Application of flavonoids in reducing pollution of antibiotic resistance genes in plants

Abstract

This invention discloses the application of flavonoids in reducing antibiotic resistance gene pollution in plants. Adding 0.05%–0.2% (by weight of soil) of rutin or neohesperidin to the soil, adjusting soil moisture content, and then planting and cultivating plants for 30 days significantly reduced the abundance of antibiotic resistance genes in the soil and plant leaves compared to the system without rutin or neohesperidin. This alleviates antibiotic resistance gene pollution in the plant cultivation system to a certain extent, resulting in greener and more environmentally friendly plants. It provides a new and effective approach to controlling antibiotic resistance gene pollution in crop cultivation systems, with broad application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antibiotic resistance gene pollution control, and particularly relates to application of flavonoids in reducing antibiotic resistance gene pollution in plants. BACKGROUND

[0002] Many studies have shown that frequent agricultural activities introduce a large amount of exogenous antibiotic resistance genes (ARGs) into the agricultural environment, for example, livestock manure application, sludge application and wastewater irrigation. It is worrying that the agricultural environment as an important "repository" of ARGs, in which ARGs and ARB (antibiotic resistant bacteria) can be transmitted from the soil and other agricultural environments to the aboveground parts of plants, leading to a sharp increase in ARGs in plants and posing a threat to the quality and safety of agricultural products.

[0003] However, existing control technologies are mostly focused on reducing the abundance of antibiotic resistance genes in soil and water environments, and few studies have focused on how to prevent antibiotic resistance genes from entering plants from the soil. CN112997777A discloses a method for controlling antibiotic resistance genes from entering plant leaves using carbon-based materials and an effect evaluation method thereof. The gene control material is attached to the plant leaves, and physical means are used to adsorb the antibiotic resistance genes on the material to prevent them from entering the plant body.

[0004] Rutin (CAS No. 153-18-4), also known as rutin, is a natural flavonoid glycoside belonging to flavonoids widely present in plants, and has anti-inflammatory, antioxidant, anti-allergic, antiviral and other effects. Neohesperidin (CAS No. 13241-33-3) is a flavonoid compound present in the pericarp of citrus plants, especially oranges and lemons, and has antioxidant, anti-inflammatory, antitumor, lipid-lowering, antibacterial and other biological activities. At present, there are few control strategies for antibiotic resistance gene pollution in plants in the prior art, and it has not been found whether flavonoids can control the entry of antibiotic resistance genes into plants and prevent their transfer, which is not involved in the prior art. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide the application of flavonoids in reducing antibiotic resistance gene pollution in plants to solve the problems mentioned in the background art.

[0006] The present application provides the application of flavonoids in reducing antibiotic resistance gene pollution in plants.

[0007] Preferably, the flavonoids are any one of rutin and neohesperidin.

[0008] Preferably, the antibiotic resistance genes include antibiotic resistance genes for aminoglycosides, glycopeptides, multidrugs, chloramphenicol, sulfonamides, tetracyclines, etc.

[0009] Preferably, a method for reducing antibiotic resistance gene pollution in plants using flavonoids:

[0010] S1. Add flavonoids to the soil, mix well, and adjust the soil moisture content to 65%;

[0011] S2. Plant the plants and cultivate them.

[0012] Preferably, the amount of flavonoids added is 0.05%-0.2% of the soil weight.

[0013] Preferably, the culture time is 30 days.

[0014] Preferably, the plant is a cherry tomato.

[0015] Antibiotic resistance gene abundance refers to the copy number of various antibiotic resistance genes in the genome. The higher the abundance, the greater the number of that gene. Abundance is divided into absolute abundance and relative abundance, where relative abundance describes the percentage of a single gene out of the total number of genes.

[0016] The data measured in this invention were obtained by screening representative antibiotic resistance genes based on gene abundance, including glycopeptide antibiotic resistance genes. vanA, vanN, vanRM, vanXB, vanRO, vanM Etc.; Chloramphenicol antibiotic resistance genes include cmlA6, cmlA4, fexA, cmrA Etc.; multi-drug antibiotic resistance genes include adeG, mdtE, ceoA, mexX, smeC Etc.; Sulfonamide antibiotic resistance genes include sul1, sul2, sul4 Etc.; Tetracycline antibiotic resistance genes include tet(K), tetA(P), tet(Y), tetO, tet44 Etc.; aminoglycoside antibiotic resistance genes include AAC(3)- VIIIa, AAC(6')-If, AAC(6')-Ib9, APH(9)-Ib wait.

