CN111676045A

CN111676045A - Method for reducing antibiotic resistance genes in soil by using wood vinegar and application

Info

Publication number: CN111676045A
Application number: CN202010615642.8A
Authority: CN
Inventors: 郑浩; 李锋民; 张倩; 刘冰洁; 王瑞瑞
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2019-11-28
Filing date: 2019-11-28
Publication date: 2020-09-18
Anticipated expiration: 2039-11-28
Also published as: CN111662737A; CN111676045B; CN111662737B; CN110804453A; CN110804453B

Abstract

The invention provides a method for reducing antibiotic resistance gene pollution in soil by utilizing wood vinegar, relating to the field of environmental pollution treatment, wherein the wood vinegar used in the reduction method comprises the following steps: 1) preparing wood vinegar stock solution WV by an anaerobic slow pyrolysis method; 2) the wood vinegar stock solution is refined by a normal pressure distillation method, and the obtained rectification components are respectively a low-temperature section rectification component F1, an intermediate-temperature section rectification component F2 and a high-temperature section rectification component F3, so that the absolute abundance of total antibiotic resistance genes of rhizosphere and non-rhizosphere soil can be reduced, and each refining component has obvious selectivity.

Description

Method for reducing antibiotic resistance genes in soil by using wood vinegar and application

The application is a divisional application of application with application date of 2019, 11 and 28, application number of 2019111948458, and title of "a method for reducing antibiotic resistance genes in soil by using wood vinegar and application thereof".

Technical Field

The invention relates to the field of environmental pollution treatment, in particular to a method for reducing antibiotic resistance genes in soil by utilizing wood vinegar and application thereof.

Background

Antibiotic resistance has become one of the largest public health challenges facing humans in the twenty-first century. In recent years, abuse of ATs (Antibiotics, ATs) in medicine, livestock and aquaculture has led to a rapid increase in Antibiotic Resistance Bacteria (ARB) and Antibiotic Resistance Genes (ARGs) in the environment, seriously threatening human health and ecosystem safety. The european commission estimates that the cost of treatment associated with antibiotic-resistant infections exceeds 15 billion euros per year, while in the united states, an estimate has shown costs as high as $ 550 billion per year. The production of ARBs results in a large number of ARGs (present in the environment. at present, ARBs and ARGs have been detected in a variety of environmental media such as various bodies of water, sediments, the atmosphere and soil. soil is one of the main repositories for ARGs. ARGs are detected in various regions of the world, such as the United states, Australia, Mexico, China, etc., and are found in amounts of 10 ARGs^-7-10^-1copies/16S rRNAcopies。

ARGs have the biological property of being "replicable or transmissible", and thus can be either genetically transferred from parent to offspring, i.e., vertically transferred (VGT), or genetically transferred (HGT) both intraspecies and interspecies of microorganisms. VGT occurs between parents and progeny of microorganisms of the same species, is a normal phenomenon in nature, and has a limited spread. The HGT can transmit the ARGs among species and is considered as a main path for the propagation and diffusion of the ARGs in soil.

ARGs have become a new class of environmental pollutants, and the prior art has few strategies for controlling such pollutants. Manure or sludge applied to the soil is mainly subjected to aerobic composting or anaerobic digestion in order to reduce the ARGs at the source. However, for ARGs that have entered the soil or have accumulated for many generations, the reduction can only be done by adding soil amendments. The current soil conditioners are of a small variety and not all soil conditioners have the positive effect of reducing ARGs contamination. Therefore, in order to solve the problem of the increasingly serious pollution of the soil ARGs, the development of more efficient and environment-friendly soil conditioners for reducing the pollution of the soil ARGs is urgently needed.

The pyroligneous liquor is an organic mixture obtained by condensing, refluxing and re-separating smoke generated in the process of preparing the biochar from the biomass material. The pyroligneous liquor can neutralize harmful gas such as ammonia gas and hydrogen sulfide in environmental pollution. However, the crude wood vinegar was recovered by simple cooling and was a brownish black liquid. It contains a large amount of tar and harmful substances, and it is not known in the prior art whether it can be directly used for agricultural pollution treatment, whether it can reduce the pollution of ARGs in soil and ensure positive effects.

Disclosure of Invention

The invention aims to solve the problems that the antibiotic resistance gene accumulated in the soil is difficult to reduce in the prior art, and the like, and aims to provide a method for reducing the pollution of the antibiotic resistance gene in the soil by adopting wood vinegar and application of the method in reducing the pollution of the antibiotic resistance gene in agricultural soil, wherein the wood vinegar is used as a main raw material and is modified by refining; the absolute abundance of total ARGs of rhizosphere and non-rhizosphere soil can be reduced after the composition is used, and each refined component has obvious selectivity.

The invention provides a method for reducing the pollution of wood vinegar to antibiotic resistance genes, and the preparation of the wood vinegar comprises the following steps:

1) preparing wood vinegar stock solution by an anaerobic slow pyrolysis method: drying the plant raw materials to constant weight, pyrolyzing the plant raw materials by a pyrolysis carbonization furnace to prepare wood vinegar liquid, wherein the pyrolysis temperature is 350-550 ℃, the pyrolysis time is 3-6 h, gas generated in the pyrolysis process is condensed to obtain brownish black liquid with pungent smell, then standing for 3-6 months in the dark, and filtering the intermediate liquid with a microporous filter membrane to obtain wood vinegar stock solution WV;

2) refining wood vinegar stock solution by a normal pressure distillation method: under normal pressure, three temperature sections of (0-98) DEG C, (98-130) DEG C and (130-.

Preferably, the plant material is at least one of chaff, straw, wood chips, hardwood and bamboo.

