CN112997777A - Method for preventing and controlling antibiotic resistance genes from entering plant leaves by using carbon-based material and effect evaluation method thereof - Google Patents

Method for preventing and controlling antibiotic resistance genes from entering plant leaves by using carbon-based material and effect evaluation method thereof Download PDF

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CN112997777A
CN112997777A CN202110259591.4A CN202110259591A CN112997777A CN 112997777 A CN112997777 A CN 112997777A CN 202110259591 A CN202110259591 A CN 202110259591A CN 112997777 A CN112997777 A CN 112997777A
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CN112997777B (en
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王芳
梅芝
付玉豪
卞永荣
蒋新
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Abstract

The invention discloses a method for preventing and controlling an antibiotic resistance gene from entering plant leaves by using a carbon-based material and an effect evaluation method thereof, belonging to the technical field of resistance control genes. The method comprises the steps of utilizing a carbon-based material to prevent and control antibiotic resistance genes from entering plant leaves, attaching the gene resistance control material to the plant leaves to treat the plant leaves; the gene control material comprises a carbon-based porous material A and a sterilization material B; the porosity or comparative area of the carbon-based porous material A is 0.7m2/g～0.9m2(ii)/g; the sterilization material B is quaternary phosphonium salt; the bactericidal material B is loaded on the carbon-based porous material A, and the loading rate of the bactericidal material B is 9.7-10.3%. The invention can make the antibiotic resistance gene in the environment adsorbed by the gene resistance material before entering the leaf, and the bacterial containing the intracellular antibiotic resistance gene can be killed by the gene resistance material and changed into extracellular antibioticThe antibiotic resistance gene is adsorbed by the gene control material and is blocked outside the plant leaves.

Description

Method for preventing and controlling antibiotic resistance genes from entering plant leaves by using carbon-based material and effect evaluation method thereof
Technical Field
The invention belongs to the technical field of resistance control genes, and particularly relates to a method for preventing and controlling an antibiotic resistance gene from entering plant leaves by using a carbon-based material and an effect evaluation method thereof.
Background
Soil is an important repository for antibiotic resistance genes in the environment. Resistance of resistant bacteria to antibiotics is derived from resistance genes located on chromosomes or mobile genetic elements. Unlike traditional chemical pollutants, Antibiotic Resistance Genes (ARGs) can be transferred and spread among the same or even different bacteria, affecting the therapeutic effect of antibiotics, and have been classified as a new type of environmental pollutants. Manure farming may be an important route for ARGs to enter the soil environment. The antibiotics are widely applied in animal husbandry and breeding industry, and researches show that 40% -90% of the antibiotics for animals are discharged from animal excrement in the form of original compounds or metabolites with biological activity; in addition, the application of antibiotics can also lead to the accumulation of abundant ARGs in the intestinal tract of animals.
Plant leaves are an important place for the communication between soil microorganisms and plant microorganisms, and leaf microorganisms are often exposed to the environment with severe fluctuation and have lower diversity than soil microorganisms and rhizosphere microorganisms. Both irrigation and soil dusting can cause a portion of the soil microorganisms to inhabit the plant leaves. Meanwhile, soil microorganisms also migrate to leaves along roots and stems of plants. Leaf microorganisms are thought to be the vehicle for plant interaction with the atmospheric environment and also to interact intimately with the human microflora. Studies have found that the abundance of ARG in vegetables grown in soil treated with manure rich in antibiotic resistance genes is significantly increased, and previous studies have shown that this is probably due to the fact that ARGs in soil diffuse into the air and then deposit on the leaf surface, and that a large amount of ARG can be detected in urban air.
However, the existing resistance control technologies mostly focus on reducing the abundance of antibiotic resistance genes in soil and water environments, and few researches focus on the introduction of antibiotic resistance genes into plants from the air and resistance control technologies thereof. Therefore, there is a need to invent a technology capable of controlling antibiotic resistance genes in plant leaves, so as to reduce the risk of the antibiotic resistance genes entering the food chain through leaf vegetables.
Disclosure of Invention
1. Problems to be solved
Aiming at the problem that the antibiotic resistance gene is easy to enter plant leaves from the air and further enter a food chain in the prior art, the invention provides a method for preventing and controlling the antibiotic resistance gene from entering the plant leaves by utilizing a carbon-based material and an effect evaluation method thereof; through rationally setting up the gene and hindering accuse material to the gene hinders accuse material and handles plant leaf, thereby effectively solves the antibiotic resistance gene and gets into plant leaf, and then gets into the problem of food chain from the air easily.
