Ecological prevention and control method for constructing antibiotic resistance gene transmission barrier by using indigenous microorganism diversity

Abstract

The invention belongs to the technical field of environmental biology, and discloses an ecological prevention and control method for constructing an antibiotic resistance gene transmission barrier by using indigenous microorganism diversity. In order to overcome the defects in the prior art, the invention provides an innovative method based on soil microbial diversity regulation and control. The core of the method lies in that diverse soil microbial communities with stable functions are actively constructed, and ecological barrier functions such as internal competition rejection, resource competition and interference / degradation capability to movable genetic elements are enhanced, so that the horizontal transfer frequency and diffusion range of endogenous and exogenous antibiotic resistance genes (ARGs) of soil are effectively inhibited, and the growth of the soil is promoted. The long-term resistance and the restoring force of a soil ecological system to the ARGs are enhanced, and an eco-friendly and efficient solution is provided for sustainable control of the antibiotic resistance of the environment.

Description

Technical Field

[0001] This invention belongs to the field of environmental biotechnology, and more specifically, relates to an ecological control method for constructing a barrier to the spread of antibiotic resistance genes by utilizing the diversity of indigenous microorganisms. Background Technology

[0002] With the rapid development of my country's economy and the improvement of people's living standards, the demand for livestock and poultry products such as meat, eggs, and dairy products has been increasing, and the scale of the livestock industry has expanded rapidly, making my country a major livestock country in the world. Integrated crop and livestock farming is the main model promoted for the treatment of livestock and poultry manure in my country. In this model, antibiotic resistance genes (ARGs) from livestock and poultry manure enter the agricultural ecosystem with the application of pig manure, accumulating through the food chain and ultimately affecting human health. The expansion of the livestock scale has also led to a corresponding increase in the production and use of veterinary antibiotics. The large-scale use of antibiotics has also brought about a series of problems, among which the pollution problem of ARGs has received particular attention. Existing technologies for controlling antibiotic resistance pollution in livestock and poultry sources have the following drawbacks: 1. High physicochemical costs: Although existing technologies (such as chemical leaching) are highly efficient in treating heavily polluted soil, the large amounts of wastewater they produce containing high concentrations of antibiotics and chemical agents require expensive subsequent treatment. This can easily lead to secondary pollution and potential ecological risks (such as damage to soil ecology, production of toxic intermediates, and chemical residues), and inevitably cause problems such as a decline in soil quality; 2. Existing methods for controlling ARGs / ARBs (such as applying high doses of disinfectants, antibiotics, and heavy metals) can directly kill some target microorganisms, but the resulting community stability is poor, its function is impaired, and it loses its original competitive exclusion and inhibition capabilities. Instead, it creates ecological niche vacancies, making it easier for invasive ARBs to colonize and for surviving ARGs to be horizontally transferred; 3. Ignoring vitamin ecological interactions: Existing technologies (such as introducing a single antagonistic strain) often focus on the direct antagonistic effect of a single strain or its metabolites, neglecting the core role of the microbial community as a whole (network interactions, functional redundancy, resource competition) in inhibiting invaders and horizontal gene transfer. The effect of a single strain may be unstable, easily fail, and cannot simulate the complex and diverse natural barrier effects.

[0003] A review of existing technologies reveals a lack of effective strategies targeting horizontal gene transfer (HGT), and a lack of effective ecological interventions to address the horizontal transfer of ARGs among bacteria via plasmids, integrons, and transposons. Even if existing methods eradicate some ARBs, ARGs can still rapidly spread and persist in soil microbial communities via HGT, becoming a long-term resistance pool. Furthermore, existing remediation or inhibition technologies are difficult to maintain long-term effectiveness, are susceptible to environmental fluctuations, and are costly and complex to implement. Summary of the Invention

[0004] In view of the above-mentioned defects in the existing technology, the present invention first provides a soil microbial community restorer.

[0005] A second objective of this invention is to provide the application of the aforementioned soil microbial community restorer.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A soil microbial community restorer is obtained by mixing local normal soil suspension and bamboo biochar in a certain proportion, wherein the mass ratio of local normal soil suspension to biochar is 1:3.

[0008] Preferably, the preparation method of the moso bamboo biochar is as follows: the moso bamboo is first screened through a 200-mesh sieve, then pyrolyzed at high temperature, and prepared under a nitrogen atmosphere.

[0009] More preferably, the high temperature refers to 600-700℃, and the high temperature reaction time is 2.5 h.