Further, the wood vinegar stock solution and the refined distillation component are respectively applied to a bacteria liquid culture medium, the bacteriostasis of various wood vinegar liquids is measured, and the bacteriostasis rate is calculated according to the following formula:

the bacteriostatic rate (%) is (diameter of bacteriostatic circle of treatment group-diameter of bacteriostatic circle of control group)/diameter of bacteriostatic circle of treatment group x 100%.

Preferably, the wood vinegar stock solution WV and the rectification components thereof are subjected to component analysis through high performance gas chromatography-mass spectrometry, and then the relative abundance area is obtained by dividing the area of each peak spectrum by the total area of all peaks to calculate the relative content of each substance.

Further, the total content of organic acid and phenols is (50-85) wt%; in the wood vinegar stock solution WV, the content of organic acid is (30-50) wt%, and the content of phenols is (30-50) wt%; in the rectification component F1, the content of organic acid is (25-30) wt%, and the content of phenols is (20-27) wt%; in the rectification component F2, the content of organic acid is (50-65) wt%, and the content of phenols is (15-25) wt%; in the rectification component F3, the content of organic acid is (65-80) wt%, and the content of phenols is (8-15) wt%.

The invention also provides application of the reduction method in the antibiotic resistance gene contaminated soil, and specifically at least one of the wood vinegar stock solution WV, the rectification component F1, the rectification component F2 and the rectification component F3 is applied to the antibiotic resistance gene contaminated soil as a soil conditioner.

Preferably, the experimental methods applied include:

firstly, using vegetables as tested plants, adding wood vinegar stock solution WV and rectification components F1-F3 thereof into potting soil in proportion, collecting rhizosphere soil and non-rhizosphere soil after culturing for 65d, secondly, analyzing the correlation between the ARGs and the MGEs by measuring the abundance of the ARGs and Mobile Genetic Elements (MGEs) of the rhizosphere soil and the non-rhizosphere soil of different treatment groups, thirdly, comparing the difference of the changes of the ARGs of the rhizosphere soil and the non-rhizosphere soil added with the wood vinegar stock solution WV, and fourthly, obtaining the reduction evaluation of the soil ARGs by the WV and the rectification components thereof.

More preferably, in step ①, the wood vinegar stock solution WV and its distillation components F1-F3 are added in the ratio of WV (140-^-1mu.L kg of the rectification component F1(100-120)^-1mu.L kg of the rectification component F2(120-130)^-1The rectification component F3(10-30) is mu L kg^-1The diluted (200-500) times is added into soil; and proportionally adding the mixture for three times respectively according to the proportion of 15 days before sowing, 15 days after sowing and 30 days after sowing.

More preferably, in step (i), rhizosphere bags are used to distinguish rhizosphere from non-rhizosphere soil, and the steps are as follows: a60-mesh nylon screen is used for manufacturing a cylindrical bag with the diameter of 10cm and the height of 9cm and an opening at one end to serve as a rhizosphere bag, air-dried soil is filled for about 500g and placed into a flowerpot, and then the remaining soil is filled into the flowerpot to keep the height of the soil in the rhizosphere bag consistent with that of the flowerpot soil.

Preferably, the absolute abundance of total ARGs in rhizosphere and non-rhizosphere soil is reduced by using wood vinegar stock solution WV and rectification component F3.

More preferably, the formula for calculating the absolute abundance of ARGs is:

in the formula, C₀Represents the concentration of plasmid DNA in ng/. mu.L; x represents the fragment length of the target gene in bp.

Preferably, wood vinegar stock solution WV and rectification components F2 and F3 thereof are used for treating and reducing the absolute abundance of sulfonamides, aminoglycosides and macrolide ARGs in rhizosphere and non-rhizosphere soil; the wood vinegar stock solution WV and rectification components F1 and F2 thereof are used for treating and reducing the absolute abundance of tetO-type ARGs in rhizosphere and non-rhizosphere soil; and (3) treating and removing MGEs in rhizosphere and non-rhizosphere soil by using wood vinegar stock solution WV and a rectification component F3 thereof.

Further, the total ARGs preferably specifically include tetO, sul1, sul2, ermF, ermB, aadA5, and strA; MGEs preferably include intI1 and Tn 916/1545.

The comprehensive effects brought by the invention comprise:

the wood vinegar contains a large amount of phenols and organic acid compounds, and can inhibit the reproduction and activity of pathogenic microorganisms; when high-concentration pyroligneous liquor is added, the quantity of phenols and organic acid compounds is large, the toxicity and inhibition on microorganisms are strong, and the reproduction and the biological activity of the microorganisms are selectively inhibited, so that specific antibiotic resistance genes are reduced. Secondly, the wood vinegar stock solution and the specific rectification components can reduce the absolute abundance of total ARGs, and the reduction application of each rectification component has obvious selectivity, thereby providing conditions for manually and selectively removing specific resistance genes in specific environments and further avoiding the generation of ultra-antibacterial agents. Thirdly, discloses the selective application of the wood vinegar stock solution and the corresponding genes of the rectification components thereof and a preliminary principle thereof, and lays a foundation for the artificial synthesis or the combination of the non-natural wood vinegar resistance gene cutting agent and the industrial production thereof.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

In the drawings:

FIG. 1 is a photograph showing wood vinegar stock solution WV and its rectification components F1-F3 in accordance with an embodiment 1 of the present invention, using a method for reducing contamination of soil with antibiotic resistance genes by wood vinegar;

FIG. 2 is the pH (a) and density (b) of wood vinegar stock solution WV and its distillation components F1-F3 in the method for reducing the contamination of soil with antibiotic resistance genes by wood vinegar in example 1 of the present invention;

