depth was 3cm, 3 seeds were sowed in each hole, and the hole distance was 20cm. When strip sowing was adopted, the row distance was 30cm, the sowing depth was 3cm, and the soil covering depth after sowing was 3cm.

[0027] Base fertilizer application: Apply 150 kg / acre of commercial organic fertilizer (main components are fulvic acid and potassium elements), and apply 30 kg / acre of compound fertilizer (P2O5-K2O = 20-10) at the same time.

[0028] During the growth period of soybeans, the soil moisture is maintained at 70% of the field water holding capacity.

[0029] Test Example 1

[0030] Step one: After the strain is activated, the strain is subjected to functional identification by the following method:

[0031] ACC deaminase activity determination: After the bacterial cells are collected by centrifugation at 4°C and washed three times, they are resuspended in a culture medium containing 1-aminocyclopropane-1-carboxylic acid (ACC) as the sole nitrogen source and cultured for 48 hours. The bacterial cells are collected by centrifugation at 4°C. They are washed three times with 0.1 mol / L Tris-HCl buffer (pH = 7.6) and resuspended in 600 μL of 0.1 mol / L Tris-HCl buffer (pH = 8.0). 30 μL of toluene is added to break the cells. 200 μL of cell extract is mixed with 20 μL of 0.5 mol / L ACC. After 30°C reaction for 15 minutes, 1 mL of 0.56 mol / L HCl is added to terminate the reaction, and centrifugation is performed at 16000g for 5 minutes. 1 mL of supernatant is taken, 800 μL of 0.56 mol / L HCl and 300 μL of 2,4-dinitrophenylhydrazine are added, and color development is performed at 30°C for 30 minutes. Finally, 2 mL of 2 mol / L NaOH is added, and the absorbance of the reaction product α-ketobutyric acid at 540 nm is measured. The ACC deaminase activity is calculated by a standard curve.

[0032] IAA yield determination: After the bacterial cells are collected by centrifugation at 4°C and washed three times, they are resuspended in LB liquid medium containing tryptophan (500 mg / L) and shaken for 48 hours. 1 mL of supernatant is taken by centrifugation, 50 μL of 10 mmol / L orthophosphoric acid and 2 mL of Salkowski's color developing agent are added, color development is performed in the dark at 25°C for 30 minutes, and the absorbance is measured at 530 nm. The IAA concentration is calculated by a standard curve.

[0033] Iron carrier production determination: The strain is inoculated into MSA liquid medium (sucrose 20 g / L, asparagine 2 g / L, K2HPO4 1 g / L, MgSO4·7H2O 0.5 g / L) and shaken at 28°C for 48 hours. The bacterial liquid is centrifuged at 4°C at 16000g for 5 minutes, and the supernatant is scanned by ultraviolet-visible spectrophotometry at a wavelength range of 400-450 nm to detect whether there is an absorption peak at 405 nm.

[0034] Phosphorus solubilization assay: After centrifugation at 4℃, the bacterial cells were washed three times and resuspended in 0.5% Ca3(PO4)2-modified SRSM liquid medium. After 72 h, the inorganic phosphorus solubilization capacity of the strains was determined: first mix the bacterial solution, take 1 mL of bacterial solution, centrifuge at 10000 r / min for 10 min, and then measure the phosphorus solubilization capacity of the supernatant by molybdenum-antimony anti-colorimetric method, with three repeats.

[0035] Acid-base tolerance assay: The strains were inoculated into LB medium with pH 3-11, and cultured at 28℃ for 24 h with shaking. The OD value (biomass) at 600 nm was measured.