2. Technical scheme
In order to solve the problems, the technical scheme adopted by the invention is as follows:
the invention relates to a method for preventing and controlling antibiotic resistance genes from entering plant leaves by utilizing carbon-based materials, which is characterized in that the gene prevention and control materials are attached to the plant leaves to be processed; the gene control material comprises a carbon-based porous material A and a sterilization material B; the porosity or comparative area of the carbon-based porous material A is 0.7m2/g～0.9m2(ii)/g; the sterilization material B is quaternary phosphonium salt; the bactericidal material B is loaded on the carbon-based porous material A, and the loading rate of the bactericidal material B is 9.7-10.3%.
Preferably, the area density of the gene control material attached to the plant leaves is 195mg/m2～250mg/m2。
Preferably, the carbon-based porous material A is magnetic biomass charcoal and/or the bactericidal material B is tributyl dodecyl phosphonium bromide. The magnetic biomass charcoal is prepared by coprecipitating Fe3+And Fe2+The magnetic material is loaded on the biomass charcoal, and is convenient to recycle after being used, so that the magnetic material has the advantages of environmental protection and saving.
Preferably, the specific treatment steps are as follows:
(1) preparing a gene control material: mixing the carbon-based porous material A and the sterilization material B, carrying out hydrothermal reaction, and cooling to room temperature to obtain a gene resistance control material;
(2) preparing an adhering liquid: dispersing the gene resistance control material in the step (1) in deionized water to obtain an attachment solution;
(3) spraying treatment: and (3) spraying the adhering liquid in the step (2) on plant leaves to treat the plant leaves. The magnetic biomass carbon-quaternary phosphonium salt composite material (gene resistance control material) can slowly release the loaded quaternary phosphonium salt in aqueous solution, so that the slow release process can be generated on the surface of plant leaves as much as possible, and the resistance control efficiency of antibiotic resistance genes is improved.
Preferably, the hydrothermal reaction temperature in the step (1) is 80-90 ℃.
Preferably, the concentration of the attachment solution in the step (3) is 195 mg/L-205 mg/L, and the specific spraying rate is 0.7 mL/s-0.9 mL/s. 195-plus 205mg/L of attachment liquid is used for spraying and coating the plant leaves, so that the absolute abundance of the antibiotic resistance genes on the plant leaves can be obviously reduced, the human pathogenic bacteria on the leaves can also be inhibited, and the method is suitable for reducing the antibiotic resistance genes of the leaves when the leaf vegetables are planted in agriculture, thereby controlling the antibiotic resistance genes in the external environment to enter the plant leaves through the leaves.
According to the method for evaluating the effect of inhibiting the plant leaves from being polluted by the antibiotic resistance genes, the antibiotic resistance genes are inoculated on the plant leaves subjected to antibiotic resistance gene resistance control treatment, and DNA extraction and analysis are carried out on the inoculated plant leaves; the method for carrying out antibiotic resistance gene resistance treatment on the plant leaves is the resistance treatment method disclosed by the invention.
Preferably, the plant leaves are pakchoi leaves; wrapping the stem of the pakchoi with a preservative film before the antibiotic resistance gene control treatment is carried out on the pakchoi leaves. The stem of the pakchoi is wrapped to control the gene resistance and control material to only process the leaves of the pakchoi.
Preferably, the inoculation adopts a water-soluble fertilizer rich in antibiotic resistance genes and antibiotic resistance bacteria, and the inoculation time is 2-14 days.
Preferably, the inoculated pakchoi leaves are washed with deionized water. The cleaning with the deionized water can simulate the cleaning process of the edible pakchoi before eating, and has obvious practical significance for DNA extraction and test of the cleaned pakchoi.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention relates to a method for preventing and controlling antibiotic resistance genes from entering plant leaves by utilizing carbon-based materials, which is characterized in that the gene prevention and control materials are attached to the plant leaves to be processed; the gene control material comprises a carbon-based porous material A and a sterilization material B; the porosity or comparative area of the carbon-based porous material A is 0.7m2/g～0.9m2(ii)/g; the sterilization material B is quaternary phosphonium salt; the bactericidal material B is loaded on the carbon-based porous material A, and the loading rate of the bactericidal material B is 9.7-10.3%; through the treatment, the gene resistance control material is attached to the plant leaves, the antibiotic resistance genes in the environment can be adsorbed by the gene resistance control material before entering the leaves, further, strains containing intracellular antibiotic resistance genes can be killed by the gene resistance control material, and the intracellular antibiotic resistance genes are changed into extracellular antibiotic resistance genes, so that the intracellular antibiotic resistance genes are further adsorbed by the gene resistance control material and are blocked outside the plant leaves.