FIG. 3 is an FTIR spectrum of wood vinegar stock solution WV and its distillation components F1-F3 of the method for reducing the contamination of soil with antibiotic resistance gene by wood vinegar in example 1 of the present invention;

FIG. 4 is a graph showing the bacteriostatic effects of wood vinegar stock solution WV and its rectification components F1-F3 on Escherichia coli NK5449 in example 1 of the present invention using a method for reducing the contamination of wood vinegar with antibiotic resistance genes in soil, wherein (a) 100% WV-3.75% WV; (b) 100% F1-3.75% F1; (c) 100% F2-3.75% F2; (d) 100% F3-3.75% F3;

FIG. 5 is a diagram showing the bacteriostatic effects of wood vinegar stock solution WV and its rectification components F1-F3 on Escherichia coli HB101 in example 1 of the present invention, wherein (a) 100% WV-3.75% WV, using a method for reducing the contamination of wood vinegar with antibiotic resistance genes in soil; (b) 100% F1-3.75% F1; (c) 100% F2-3.75% F2; (d) 100% F3-3.75% F3;

FIG. 6 shows the bacteriostatic rate of wood vinegar stock solution WV and its rectification components F1-F3 against Escherichia coli NK5449(a) and HB101(b) in the method for reducing the contamination of soil with antibiotic resistance gene by wood vinegar in example 1 of the present invention.

FIG. 7 is a method for reducing the contamination of the antibiotic resistance genes in the soil by the wood vinegar in example 1 of the present invention, and the wood vinegar stock solution WV and its rectification components F1-F3 have absolute abundance (unit copies g) of total ARGs in the rhizosphere soil (a) and the non-rhizosphere soil (b) in the application of the wood vinegar in the soil contaminated by the antibiotic resistance genes^-1) The influence of (a);

FIG. 8 is a method for reducing the contamination of the antibiotic resistance genes in the soil by the wood vinegar in example 1 of the present invention, and in the application of the wood vinegar stock solution WV and its rectification components F1-F3 to the absolute abundance (unit of copies g) of each ARGs in the soil contaminated by the antibiotic resistance genes^-1) Wherein the ARGs are sul1, sul2, ermF, ermB, aadA5, strA and tetO in this order;

FIG. 9 is a method for reducing the contamination of the antibiotic resistance genes in the soil by the wood vinegar in example 1 of the present invention, and in the application of the wood vinegar stock solution WV and its rectification components F1-F3 to the absolute abundance of soil MGEs (unit copies g) in the soil contaminated by the antibiotic resistance genes^-1) The influence of (c).

FIG. 10 is a block diagram of the technical scheme of the method for reducing the contamination of the antibiotic resistance genes in the soil by the wood vinegar and the application of the method in the soil contaminated by the antibiotic resistance genes in example 1 of the present invention.

Detailed Description

HGT mainly comprises three pathways: transformation (Transformation), Transduction (Transduction), and Conjugation (Conjugation). Transformation means that foreign DNA fragments present in the environment are taken up by the microorganism and integrated into the genome of the microorganism by homologous or abnormal recombination. Transduction is the process by which a bacteriophage injects DNA into a bacterial cell, binding to the recipient cell DNA. Conjugation is the transfer of a plasmid or a Mobile Genetic Element (MGEs) encoding resistance between cells. Transduction or conjugation of MGEs carrying ARGs is the main process of the present invention for the reduction of HGT in soil. MGEs include plasmids (plasmids), Transposons (Transposons), integrants (Integron), and transducible phages, among others. Plasmids are a class of self-replicating, double-stranded, extra-chromosomal genetic elements, typically encoding an optional functional helper gene, that confer properties on the host that enhance its adaptability to adverse conditions.

Wood Vinegar (WV) is a by-product in the process of preparing charcoal, and has a variety of effects of promoting plant growth, inhibiting bacteria, weeding, preventing corrosion and the like, and is widely used as a soil conditioner. However, the mechanism by which WV can reduce the pollution of ARGs in soil and the mechanism thereof have not been clarified in the prior art. Therefore, as shown in fig. 1, the invention prepares WV from waste wood, refines the WV by atmospheric distillation to obtain 3 different rectification components (F1, F2 and F3), compares and analyzes the differences of the physicochemical properties of the WV and its rectification components, particularly illustrates the influence of the differences on the abundance of typical ARGs and mobile genetic elements in soil by applying to the potting experiment of the contaminated soil, and applies to WV and its rectification components to reduce the ARGs contamination by using the relationship and action mechanism between the microbial community structure and soil physicochemical properties and the types and abundances of ARGs. The invention is beneficial to expanding the application of WV in agriculture and lays a foundation for establishing the soil ARGs resistance control technology based on WV.

The invention is further explained below with reference to the drawings and the specific examples of the specification, but it is to be understood that the scope of the invention is not limited thereto.

Examples 1 to 4

A method for reducing the pollution of antibiotic resistance genes by adopting wood vinegar, wherein the preparation of the wood vinegar comprises the following steps:

1) preparing wood vinegar stock solution by an anaerobic slow pyrolysis method: drying the plant raw materials to constant weight, pyrolyzing the plant raw materials by a pyrolysis carbonization furnace to prepare wood vinegar liquid, wherein the pyrolysis temperature is 450 ℃, the pyrolysis time is 6 hours, gas generated in the pyrolysis process is condensed to obtain brownish black liquid with pungent smell, then standing for 6 months in the dark, and filtering the intermediate liquid through a 0.22 mu m microporous filter membrane to obtain wood vinegar stock solution WV;

The obtained wood vinegar stock solution WV, the rectification component F1, the rectification component F2 and the rectification component F3 are respectively taken as example 1, example 2, example 3 and example 4 of the invention for property characterization.