[0010] This invention utilizes a soil microbial restorer in soils under drought or antibiotic stress, finding that it significantly improves microbial diversity in stressed soils and simultaneously builds a healthy soil microbial barrier. This barrier naturally resists the invasion of exogenous drug-resistant bacteria (ARBs) and significantly reduces the frequency of horizontal transfer of drug-resistant genes (ARGs) between bacteria. It avoids the drawbacks of chemical disinfectants killing beneficial bacteria and disrupting soil balance, which could lead to even more severe drug resistance, thus protecting the soil's own "resistance."

[0011] Therefore, the present invention also protects the application of the soil microbial community restorer in improving the diversity of microbial communities in soil.

[0012] Preferably, the above-mentioned improvement of soil microbial community diversity refers to:

[0013] (1) Increase the number of beneficial microorganisms in soils under drought and / or antibiotic stress;

[0014] (2) Restore α- and / or β-diversity of microorganisms in soils under drought and / or antibiotic stress;

[0015] This invention also protects the application of the soil microbial restoration agent in inhibiting the spread of ARGs in soil contaminated by pig manure.

[0016] Preferably, in the above application, the soil microbial community restorer is mixed into the soil under drought and / or antibiotic stress at a mass ratio of 10-50% and cultured to obtain restored soil. The restored soil is then made into a suspension to obtain a normal soil suspension, which can be used for soil under drought and / or antibiotic stress.

[0017] The present invention also protects the application of the normal soil suspension in inhibiting the spread of ARGs in soil containing pig manure.

[0018] Preferably, inhibiting the spread of ARGs in soil containing pig manure refers to:

[0019] (1) Reduce the abundance of antibiotic resistance genes tetG, sul1, strB, ermA, intl1, and Tn916 / 1545 in pig manure-sourced soil;

[0020] (2) Increase the number of culturable bacteria in pig manure-sourced soil.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention aims to overcome the shortcomings of existing technologies and provide an innovative method based on the regulation of soil microbial diversity. The core of this method lies in actively constructing diverse and functionally stable soil microbial communities. By strengthening their inherent ecological barrier functions, such as competitive exclusion, resource competition, and interference / degradation of mobile genetic elements, this effectively inhibits the horizontal transfer frequency and spread of endogenous and exogenous antibiotic resistance genes (ARGs) in the soil. Furthermore, it enhances the long-term resistance and resilience of the soil ecosystem to ARGs, providing an eco-friendly and efficient solution for the sustainable control of environmental antibiotic resistance.

[0023] Its advantages are as follows:

[0024] (1) Addressing the shortcomings of destructive interventions: Utilizing native soil microbiota for "microbial suppression". By adding / activating diverse native beneficial microorganisms, a healthy soil microbial barrier is constructed. This barrier can naturally resist the invasion of exogenous drug-resistant bacteria (ARBs) and significantly reduce the frequency of horizontal transfer of drug-resistant genes (ARGs) between bacteria. This avoids the drawbacks of chemical disinfectants killing beneficial bacteria and disrupting soil balance, which could lead to more severe drug resistance, thus protecting the soil's own "resistance".

[0025] (2) Addressing the shortcomings of neglecting microbial ecological interactions: Instead of relying on a single "bactericidal" strain, it regulates the entire soil microbial community. By optimizing network cooperation, resource competition (competitive exclusion), and functional complementarity (functional redundancy) within the microbial community, the entire "community" spontaneously and more stably suppresses the colonization of drug-resistant bacteria and the spread of drug-resistant genes. This solves the problem of unstable effects and easy failure of single bacterial agents, and utilizes the overall power of the microbial community to achieve longer-lasting and more reliable inhibition.

[0026] (3) Addressing the shortcomings of the lack of HGT strategy: specifically interfering with the horizontal gene transfer of drug resistance genes among bacteria. Methods include: enhancing bacterial community competition to prevent bacteria from having "free time" to exchange genes; enriching microorganisms that can "dismantle" MGEs; or creating a microenvironment unfavorable to gene exchange. This breaks through the bottleneck of existing technologies that only kill drug-resistant bacteria but cannot control the "diffusion" of drug resistance genes among live bacteria, thus curbing the spread of drug resistance from the transmission route.

[0027] (4) Addressing the shortcomings of continuous selection pressure: Actively remove the "pollutants" (selective pressure) driving the development of drug resistance in the soil. By introducing or activating functional microorganisms that can decompose residual antibiotics, heavy metals, or disinfectants, and optimizing their cooperation with other bacteria, the concentration of these pollutants can be effectively reduced or even eliminated. At the same time, the competition from healthy bacteria can squeeze the living space of drug-resistant bacteria. This reduces the chances of drug-resistant bacteria being "selected" and growing from the source, and is particularly suitable for soils that have been contaminated for a long time (such as the area around farms and former pharmaceutical factory sites).