(2) According to the method for evaluating the effect of inhibiting the plant leaves from being polluted by the antibiotic resistance genes, the antibiotic resistance genes are inoculated on the plant leaves subjected to antibiotic resistance gene resistance control treatment, and DNA extraction and analysis are carried out on the inoculated plant leaves; the method for carrying out antibiotic resistance gene resistance treatment on the plant leaves is the resistance treatment method disclosed by the invention; through the tests, the Antibiotic Resistance Genes (ARGs) and the mobile genetic factors (MGEs) in the leaves of the plants which are not treated are remarkably reduced after the leaves of the plants are treated by the gene resistance control material, wherein the removal rate reaches the maximum at the 7 th day, and the removal rates of the ARGs and the MGEs can reach 93.5 percent and 93.7 percent respectively.
Drawings
FIG. 1 is a graph showing the distribution of microbial communities at the level of each phylum of bacteria in the leaf of Brassica campestris at different treatment times in the five cases of the present invention;
FIG. 2 is a Shannon index distribution of microbial communities at the level of each bacterial phylum in the leaves of the pakchoi, under the five conditions of the invention and with different treatment times;
FIG. 3 shows the surface environment scanning electron microscope results of the leaf surface of pakchoi at different processing times under five conditions according to the present invention;
FIG. 4-1 is a graph showing the absolute abundance of the total antibiotic resistance genes (ARGs + MGEs) in leaves as a function of time without washing the pakchoi in the present invention;
FIG. 4-2 shows the absolute abundance of the total antibiotic resistance genes (ARGs + MGEs) in leaves as a function of time for a treatment of a pakchoi of the present invention with deionized water washing;
FIG. 5-1 is a graph showing the absolute abundance of Antibiotic Resistance Genes (ARGs) in leaves of a pakchoi in accordance with the present invention as a function of time without washing;
FIG. 5-2 shows the absolute abundance of Mobile Genetic Elements (MGEs) in leaves over time without washing the pakchoi in the present invention;
FIG. 6-1 shows the residual rates of ARGs and MGEs on the leaf surfaces of the pakchoi rich in resistant bacteria and resistant gene water-soluble fertilizer after spraying three materials (MB, QPS and MBQ) at different time points under the condition that the pakchoi is not cleaned;
FIG. 6-2 shows the residual rates of ARGs and MGEs on the leaf surfaces of the pakchoi rich in resistant bacteria and resistant gene water-soluble fertilizer after spraying three materials (MB, QPS and MBQ) at different time points under the deionized water cleaning treatment of the pakchoi in the invention;
FIGS. 6-3 show the removal rate of ARGs and MGEs from pakchoi leaves on day 7 with and without washing after spraying three barrier materials according to the present invention;
FIG. 7-1 is a graph showing the absolute abundance of Antibiotic Resistance Genes (ARGs) in leaves as a function of time, in the deionized water washing treatment of the present invention;
FIG. 7-2 is a graph showing the absolute abundance of Mobile Genetic Elements (MGEs) in leaves as a function of time, in the deionized water washing treatment of the present invention;
FIG. 8 shows the effect of different treatment times on the relative abundance of human pathogens in pakchoi leaves in five cases of the present invention.
Detailed Description
The structure, proportion, size and the like shown in the drawings are only used for matching with the content disclosed in the specification, so that the person skilled in the art can understand and read the description, and the description is not used for limiting the limit condition of the implementation of the invention, so the method has no technical essence, and any structural modification, proportion relation change or size adjustment still falls within the scope covered by the technical content disclosed by the invention without affecting the effect and the achievable purpose of the invention. Meanwhile, the embodiments of the present invention are not independent of each other, but may be combined.