The wood vinegar stock solution and the rectification component F1-F3 thereof have the following characteristic properties:

the property change before and after WV refining is analyzed by measuring the pH, the density, the surface functional group and the chemical composition of the WV and the rectification component thereof, and the specific measuring method is as follows:

(1) pH: measured directly with a pH meter (AB150, Thermo Fisher Scientific, USA).

(2) Density: and (4) measuring by a pycnometer method. The original weight (M1) of a 1mL volumetric flask and the weight (M2) of the sample after the WV or the rectification component is added are respectively weighed, and the sample density is determined by dividing the difference between the front and back masses (equal to the mass of the sample) of M1 and M2 by the volume of the volume.

(3) ATR-FTIR Spectroscopy: measured using a Fourier Infrared spectrometer (Tensor 27, Bruker, Germany). WV or its rectification component (10 μ L) was coated evenly on ATR wafers with a pipette gun and after drying out scanning was started. The instrument parameters are as follows: scanning area 4000-^-150 scans with a resolution of 4cm^-1。

Respectively applying the wood vinegar stock solution and the refined distillation component to a bacteria solution culture medium, measuring the bacteriostasis of various wood vinegar solutions, and calculating the bacteriostasis rate according to the following formula:

Test strains: coli HB101(E.coli HB101) and Escherichia coli NK5449(E.coli NK5449) are a pair of donor bacteria for conjugal transfer studies which are currently widely commercially available, and the strains of this example are purchased from Shanghai Dingqing Biotech Co., Ltd. Stored in a-80 ℃ freezer and activated by transferring to fresh medium before use.

The detection results and properties of various types of pyroligneous liquor are as follows:

the apparent morphology of WV and its rectification components is shown in fig. 1. WV is a dark brown liquid, with lower transmittance; the rectification components F1, F2 and F3 are yellow brown liquid which is relatively clear; and the color of the rectification component is gradually deepened along with the increase of the rectification temperature, and the rectification component is changed from light yellow to light brown. In addition, the smoke smell of WV is stronger, and the smoke smell is weakened after refining. Mainly because part of impurities, tar and other oily components in the WV are removed by distillation.

The pH value is an important index for evaluating the WV, and the acidity and the alkalinity of the WV can be visually judged. As shown in FIG. 2(a), the pH of WV is 2.81, the pH of the rectification components F1-F3 is between 1.81-2.36, both WV and its rectification components are strongly acidic, the pH of the rectification components is significantly lower than WV, and the pH of F3 is the lowest. This is because WV is an unstable organic mixture rich in organic acids and phenols, and as the temperature increases, the moisture content in WV decreases and the organic acid component gradually separates out.

The densities of WV and the rectification component are shown in FIG. 2 (b). The density of WV was 1.08g mL^-1The densities of the rectification components F1, F2 and F3 are 0.99g mL respectively^-1、1.06g mL^-1And 1.05g mL^-1The densities of the distillate components are all significantly lower than WV. In addition, except for F1, WV, F2 and F3 have higher density than water, because the density of components such as tar in WV is higher, partial tar is removed after refining and partial macromolecular substances are decomposed, so that the density of the rectified component is reducedAnd most of distilled water at 0-98 ℃ is water and some small molecular substances, so that the density of F2 is minimum.

The ATR-FTIR spectra of WV and its rectified fractions are shown in fig. 3. It can be seen that WV contains abundant oxygen-containing functional groups and carbon-carbon double bonds as well as aromatic groups, and the main absorption peak positions are: 3359cm^-1(O-H)、1665cm^-1(ester group C ═ O), 1380-^-1(C-O/C-O-C，C＝C)，1088-1014cm^-1(aliphatic C-O/C-O-C), 604cm^-1(aromatic C-H). These results indicate that WV contains organic acids, alcohols, phenols, and aromatics. In addition, there was essentially no change in the functional groups of the rectification components from F1 to F3 as compared to WV, indicating that there was no significant change in the type of WV compound before and after purification.

Full component analysis of wood vinegar stock solution and rectification components F1-F3 thereof

The WV and the rectification component thereof are subjected to component analysis according to GC-MS measurement, and then the relative abundance area is obtained by dividing the area of each peak spectrum by the total area of all peaks so as to calculate the relative content of each substance.

Chemical component analysis method: extracting with dichloromethane, concentrating, and measuring by high performance gas chromatography-mass spectrometry (GCMS, Shimadzu, Japan) by the following method: 2mL of WV was taken, an equal volume of dichloromethane was added, vortexed for 10min, allowed to stand for 10min, and the organic phase and the remaining aqueous phase were collected separately. This procedure was repeated four times, the organic phase was collected four times and the remaining aqueous phase was concentrated using a PPL column and eluted with 10mL of dichloromethane. Mixing the water phase eluent and the organic phase obtained by the extraction, concentrating to 2mL by using a rotary evaporator, blowing nitrogen to be nearly dry, transferring to a sample injection vial by using dichloromethane, and fixing the volume to 1 mL. The chromatographic temperature rise conditions are as follows: keeping the temperature at 50 ℃ for 3min, heating to 200 ℃ at 4 ℃/min, and keeping the temperature at 200 ℃ for 10 min. The mass spectrometer was equipped with an electron bombardment source with 70eV ionization energy and an ion source temperature of 250 ℃ set to scan from 45 to 500Da in 0.71 s. The carrier gas was helium, flow rate: 1 mL/min. The injection volume is 1 mul and the split ratio is 80: 1.

TABLE 1 WV and relative amounts of the major components in the rectified fractions thereof

Note: "nd" indicates no detection.