[0036] Table 1 Functional identification of Klebsiella sp. D5A

[0037] Function type Function level Unit Activity of ACC deaminase 0.083 U / mg IAA 112 mg / L Solubilization of phosphate 130 mg / L Production of siderophore No obvious ability to produce siderophore Resistance to acid and alkali 4-11 pH

[0038] Test Example 2

[0039] The following six groups of experiments were set up according to the method of Example 1, namely, conventional soybean variety (Heinong 48) without using bacterial agent for seed dressing, Jiyu 100 without using bacterial agent for seed dressing, Heishang 54 without using bacterial agent for seed dressing, conventional soybean variety (Heinong 48) using bacterial agent for seed dressing, Jiyu 100 using bacterial agent for seed dressing, and Heishang 54 using bacterial agent for seed dressing. Among them, the experimental group without additional addition of acetochlor was set as the control group (CK).

[0040] During the growth of soybeans, the growth state of soybean plants on the 30th day (as shown in Figure 1 ) was observed, the fresh weight of plants (as shown in Figure 5 ) and plant phytotoxicity symptoms (leaf yellowing, dwarfing, root browning, as shown in Figure 2 ) were counted.

[0041] The relative abundance of Klebsiella sp. D5A in the rhizosphere of soybeans was detected by 16s rDNA high-throughput sequencing (as shown in Figure 3 ) to understand the colonization of the strain.

[0042] During the repair period (soybean growth period), soil samples were collected regularly, the content of acetochlor in the soil was detected, and the acetochlor reduction rate was calculated (as shown in Figure 4 ).

[0043] At the harvest period of soybeans, the soybean yield was detected (as shown in Figure 6 ).

[0044] From Figures 1-6It can be seen that the yield of soybean was increased by 28-45% in the soil with <15 mg / kg of acetochlor pollution, and the yield of soybean was restored and exceeded the level of non-pollution in the repair period, when the acetochlor reduction rate was ≥80% and the acetochlor pollution rate was reduced by ≥40% by relying on the resistant soybean variety and the microbial agent.

[0045] The above description of disclosed embodiments enables one skilled in the art to make or use the application. Numerous modifications to these embodiments will be apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Therefore, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for promoting the growth of bacteria-soybean complex to repair the soil contaminated by acetochlor, characterized in that, Mix the growth promoting bacteria agent with the seeds of the acetochlor tolerant soybean variety according to the mass ratio (2-3):100, and sow them in the acetochlor contaminated soil; The growth promoting bacteria are Klebsiella sp. D5A with the preservation number of CGMCC NO. 7248.

2. The method for repairing the ethion contaminated soil by the bacteria-soybean complex according to claim 1, characterized in that, The preparation method of the growth promoting bacteria agent is as follows: inoculate the activated Klebsiella sp. D5A single colony in LB liquid culture medium, and ferment it at 28-30°C and 180 r / min for 48 h to obtain the fermentation liquor; then mix the fermentation liquor with bran according to the mass ratio (10-30):(70-90) to obtain the growth promoting bacteria agent.

3. The method for repairing the ethion contaminated soil by the bacteria-soybean complex according to claim 1, characterized in that, The activation step of the Klebsiella sp. D5A is as follows: inoculate the Klebsiella sp. D5A in LB solid plate culture medium, and cultivate it in a constant temperature incubator at 28-30°C; then select the single colony with typical morphology.

4. The method for repairing the ethion contaminated soil by the bacteria-soybean complex according to claim 1, characterized in that, The acetochlor tolerant soybean variety is Hei Nong 54 or Ji Yu 100.

5. The method for repairing the ethion contaminated soil by the bacteria-soybean complex according to claim 1, characterized in that, Sow the seeds in the way of hole sowing, with the hole depth of 3-4 cm, 3-4 seeds per hole, and the hole distance of 20-25 cm; and cover the soil with a thickness of 2-3 cm after sowing.

6. The method for repairing the ethion contaminated soil by the bacteria-soybean complex according to claim 1, characterized in that, Sow the seeds in the way of strip sowing, with the row distance of 30-40 cm, the sowing depth of 3-4 cm, and the soil covering thickness of 2-3 cm after sowing.

7. The method for repairing the ethion contaminated soil by the bacteria-soybean complex according to claim 1, characterized in that, During the growth period of the soybean, keep the soil humidity at 60-70% of the field water holding capacity.

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