The CK in the invention is not sprayed with a gene control material and then inoculated with an antibiotic resistance gene; MB represents spraying magnetic biomass charcoal and then inoculating antibiotic resistance genes; MBQ represents spraying gene control material and then inoculating antibiotic resistance gene; QPS stands for spray-on quaternary phosphonium salt, reseeding antibiotic resistance gene.
The invention is further described with reference to specific examples.
Example 1
The embodiment provides a method for preventing and controlling an antibiotic resistance gene from entering plant leaves by using a carbon-based material, which comprises the following processing steps:
(1) preparing a gene control material: mixing magnetic biomass charcoal and quaternary phosphonium salt, carrying out hydrothermal reaction at 85 ℃, and cooling to room temperature to obtain a gene resistance control material; the loading rate of the quaternary phosphonium salt in this example was 10%;
(2) preparing an adhering liquid: dispersing the gene resistance control material in the step (1) in deionized water to obtain attachment liquid with the concentration of 200 mg/L;
(3) spraying treatment: spraying the adhering liquid obtained in the step (2) on plant leaves to treat the plant leaves, wherein the specific spraying parameters are as follows; the area density of the gene control material attached to the plant leaf in this example was 220mg/m2. Magnetic biomass charcoal-quaternary phosphonium salt complexThe composite material (gene control material) can slowly release the loaded quaternary phosphonium salt in aqueous solution, so that the slow release process can be generated on the surface of the plant leaves as much as possible, thereby improving the control efficiency of the antibiotic resistance gene.
In order to detect the resistance control treatment effect of the embodiment, the embodiment also provides an effect evaluation method for inhibiting the plant leaves from being polluted by the antibiotic resistance genes, the antibiotic resistance gene resistance control treatment is firstly carried out on the plant leaves, and the method for carrying out the antibiotic resistance gene resistance control treatment on the plant leaves is the resistance control treatment method disclosed by the invention; and then inoculating an antibiotic resistance gene on the plant leaves, extracting DNA and measuring high-flux (96 pairs of primers) qPCR (quantitative polymerase chain reaction) on the inoculated plant leaves, analyzing and comparing the inhibition control effect of different treatments on the antibiotic resistance gene of the leaves, and focusing on the absolute abundance, the relative abundance and the number of gene types of the antibiotic resistance gene and the microbial community structure of the sample.
The plant leaves in the embodiment adopt pakchoi leaves, and the specific operation steps are as follows: carrying out dark treatment on the pakchoi seeds for 7 days, transferring the pakchoi seeds to a hydroponic box filled with a Hoagland nutrient solution in an illumination incubator for 25 days, completing seedling culture of the pakchoi, and dividing the pakchoi seeds into hydroponic tanks; respectively spraying the prepared solutions of different materials to the leaves of the pakchoi in the water culture tank, and wrapping the stems of the pakchoi with a preservative film during spraying so as to prevent the materials from contacting; inoculating a water-soluble fertilizer rich in antibiotic resistance genes and antibiotic resistance bacteria on the leaf surfaces of the pakchoi sprayed with the gene resistance control material; sampling the Chinese cabbages treated differently on days 0, 2, 7 and 14 respectively; and finally, the taken sample is treated in two modes of deionized water cleaning and non-cleaning.
Comparative example 1
The present comparative example, which is basically the same as the method of example 1 and mainly differs therefrom, provides a method for controlling the entrance of an antibiotic resistance gene into a plant leaf using a carbon-based material and a test method thereof: treating pakchoi without using a gene resistance and control material, spraying 200mg/L Magnetic Biochar (MB) solution on the pakchoi, inoculating water-soluble fertilizers rich in antibiotic resistance genes and antibiotic resistance bacteria on the leaves of the pakchoi, and sampling the pakchoi subjected to different treatments on 0 th, 2 th, 7 th and 14 th days respectively; and finally, treating the obtained sample in two modes of deionized water cleaning and non-cleaning, and finally, extracting DNA and measuring high-throughput (96 pairs of primers) qPCR on the plant leaf.
Comparative example 2
The present comparative example, which is basically the same as the method of example 1 and mainly differs therefrom, provides a method for controlling the entrance of an antibiotic resistance gene into a plant leaf using a carbon-based material and a test method thereof: the method comprises the following steps of (1) not using a gene resistance control material to process the pakchoi, but using a 20mg/L Quaternary Phosphonium Salt (QPS) solution to spray the pakchoi, inoculating water-soluble fertilizers rich in antibiotic resistance genes and antibiotic resistance bacteria on the leaves of the pakchoi, and sampling the pakchoi subjected to different treatments on 0 th, 2 th, 7 th and 14 th days respectively; and finally, treating the obtained sample in two modes of deionized water cleaning and non-cleaning, and finally, extracting DNA and measuring high-throughput (96 pairs of primers) qPCR on the plant leaf.