The relative contents of the main components in WV and its rectified components are shown in table 1. The WV and the components F1, F2 and F3 detect 41, 35, 41 and 43 compounds respectively, mainly comprise 9 compounds such as organic acids, phenols, ketones, furans, aromatic ethers, aldehydes, esters, alcohols, saccharides and the like, and main functional groups are methoxy, hydroxyl and carbonyl. Wherein, 52.6 percent to 81.8 percent of organic acid and phenols are main components. However, the relative abundance of each component is not as different as compared to the prior art, depending on the preparation raw materials and conditions. The organic acid and the phenols are effective components for inhibiting bacteria of the wood vinegar. Compared with the wood vinegar stock solution WV, the organic acid content of the rectification component F1 is reduced, and the organic acid content of F2 and F3 is obviously increased. The content of the rectification component is reduced for the phenolic substances. The change of the total abundance of the organic acid and the phenols is F3> WV > F2> F1, so the component F3 has better bacteriostasis. In addition, the rectification component F3 has lower furan substance content and higher safety.

Wood vinegar stock solution WV and antibacterial property of rectification component F1-F3 thereof

The bacteriostatic effect of wood vinegar stock solution WV and its rectification components on Escherichia coli HB101 and Escherichia coli NK5449 is shown in FIG. 4-2-5. As can be seen from the figure, WV and its rectification component have inhibition effect on the growth of Escherichia coli NK5449H and HB101, and the diameter of inhibition zone decreases with the concentration of WV and rectification component. The bacteriostatic rates of WV and its rectified fractions on two escherichia coli are shown in fig. 6. With the increase of WV and the concentration of the rectification component, the bacteriostasis rates of Escherichia coli NK5449H and HB101 are constantly increased, and the bacteriostasis effect is gradually enhanced. For E.coli NK5449, bacteriostatic effect: WV > F3> F2> F1; and the bacteriostasis rates of the WV and the rectification component with the concentration of 100 percent and 50 percent are obviously higher than those of other concentrations, and the bacteriostasis rates of 100 percent WV and 100 percent F3 are respectively as high as 45.9 percent and 43.1 percent. For E.coli HB101, the bacteriostatic effect was: f3> WV > F2> F1; similarly, when the concentration of WV and the concentration of the rectification component are more than 25.0%, the bacteriostasis rate is obviously increased, and the bacteriostasis rate of 100% WV and 100% F3 is respectively as high as 46.8% and 50.3%.

In conclusion, the wood vinegar stock solution WV and the rectification component F3 have the best bacteriostatic effect. In addition, organic acids and phenols in WV have inhibitory effect on pathogenic bacteria and fungi. The GC-MS result shows that more than 52% of WV and rectification components are organic acid and phenolic compounds, wherein 2 main components: 2, 6-dimethoxyphenol and 2-methoxyphenol have antibacterial activity. Thus, the bacteriostatic activity of WV and its rectification components is a result of the combined action of the phenolic substances and the organic acid.

Examples 5 to 8

In the series of embodiments, the wood vinegar stock solution WV obtained by the reduction method of the embodiment 1-4 and the rectification component F1-F3 thereof are adopted to illustrate the application of the wood vinegar stock solution in the soil polluted by antibiotic resistance genes, and the application method comprises the following steps:

firstly, rape (Brassica napus L.) is taken as a tested plant, wood vinegar stock solution WV and a rectification component F1-F3 thereof are added to potting soil in proportion, rhizosphere and non-rhizosphere soil is collected after 65d of culture, secondly, the difference of the change of the rhizosphere and non-rhizosphere soil ARGs after the wood vinegar stock solution WV is added is compared by measuring the abundance of the rhizosphere and non-rhizosphere soil ARGs and MGEs of different treatment groups, analyzing the correlation between the ARGs and MGEs, and obtaining the reduction evaluation of the WV and the rectification component thereof on the soil ARGs.

Test soil: the experiment is collected in a Tokyanlan Zhenyuan pig farm (120.35 DEG E,36.60 DEG N) in Shandong, namely Mo city in 8 months in 2017. The plant has been built for 15 years, and 100 pigs are available. Pig manure is typically used for anaerobic fermentation in biogas digesters, for open-air stacking or for direct application to surrounding soil. Collecting surface soil 0-20cm above ground, mixing at multiple points, sampling, air drying, and sieving with 2mm sieve.

The basic chemistry of the soil is as follows: pH 6.68, maximum water holding capacity 42.3%, organic carbon (SOC) content 22.6 kg^-11.65g kg of Available Phosphorus (AP)^-1Effective Nitrogen (AN) content 21.1 μ g g^-10.42g kg of available zinc (AZn)^-148mg kg of available copper (ACu)^-1169mg kg of available lead (APb)^-1。

The test plants: the test plant is rape (Brassica napus L.), which is a cruciferous plant originally produced in China and mainly distributed in Anhui, Henan, Sichuan and the like. The vegetable is rich in nutrition and high in vitamin C content, and is one of the vegetables which are frequently eaten by people. Rape seeds were purchased from fuxin agro-seed shop, Qingdao, Shandong province.