[0017] The comprehensive effects of this invention include: In crop cultivation systems, organic fertilizers are usually applied, which contain a large amount of antibiotics. This invention uses flavonoids to reduce antibiotic resistance gene pollution in cherry tomato cultivation systems to a certain extent, inhibit the spread of antibiotic resistance genes from soil to plants, and significantly reduce the abundance of antibiotic resistance genes such as tetracyclines and sulfonamides. The method is simple and saves time. Attached Figure Description

[0018] Figure 1 To investigate the reduction of antibiotic resistance gene contamination in soil and cherry tomato leaves by rutin after 30 days of cultivation;

[0019] Figure 2 To investigate the reduction of antibiotic resistance gene contamination in soil and cherry tomato leaves by neohesperidin after 30 days of cultivation; Detailed Implementation

[0020] The technical solution of this patent will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments below are the preferred embodiments of this patent, but this patent is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the scope of this patent technology.

[0021] In embodiments of the present invention, the terms "exemplary" or "for example" are used to indicate that something is an example, illustration, or description. Any embodiment or design described as "exemplary" or "for example" in embodiments of the present invention should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of the terms "exemplary" or "for example" is intended to present the relevant concepts in a specific manner.

[0022] Rutin, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0023] Neohesperidin was purchased from Shanghai Dipo Chemical Technology Co., Ltd.

[0024] The indoor potted plant experiment used cherry tomatoes as the test material. The seeds were provided by Shouhe Seed Industry in Weifang City, Shandong Province. The seeds have advantages such as strong growth and a growth period of about 100-120 days.

[0025] Soil for the indoor pot experiment was collected from the agricultural base of Hancheng Village, Qingjiangpu District, Huai'an City, Jiangsu Province (33°33′N, 118°58′E). Multiple 20×20 cm quadrats were randomly set up to collect fresh soil from the top 0-15 cm of the field. The soil type was silty loam.

[0026] Before indoor cultivation begins, chicken manure is added to the soil and mixed evenly. The soil moisture content is adjusted to 60% of the maximum water holding capacity. The soil is then placed in an artificial climate chamber and cultured for 7 days at 25°C and 60% humidity to ensure that the pre-cultured soil contains multiple antibiotic resistance genes. Example

[0027] S1, Seedling raising

[0028] Evenly fill the seedling trays with nutrient substrate, sow two cherry tomato seeds in each hole, lightly cover with a layer of substrate, compact slightly, water thoroughly, and cover with a film to maintain the temperature and humidity required for germination. After the cherry tomato seeds emerge, thin out the seedlings, leaving one seedling per hole. During this period, keep the seedling trays moist. Once the plants have grown to four leaves and a bud, retain the healthy seedlings and transplant them to seedling pots. Place them in an artificial climate chamber and continue cultivation for three weeks before use.

[0029] S2, Processing

[0030] Accurately weigh 800g of pre-cultured soil, add 0.05% rutin, and mix thoroughly. Then adjust the soil moisture content to 65% of its maximum saturated water holding capacity. Transfer the soil into small flower pots (12 cm inner diameter, 14 cm height). Set up a blank control without rutin, with three replicates.

[0031] S3. Construction of Indoor Soil-Tomato Cultivation System

[0032] Select healthy, uniformly growing cherry tomato seedlings (approximately 15 cm tall) and transplant them into plastic pots filled with treated soil in the early morning. Once the leaves have fully unfolded, place them in a 25±1℃ light incubator with a relative humidity of 70% and a light cycle of 16 hours of light / 8 hours of darkness. During the growing period, regularly observe plant growth and the occurrence of pests and diseases, weigh the plants periodically, and replenish the soil with sterilized deionized water to maintain soil moisture and prevent dehydration and wilting.

[0033] S4, Sampling

[0034] Soil and tomato leaf samples were collected at 30 days.

[0035] S5. Total DNA extraction and sequencing from soil and tomato leaves.

[0036] Tomato leaf samples were pretreated in a clean bench as follows: 10g of plant tissue (leaf sample) was weighed and transferred to a beaker. The tissue was manually chopped and 90 mL of 1×PBS (containing 0.1% glycerol, 0.15% Tween 80, pH 7.0) was added. The mixture was homogenized at medium to low speed for 2 min using a tissue disperser. The homogenate was then passed through 150μm and 26μm mesh sieves to remove large pieces of plant tissue. The filtered homogenate was aliquoted into 50 mL centrifuge tubes. The mixture was centrifuged at 8000 rpm for 5 min to obtain a precipitate. The precipitate was washed and resuspended with PBS and centrifuged at 3200 rpm for 10 min. The supernatant was removed, and the precipitate was retained. The precipitate surface was gently washed with PBS buffer using a pipette, repeated three times. All washings were combined in centrifuge tubes. Finally, the mixture was centrifuged at 3200 rpm for 10 min, and the precipitate was retained for total DNA extraction (Nycodenz density gradient centrifugation was used for all samples, depending on their condition). DNA was subsequently extracted from tomato leaves following the extraction procedure of the FastDNA Spin Kit for Soil. DNA was also extracted from soil samples following the extraction procedure of the FastDNA Spin Kit for Soil.