Comparative example 3
The present comparative example, which is basically the same as the method of example 1 and mainly differs therefrom, provides a method for controlling the entrance of an antibiotic resistance gene into a plant leaf using a carbon-based material and a test method thereof: the method comprises the following steps of (1) directly inoculating a water-soluble fertilizer rich in antibiotic resistance genes and antibiotic resistance bacteria on the leaves of the pakchoi without using a gene resistance control material to process the pakchoi, and sampling the pakchoi subjected to different treatments on 0 th day, 2 th day, 7 th day and 14 th day respectively; and finally, treating the obtained sample in two modes of deionized water cleaning and non-cleaning, and finally, extracting DNA and measuring high-throughput (96 pairs of primers) qPCR on the plant leaf.
Comparative example 4
The present comparative example, which is basically the same as the method of example 1 and mainly differs therefrom, provides a method for controlling the entrance of an antibiotic resistance gene into a plant leaf using a carbon-based material and a test method thereof: the method is a pure negative control group, the pakchoi is not treated by using a gene resistance and control material, and water-soluble fertilizers rich in antibiotic resistance genes and antibiotic resistance bacteria are not inoculated on the leaves of the pakchoi, and the pakchoi subjected to different treatments are respectively sampled on days 0, 2, 7 and 14; and finally, treating the obtained sample in two modes of deionized water cleaning and non-cleaning, and finally, extracting DNA and measuring high-throughput (96 pairs of primers) qPCR on the plant leaf.
Comparing example 1 with comparative examples 1 to 4, as shown in FIG. 1, 16S rRNA gene amplicon sequences of leaf discs of pakchoi treated with three material (MB, QPS and MBQ) solutions were divided into 5 to 15 phyla. The three of Proteobacteria (Proteobacteria), Firmicutes (Firmicutes) and Bacteroides (Bacteroides) account for 91.0% -99.9% of the total sequence. After inoculation with a water-soluble fertilizer containing antibiotic-resistant bacteria and antibiotic-resistant genes, the relative abundance of proteobacteria (proteobacteria) in endophytes of the leaves gradually decreased (from 99% to 16%), while the relative abundance of Firmicutes (Firmicutes) gradually increased (from 1% to 73%) on days 2-14. Spraying solutions of MB, MBQ and QPS 3 materials attenuates this amplitude of variation. The influence of spraying water soluble fertilizer on the bacterial community of the leaf of the pakchoi is weakened by spraying the three materials. In addition, as shown in fig. 2, in the QPS, MB and MBQ treatments, Shannon index (diversity) of endogenous bacteria at day 2 and day 7 was significantly lower than at day 14, which indicates that bacterial diversity in pakchoi leaves after treatment can be effectively controlled within two weeks, preventing the migration of antibiotic resistance genes from the leaf surface into the leaves, thereby reducing the risk of the intake of leaf vegetable antibiotic resistance genes.
As shown in fig. 3, environmental Scanning Electron Microscopy (SEM) results showed that microbial communities on the leaf of pakchoi increased rapidly within 2 days after inoculation with water-soluble fertilizers containing resistant bacteria and resistant genes. Spraying Quaternary Phosphonium Salt (QPS), Magnetic Biochar (MB) and magnetic biochar-quaternary phosphonium salt (MBQ) can obviously reduce microorganisms on the surface of the leaves within 2 days. The results of environmental scanning electron microscopy are compared to find that the QPS and MBQ spraying has the best bacteriostatic effect on the 7 th day.
In the embodiment, the diversity and abundance of ARGs and MGEs in the leaves of the pakchoi are also detected, and 73 unique ARGs and 19 MGEs are detected in the leaf circle of the pakchoi. These genes encompass 12 antibiotic resistance types (aminoglycosides, mycins, -lactams, fluoroquinolones, macrolide lincosamides-streptogramins-b (mlsb), Multidrug (MDR), sulfonamides, tetracyclines, vancomycin, phenols, plasmids, etc.) and 6 MGEs (transposase, integrase, plasmid incompatibility, insertion sequence, plasmid replication, trimethoprim).