Pot experiment: resin plastic flowerpots with the diameter of 12.5cm and the height of 16.5cm are adopted, and 3kg of soil is filled in each flowerpot. To study the effect of rhizosphere secretions on soil ARGs abundance, rhizosphere bags were used to distinguish between rhizosphere and non-rhizosphere soils. The method comprises the following specific steps:

a60-mesh nylon screen is used for manufacturing a cylindrical bag with the diameter of 10cm and the height of 9cm and an opening at one end to serve as a rhizosphere bag, air-dried soil is filled into the bag, about 500g of the air-dried soil is put into a flowerpot, and then the remaining soil is put into the flowerpot to keep the height of the soil in the rhizosphere bag consistent with that of the flowerpot soil. Then adding 167 microliter kg of WV and rectification components thereof^-1The rectification component F1113 mu L kg^-1The rectified component F2123 mu L kg^-1The rectification component F313.3 mu L kg^-1The ratio of (1) to (2) is diluted by 300 times and added to soil, and is respectively marked as treatment groups WV, F1, F2 and F3, and a control group is marked as CK without adding any wood vinegar component. And (5) sowing rape after standing for 72 h. 3 seeds are sowed in each pot by adopting a hole sowing mode. And respectively adding the WV or the rectification components F1-F3 into the rape seeds 15d and 30d after the rape seeds are sown according to the proportion. Each process set 5 replicates. The experiment adopts a weighing method to water, and the maximum field water capacity of the soil with the water content of 60 wt% is obtained. The placement position of the potted plant is changed regularly, and the system error caused by illumination is reduced as much as possible.

Collecting samples: after 65 days of cultivation, plant and soil samples were collected. The rape is respectively collected at the upper part and the lower part of the root, and the fresh weight of the rape is measured. The soil sample is divided into rhizosphere soil and non-rhizosphere soil which are collected respectively, the rhizosphere soil is the soil adhered to the roots of the rapes in the rhizosphere bag, and the non-rhizosphere soil is flowerpot soil far away from the roots outside the rhizosphere bag. Storing part of the collected soil in a sterilized centrifugal tube at-20 ℃ in a refrigerator for measuring the abundance and microbial community structure of the soil ARGs; and (3) after being air-dried, a part of soil sample is placed in a self-sealing bag for sealing and storage, and the soil sample is used for measuring the physical and chemical properties of the soil.

The present example was carried out by using conventional DNA extraction and quantitative PCR assay techniques, the target gene primer information is shown in table 2, and the specific method steps are as follows:

DNA extraction: DNA was extracted using PowerSoil DNA extraction kit (Mobio, USA), and the specific procedures were as described in the kit instructions. The extracted DNA was detected by electrophoresis on a 1% agarose gel. After electrophoresis, the electrophoresis bands were observed with a gel imaging system (azure biosystems C150, usa) and the DNA bands were single band without tailing. The concentration and purity of the DNA was then determined using a ultramicro spectrophotometer (Colibri, Shandong, China). The pure DNA sample A260/A280 is 1.8-2.0. A ratio of less than 1.8 indicates the effect of the presence of protein or phenolic material; ratios above 2.0 indicate RNA contamination.

Construction of a standard curve: the ARGs and MGEs of interest are first amplified using a common PCR instrument (Analytikjena, Germany). The reaction system, 20. mu.L in total, included: 2 × EasyTaq PCR Supermix (TransGen Biotech, Beijing) 10 μ L, 10 μ M forward and reverse primers (Shanghai, Japan) each 1 μ L, template DNA 2 μ L, and sterilized ultrapure water 6 μ L. The amplification product obtained was subjected to gel electrophoresis using 1% agarose (110V, 30min), after which the position of the target gene was observed using a gel imaging system (Azure Biosystems C150, USA), the target fragment was cut into gel, recovered and purified (TransGen Biotech, Beijing), and the concentration of the gel-recovered product was determined.

The gene fragment of interest was then ligated into pEASY-T1(TransGen Biotech, Beijing) vector, introduced into Trans1-T1 competent cells, and the competent cells were plated on LB solid medium containing X-gal and IPTG. The white single clone was then picked and inoculated with benzyl-containing ammonia (10mg mL)^-1) The LB liquid medium of (1), and streaked on an LB solid plate. And (3) culturing the strain in an LB liquid culture medium at 37 ℃ for 6-12h, sending the strain liquid to a company (living creatures, Shanghai) for sequencing, comparing a sequencing result with an NCBI database, and determining that the selected clone contains a target fragment. Then, the plasmid was extracted using a plasmid extraction kit (TransGenBiotech, Beijing), and the concentration and purity of the plasmid were measured using a ultramicro spectrophotometer (Colibri, Shandong, China). Plasmid was diluted in 10-fold gradient series of concentrations to make a standard curve. The standard curve amplification efficiency meets 90-110%.

Quantitative detection of ARGs: selecting 7 common ARGs in soil as tetO, sul1, sul2, ermF, ermB, aadA5 and strA, and 2 common MGEs as int 1 and Tn916/1545, and carrying out quantitative detection by using a real-time quantitative PCR instrument (applied biosystems, USA). The primer information of the target gene is shown in Table 2. The reaction system, 20. mu.L in total, included: mu.L of qPCR Supermix (TransGen Biotech, Beijing), 2. mu.L of DNA template, 0.5. mu.L of forward and reverse primers (10. mu.M, Biotech, China), and 7. mu.L of sterilized ultrapure water were all commercially available. In addition, the presence or absence of non-specific amplification was checked by dissolution curve conditions.

TABLE 2 primer information for target genes

Influence of wood vinegar stock solution and rectification components thereof on abundance of soil ARGs

The abundance changes of the total ARGs of the soil under the treatment of the wood vinegar stock solution WV and the rectification components thereof are shown in figure 7, and the absolute abundances of the total ARGs of the rhizosphere soil and the non-rhizosphere soil of the control group are respectively 1.14 × 10⁸And 7.29 × 10⁷copies g^-1Absolute abundance of various ARGs is between 10⁵-10⁷copies g^-1. The two ARGs with the highest relative abundance are sul1 and ermF, the two genes in the F3 treatment group account for 60.8 percent and 56.0 percent of the total ARGs in rhizosphere and non-rhizosphere soil respectively, and the ratio of the two genes in the WV treatment group and the F1 and F2 treatment groups is more than 88.0 percent.