[0037] After quality control, the extracted DNA data underwent metagenomic sequencing and antibiotic resistance gene analysis. Considering the large amount of experimental data, the attenuation rate of antibiotic resistance genes was calculated using the following formula: (Abundance of resistance genes in the control group - Abundance of resistance genes with added flavonoids) / Abundance of resistance genes in the control group × 100%. The analysis results are shown below. Figure 1 As shown:

[0038] The flavonoid rutin significantly reduced the abundance of antibiotic resistance genes in the soil where cherry tomatoes were grown. After 30 days of cultivation, the abundance of aminoglycoside resistance genes decreased by 25.09%, glycopeptide resistance genes by 45.04%, multidrug resistance genes by 33.15%, chloramphenicol resistance genes by 24.79%, sulfonamide resistance genes by 16.64%, and tetracycline resistance genes by 50.92% in the soil with added rutin.

[0039] The flavonoid rutin significantly reduced the abundance of antibiotic resistance genes in cherry tomato leaves. After 30 days of culture, the abundance of aminoglycoside resistance genes, glycopeptide resistance genes, multidrug resistance genes, chloramphenicol resistance genes, sulfonamide resistance genes, and tetracycline resistance genes was reduced by 49.75%, 93.09%, 58.09%, 98.44%, 94.71%, and 95.34% in the leaves of the rutin-treated group. Example

[0040] The amount of rutin added in step S2 of Example 1 was changed to 0.1%, and the remaining steps were the same as in Example 1. Example

[0041] The amount of rutin added in step S2 of Example 1 was changed to 0.2%, and the remaining steps were the same as in Example 1. Example

[0042] In Example 1, the rutin used in step S2 was replaced with neohesperidin at an addition rate of 0.05%, while the remaining steps were the same as in Example 1.

[0043] After quality control, the extracted DNA data underwent metagenomic sequencing and antibiotic resistance gene analysis. Considering the large amount of experimental data, the reduction rate of antibiotic resistance genes was calculated using the following formula: (Abundance of resistance genes in the control group - Abundance of resistance genes in the flavonoid-added group) / Abundance of resistance genes in the control group × 100%. The analysis results are shown below. Figure 2 AAC(3)- VIIIa, AAC(6')-If, AAC(6')-Ib9, APH(9)-Ib Figure 1 Figure 2 Figure 1 Figure 2 As shown:

[0044] The flavonoid compound neohesperidin significantly reduced the abundance of antibiotic resistance genes in tomato-growing soil. After 30 days of cultivation, the abundance of aminoglycoside resistance genes decreased by 6.89%, glycopeptide resistance genes by 51.84%, multidrug resistance genes by 16.13%, chloramphenicol resistance genes by 18.75%, sulfonamide resistance genes by 23.61%, and tetracycline resistance genes by 37.57% in soil supplemented with neohesperidin.

[0045] The flavonoid compound neohesperidin significantly reduced the abundance of antibiotic resistance genes in tomato leaves. After 30 days of culture, the abundance of aminoglycoside resistance genes, glycopeptide resistance genes, multidrug resistance genes, chloramphenicol resistance genes, sulfonamide resistance genes, and tetracycline resistance genes was reduced by 35.3%, 33.97%, 37.76%, 74.35%, 39.68%, and 59.55% in the tomato leaves of the neohesperidin-treated group. Example

[0046] In Example 1, the rutin used in step S2 was replaced with neohesperidin at an addition rate of 0.1%, while the remaining steps were the same as in Example 1. Example

[0047] In Example 1, the rutin used in step S2 was replaced with neohesperidin at an addition amount of 0.2%, and the remaining steps were the same as in Example 1.

[0048] The experimental results show that rutin and neohesperidin, as flavonoids, can significantly reduce antibiotic resistance gene pollution in cherry tomato cultivation systems, inhibit the spread of antibiotic resistance genes from soil to plants, and make the grown vegetables greener and more environmentally friendly.

[0049] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If these modifications and variations fall within the scope of the claims of this invention and their equivalents, then this invention is also intended to include these modifications and variations.

Claims

1. The application of flavonoids in reducing antibiotic resistance gene pollution in plants, characterized in that, Add flavonoids to the soil, mix well, adjust the soil moisture content to 65%, and plant cherry tomatoes for 30 days. The flavonoid compound is either rutin or neohesperidin; The amount of flavonoids added is 0.05%-0.2% of the soil weight; The flavonoids mentioned above reduce antibiotic resistance gene contamination in cherry tomato cultivation systems to a certain extent and inhibit the spread of antibiotic resistance genes from soil to plants. The antibiotic resistance genes include aminoglycosides, glycopeptides, multidrugs, chloramphenicol, sulfonamides, and tetracyclines.

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