As shown in FIGS. 4-1 and 4-2, the absolute abundance of the total antibiotic resistance genes (ARGs + MGEs) in the leaf of Brassica campestris was changed with time without washing and after washing with DI water, and the inhibition control effect of QPS was the best by the seventh day without washing, but the inhibition control effect of MBQ solution was the best for food safety after washing with DI water. Therefore, the MBQ has the best effect on reducing the edible safety of the pakchoi.
As shown in FIGS. 5-1 and 5-2, in the case of unwashed pakchoi, the application of water-soluble fertilizer rich in antibiotic-resistant bacteria caused the abundance of the resistance genes on the leaf surfaces of pakchoi to increase rapidly in the first two days, and the total absolute abundance of ARGs and MGE showed a tendency to decrease continuously in the first 7 days. On the seventh day, the absolute abundance of ARGs and MGEs under the treatment of the three materials is obviously lower than that of the treatment without spraying material in the comparative example 3, wherein the treatment effects of the example 1 and the comparative examples 1-2 are not greatly different. In addition, as shown in fig. 6-3, the removal rate of ARGs and MGEs on the leaves of pakchoi by the three materials can reach about 93.5% and 93.7%, the three materials have better reduction effect on ARGs and MGEs in the first seven days, wherein the treatment effect of MBQ and QPS is better than that of MB.
Figure BDA0002969274200000071
Wherein A1 refers to the total absolute abundance of ARGs (MGEs) in the leaves of the Chinese cabbage after the spraying of the resistance control material; a0 indicates the total absolute abundance of ARGs (MGEs) in the leaf of pakchoi in the control group, i.e. in the treatment of spraying only the water soluble fertilizer without spraying any barrier and control material.
For the purpose of simulating treatment of vegetables before eating, ARGs and MGEs were measured after washing the leaves of pakchoi with deionized water. Results as shown in fig. 7-1 and 7-2, the absolute abundance of ARGs and MGEs after leaf washing the next day appeared between treatments: the rule of spraying the MB solution treatment > not spraying the resistance control material ═ spraying the QPS solution treatment > spraying the MBQ solution treatment (p <0.05), it can be seen that spraying the MB material alone is not favorable for deionized water cleaning to remove the ARGs and MGEs on the leaves, while spraying the gene resistance control material MBQ of the present invention still has excellent genome control effect after cleaning, and on the next and seventh days, the absolute abundance of the ARGs and MGEs in the treatment of spraying the MBQ material is the lowest, and the resistance control effect on the resistance gene is also the best. In addition, as shown in FIGS. 6-1, 6-2 and 6-3, the residual rates of the three materials on the ARGs and MGEs on the leaves of the pakchoi are the minimum on the 7 th day, and the removal rates are the maximum on the 7 th day, and can reach about 93.5 percent and 93.7 percent. The three materials all have better reducing effect on ARGs and MGEs in the first seven days, wherein MBQ and QPS treatment have better effect than MB. It can also be seen from fig. 6-3 that the removal rates of ARGs and MGEs from leaves of MBQ-treated pakchoi, which are still high after washing the leaves with deionized water, can reach 92.4% and 90.2%, respectively, so MBQ is the most effective of the three materials in preventing ARGs and MGEs from entering the food chain from the viewpoint of food safety.
In addition, as shown in fig. 8, 22 Human Pathogenic Bacteria (HPB) were detected in the leaf of pakchoi, among which Pseudomonas (Pseudomonas), Clostridium (Clostridium _ sensu _ stricoto _1), Bacillus (Bacillus), vibrio cellulans (Cellvibrio) and streptococcus (streccocus) were mainly used. In the network analysis, 8 HPBs were significantly associated with other ARGs, MGEs or bacterial genera (n >5, p > 0.05). As can be seen from the figure, the dominant genera of all samples are Pseudomonas (HPB), Chryseobacterium and Clostridium _ sensu _ stricoto _1 (HPB). On day 2, the relative abundance of the pseudomonas subjected to MBQ treatment is remarkably reduced, which is different from CK treatment, MB treatment and QPS treatment of non-sprayed materials, and on day 7, the relative abundance of the clostridium in the pakchoi leaves can be effectively reduced by the MBQ material provided by the invention. Therefore, the resistance control method of the invention can obviously inhibit the activity of human pathogenic bacteria in the leaf of the pakchoi when the pakchoi leaf is treated, thereby reducing the risk of the human pathogenic bacteria entering the human body through the food chain.