Compared with a control group, the abundance of total ARGs in rhizosphere soil is reduced by the treatment of WV, F2 and F3, the reduction effect of F3 is the best, and the removal rate of the total ARGs in the rhizosphere soil reaches 83.8%; secondly, WV reduces the abundance of total ARGs in rhizosphere soil by 75.5 percent after treatment; f2 had the weakest effect of reducing total ARGs in rhizosphere soil, and the removal rate was 50.5%. The abundance of total ARGs in rhizosphere soil is increased by 25.5% after F1 is added, which is related to the increase of the abundance of most ARGs (sul1, sul2, ermF, ermB, aadA5, andstrA) after F1 is added.

The change trend of the total ARGs of the non-rhizosphere soil of different treatment groups is the same as that of the rhizosphere soil, the total ARGs of the F1 treatment group are increased by 50.4%, the abundance of the total ARGs of the non-rhizosphere soil is reduced by the treatment of WV, F2 and F3, and the removal effect is F3 (79.7%) > WV (65.3%) > F2 (19.2%).

In conclusion, WV and F3 both had a better reduction effect (removal rate > 65%) on total ARGs in rhizospheric and non-rhizospheric soils, which is consistent with the above bacteriostatic data, and therefore, WV and F3 reduced the number of potential host bacteria in ARGs in soils was part of the reason for the reduced abundance of ARGs. And the rectification component in a specific temperature range does not influence the survival of part of host bacteria, so that the abundance of the ARGs corresponding to the flora is increased.

In particular, sulfonamides, aminoglycosides and macrolides ARGs have a similar tendency to change under the influence of WV and its rectification components as total ARGs. The absolute abundance of ARGs in rhizosphere and non-rhizosphere soils was significantly reduced by WV and F2, F3 treatments (19.8% and 2.56% for the absolute content of rhizosphere and non-rhizosphere soil ermF, respectively, and 36.8% for the absolute content of non-rhizosphere soil strA) for the F2 treatment group, while the absolute abundance of ARGs in rhizosphere and non-rhizosphere soil was significantly increased by the rectification component F1 treatment (39.2% for the absolute content of rhizosphere soil ermB, respectively, for the F1 treatment group).

The effect of WV and its rectification components on tetO is different from that of other 6 ARGs. The WV treatment, the F1 treatment and the F2 treatment obviously reduce the absolute abundance of tetO in rhizosphere soil, the removal rate is WV (58.8%) > F2 (55.5%) > F1 (37.8%), and the reduction effect of WV on tetO in rhizosphere soil is the best. For non-rhizosphere soil, only treatment with WV and F2 reduced the absolute abundance of its tetO, and the removal rates were only 2.27% and 14.4%, respectively, indicating that WV and F2 had better effect on the reduction of the tetO in rhizosphere soil.

Furthermore, the abundance of both rhizospheric and non-rhizospheric soil tetO was significantly increased in the F3-treated group, probably because component F3, which is the most acidic, lowered soil pH early in the addition, and further affected the soil microbial community. The abundance of the tetracyclines ARGs (tetO, tetC, tetQ, tetX) increases under acidic conditions, since the acidic pH favors the propagation of tetracycline-resistant bacteria such as Proteobacteria (Proteobacteria), calidisceria and Firmicutes (Firmicutes), whereas the increase in abundance of the F3-treated group soil Proteobacteria leads to an increase in potential hosts.

Influence of pyroligneous liquor and rectification components thereof on abundance of MGEs in soil

The effect of WV and its rectified fractions on the abundance of MGEs in soil is shown in FIG. 9 the absolute abundance of control groups intI1 and Tn916/1545 in rhizospheric and non-rhizospheric soils, respectively, is 3.84 × 10⁷、2.63×10⁷、5.70×10⁷、3.75×10⁷copiesg^-1Higher MGEs content in rhizosphere soil indicates that the rhizosphere is a hot spot of gene HGT because root exudates increase the relative abundance and metabolic activity of some bacteria and often the cell density is higher near the plant root. After WV and the rectification components thereof are added, the absolute abundance of the intI1 of rhizosphere and non-rhizosphere soil is respectively reduced by 32.6-72.1% and 6.19-68.8%, and the reduction effect of F1 on the intI1 is the worst. Similarly, treatment of WV and its rectification components reduced the absolute abundance of rhizosphere and non-rhizosphere soils Tn916/1545, with removal rates of rhizosphere and non-rhizosphere soils 28.2% -71.2% and 7.06% -47.9%, respectively. Among them, the removal rate of F1 was the lowest, and F3 was the next. In conclusion, the WV and the rectification component thereof can effectively remove MGEs in the soil

Therefore, the preferred ARGs reduction schemes in this embodiment are: the wood vinegar stock solution WV and rectification components F2 and F3 thereof are used for treating and reducing the absolute abundance of sulfonamides, aminoglycosides and macrolide ARGs in rhizosphere and non-rhizosphere soil; the wood vinegar stock solution WV and rectification components F1 and F2 thereof are used for treating and reducing the absolute abundance of tetO-type ARGs in rhizosphere and non-rhizosphere soil; and (3) treating and removing MGEs in rhizosphere and non-rhizosphere soil by using wood vinegar stock solution WV and a rectification component F3 thereof.

Correlation analysis of ARGs and MGEs

HGT of ARGs in environmental microbial communities is an important pathway for ATs resistance transmission in agricultural soils. Because the abundance of the ARGs and the abundance of the MGEs are obviously related in the long-term nickel-polluted agricultural soil, the increase of the nickel-polluted soil ARGs is probably due to the increase of the HGT frequency of the soil ARGs caused by nickel pollution. As shown in table 3-1, Spearman correlation analysis indicates that there is a significant positive correlation between the abundance of int 1 and the abundances of sulfonamides, macrolides and aminoglycosides (r ═ 0.783-0.864, P <0.01), and there is also a positive correlation between the abundance of Tn916/1545 and the abundances of these several ARGs, but the correlation is low (r ═ 0.572-0.810, P <0.01) because the proportion of int 1 in MGEs is high (more than 80%), and the abundance of Tn916/1545 is low. In addition, neither MGEs was detected to be associated with the tetracycline ARGs (tetO) (P >0.05), but because tetO is less abundant relative to total ARGs, there was also a significant association between MGEs and total ARGs (r ═ 0.810, P <0.01), indicating that soil ARGs are significantly associated with MGEs. Therefore, ARGs cause resistance exchange and spread between soil bacteria through HGT by means of plasmids, transposons, and other MGEs. However, the HGT frequency of soil ARGs is affected by many abiotic factors (e.g., temperature, soil pH, nutrients, soil type, etc.) and biological factors, which together with MGEs determine the actual frequency of HGT in complex soil environments.

Although the present invention has been described in detail, modifications within the spirit and scope of the invention will be apparent to those skilled in the art. Further, it should be understood that the various aspects recited herein, portions of different embodiments, and various features recited may be combined or interchanged either in whole or in part. In the various embodiments described above, those embodiments that refer to another embodiment may be combined with other embodiments as appropriate, as will be appreciated by those skilled in the art. Furthermore, those skilled in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention.

Claims

1. An application of wood vinegar in the reduction of the soil polluted by antibiotic resistance genes is characterized in that,

the preparation method of the wood vinegar comprises the following steps:

1) preparing wood vinegar stock solution WV by an anaerobic slow pyrolysis method: drying the plant raw materials to constant weight, pyrolyzing the plant raw materials by a pyrolysis carbonization furnace to prepare wood vinegar liquid, wherein the pyrolysis temperature is 350-550 ℃, the pyrolysis time is 3-6 h, gas generated in the pyrolysis process is condensed to obtain brownish black liquid with pungent smell, then standing for 3-6 months in the dark, and filtering the intermediate liquid with a microporous filter membrane to obtain wood vinegar stock solution WV;

2) refining wood vinegar stock solution WV by a normal pressure distillation method: setting a 15-98 ℃ low-temperature section under the normal pressure state, heating a distillation device, collecting a product of the temperature section, and taking a distillation component F1 with the distillation component being 15-98 ℃ low-temperature section and taking a wood vinegar stock solution WV or a distillation component F1 as required wood vinegar liquid respectively;

specifically, the wood vinegar stock solution WV or the rectification component F1 is respectively used as a soil conditioner to be applied to the soil polluted by antibiotic resistance genes;

wherein, the wood vinegar stock solution WV is used for treating and reducing the absolute abundance of sulfanilamide, aminoglycoside or macrolide antibiotic resistance genes sul1, sul2, ermF, ermB, aadA5 or strA in rhizosphere and non-rhizosphere soil; the wood vinegar stock solution WV or the rectification component F1 is used for treating and reducing the absolute abundance of tetO antibiotic resistance genes in rhizosphere and non-rhizosphere soil; applying wood vinegar stock solution WV to treat and remove mobile genetic elements of rhizosphere and non-rhizosphere soil;

the mobile genetic elements were either intI1 or Tn 916/1545.

2. The use of claim 1, wherein the wood vinegar stock solution WV and the refined fraction F1 are applied to a culture medium of wood vinegar, and the bacteriostatic activity of each wood vinegar is measured, and the bacteriostatic rate is calculated according to the following formula:

the bacteriostatic rate (%) (diameter of bacteriostatic circle of treatment group-diameter of bacteriostatic circle of control group)/diameter of bacteriostatic circle of treatment group x 100%; and/or

And (3) respectively carrying out component analysis on the wood vinegar stock solution WV and the rectification component F1 thereof through high performance gas chromatography-mass spectrometry, and then dividing the area of each peak spectrum by the total area of all peaks to obtain the relative abundance area and calculate the relative content of each substance.

3. The use of claim 1, wherein the wood vinegar stock solution WV and its rectification component F1 have a total organic acid and phenol content of 50-85 wt%; in the wood vinegar stock solution WV, the content of organic acid is 30-50 wt%, and the content of phenols is 30-50 wt%; in the rectification component F1, the content of organic acid is 25 wt% -30 wt%, and the content of phenols is 20 wt% -27 wt%.

4. The use according to claim 1, wherein the experimental method comprises:

firstly, using vegetables as tested plants, adding wood vinegar stock solution WV and rectification components F1 thereof to potted soil according to a proportion, collecting rhizosphere soil and non-rhizosphere soil after culturing for 65d, analyzing the correlation between antibiotic resistance genes and mobile genetic elements by measuring the abundance of the antibiotic resistance genes and the mobile genetic elements of rhizosphere soil and non-rhizosphere soil of different treatment groups, and comparing the differences of the antibiotic resistance genes of rhizosphere soil and non-rhizosphere soil added with the wood vinegar stock solution WV and the rectification components F1 respectively, and finally obtaining the evaluation of soil antibiotic resistance genes of the wood vinegar stock solution WV and the rectification components F1 thereof on reduction.

5. The use as claimed in claim 4, wherein in step ①, the wood vinegar stock solution WV and its rectification component F1 are added in a ratio of 140 μ L kg of the wood vinegar stock solution WV^-1-180μL kg^-1The rectified component F1100 mu L kg^-1-120μL kg^-1Diluting by 200-500 times, and adding the diluted solution into soil; and proportionally adding the mixture for three times respectively according to the proportion of 15 days before sowing, 15 days after sowing and 30 days after sowing.