The invention has been described in detail hereinabove with reference to specific exemplary embodiments thereof. It will, however, be understood that various modifications and changes may be made without departing from the scope of the invention as defined in the appended claims. The detailed description and drawings are to be regarded as illustrative rather than restrictive, and any such modifications and variations are intended to be included within the scope of the present invention as described herein. Furthermore, the background is intended to be illustrative of the state of the art as developed and the meaning of the present technology and is not intended to limit the scope of the invention or the application and field of application of the invention.
More specifically, although exemplary embodiments of the invention have been described herein, the invention is not limited to these embodiments, but includes any and all embodiments modified, omitted, combined, e.g., between various embodiments, adapted and/or substituted, as would be recognized by those skilled in the art from the foregoing detailed description. The limitations in the claims are to be interpreted broadly based the language employed in the claims and not limited to examples described in the foregoing detailed description or during the prosecution of the application, which examples are to be construed as non-exclusive. Any steps recited in any method or process claims may be executed in any order and are not limited to the order presented in the claims. The scope of the invention should, therefore, be determined only by the appended claims and their legal equivalents, rather than by the descriptions and examples given above.

Claims (10)

1. A method for preventing and controlling antibiotic resistance genes from entering plant leaves by utilizing carbon-based materials is characterized in that the gene prevention and control materials are attached to the plant leaves to be processed; the gene control material comprises a carbon-based porous material A and a sterilization material B; the porosity or comparative area of the carbon-based porous material A is 0.7m2/g～0.9m2(ii)/g; the sterilization material B is quaternary phosphonium salt; the bactericidal material B is loaded on the carbon-based porous material A, and the loading rate of the bactericidal material B is 9.7-10.3%。
2. The method of claim 1, wherein the gene-controlling material is attached to the plant leaves at an area density of 195mg/m2～250mg/m2。
3. The method for controlling the entering of the antibiotic resistance genes into the plant leaves by utilizing the carbon-based material as claimed in claim 1, wherein the carbon-based porous material A is magnetic biomass charcoal and/or the bactericidal material B is tributyl dodecyl phosphine bromide.
4. The method for controlling the entering of the antibiotic resistance gene into the plant leaves by the carbon-based material as claimed in claim 3, wherein the specific processing steps are as follows:
(1) preparing a gene control material: mixing the carbon-based porous material A and the sterilization material B, carrying out hydrothermal reaction, and cooling to room temperature to obtain a gene resistance control material;
(2) preparing an adhering liquid: dispersing the gene resistance control material in the step (1) in deionized water to obtain an attachment solution;
(3) spraying treatment: and (3) spraying the adhering liquid in the step (2) on plant leaves to treat the plant leaves.
5. The method for controlling the entering of the antibiotic resistance gene into the plant leaves by using the carbon-based material as claimed in claim 4, wherein the hydrothermal reaction temperature in the step (1) is 80 ℃ to 90 ℃.
6. The method for controlling the entering of the antibiotic resistance gene into the plant leaves by using the carbon-based material as claimed in claim 4, wherein the concentration of the attaching solution in the step (3) is 195mg/L to 205mg/L, and the specific spraying rate is 0.7mL/s to 0.9 mL/s.
7. An effect evaluation method for inhibiting plant leaves from being polluted by antibiotic resistance genes is characterized in that the antibiotic resistance genes are inoculated on the plant leaves subjected to antibiotic resistance gene resistance control treatment, and DNA extraction and analysis are carried out on the inoculated plant leaves; the method for the plant leaf to carry out antibiotic resistance gene resistance control treatment is the method of any one of claims 1 to 6.
8. The method of claim 7, wherein the plant leaves are pakchoi leaves; wrapping the stem of the pakchoi with a preservative film before the antibiotic resistance gene control treatment is carried out on the pakchoi leaves.
9. The method for evaluating the effect of inhibiting the leaf blade of the plant from being polluted by the antibiotic resistance gene according to claim 8, wherein the inoculation adopts a water-soluble fertilizer rich in the antibiotic resistance gene and antibiotic resistant bacteria, and the inoculation time is 2-14 days.
10. The method of claim 9, wherein the inoculated leaves of pakchoi are washed with deionized water.
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