[0028] (5) The goal is to establish an "unbreakable" soil microbial ecosystem. By restoring and maintaining a high degree of soil microbial diversity (functional redundancy), the soil will possess strong self-repair and long-term maintenance capabilities. This system can continuously resist the invasion of foreign drug-resistant bacteria and inhibit the spread of internal drug-resistant genes. It significantly reduces reliance on expensive, complex, or repetitive external interventions (such as frequent application of microbial agents or chemicals), achieving low-cost, long-lasting, and sustainable drug resistance control.

[0029] This is a highly efficient, low-consumption, environmentally friendly, and selective remediation process, offering a safer and more sustainable direction for development. For large-scale farmland pollution, it represents a gentler and lower-cost eco-friendly approach, such as bioremediation. Attached Figure Description

[0030] Figure 1 The number of culturable bacteria in soil under different stress conditions is shown in the figure. CK, DT, AT, and DA represent the control group, drought treatment group, antibiotic treatment group, and drought + antibiotic synergistic treatment group, respectively. The figure shows the differences between the treatment groups on the same day, and different letters represent significant differences (P < 0.05).

[0031] Figure 2 The changes in the α-diversity index of soil bacterial communities under different stress conditions are shown in the figure. (a), (b), (c), and (d) represent the ACE, Chao1, Shannon, and Simpson indices of bacteria, respectively. The CK group was the control group, the DT group was the drought treatment group, the AT group was the antibiotic treatment group, and the DA group was the drought + antibiotic synergistic treatment group. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference (P > 0.05).

[0032] Figure 3 β-diversity characteristics of soil bacterial communities under different stress conditions;

[0033] Figure 4 The characteristics of culturable bacteria in soil treated with different restoration ratios are shown in the figure. (a), (b), (c), (d), and (e) represent the number of culturable bacteria in soil at days 0, 15, 30, 45, and 60 of the soil diversity restoration experiment, respectively.

[0034] Figure 5 The changes in soil bacterial community α diversity index are shown for different restoration ratios. Figures (a), (b), (c), and (d) represent the ACE, Chao1, Shannon, and Simpson indices of soil bacteria, respectively. CK100 represents the 100% CK soil microbial community restoration suspension group, DT100 represents the 100% DT soil suspension group, DT90 represents the 90% DT soil suspension + 10% CK soil microbial community restoration suspension group, DT70 represents the 70% DT soil suspension + 30% CK soil microbial community restoration suspension group, DT50 represents the 50% DT soil suspension + 50% CK soil microbial community restoration suspension group, and AT100 represents... The groups are: 100% AT soil suspension group, AT90 group (90% AT soil suspension + 10% CK soil microbial regeneration suspension group), AT70 group (70% AT soil suspension + 30% CK soil microbial regeneration suspension group), AT50 group (50% AT soil suspension + 50% CK soil microbial regeneration suspension group), DA100 group (100% DA soil suspension group), DA90 group (90% DA soil suspension + 10% CK soil microbial regeneration suspension group), DA70 group (70% DA soil suspension + 30% CK soil microbial regeneration suspension group), and DA50 group (50% DA soil suspension + 50% CK soil microbial regeneration suspension group); * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference (P > 0.05).

[0035] Figure 6The soil bacterial community β diversity characteristics were shown for different recovery ratios. Figures (a), (b), and (c) show the results of PCA analysis between CK100 and the DT treatment groups (DT100, DT90, DT70, DT50), AT treatment groups (AT100, AT90, AT70, AT50), and DA treatment groups (DA100, DA90, DA70, DA50), respectively.

[0036] Figure 7 To restore the abundance of tetG during the model fertilization experiment, (a) the absolute abundance of tetG in the DT treatment group; (b) the absolute abundance of tetG in the AT treatment group; (c) the absolute abundance of tetG in the DA treatment group; (d) the relative abundance of tetG in the DT treatment group; (e) the relative abundance of tetG in the AT treatment group; and (f) the relative abundance of tetG in the DA treatment group.

[0037] Figure 8 To restore the abundance of sul1 during the model fertilization experiment, (a) the absolute abundance of sul1 in the DT treatment group; (b) the absolute abundance of sul1 in the AT treatment group; (c) the absolute abundance of sul1 in the DA treatment group; (d) the relative abundance of sul1 in the DT treatment group; (e) the relative abundance of sul1 in the AT treatment group; and (f) the relative abundance of sul1 in the DA treatment group.

[0038] Figure 9 To restore the abundance of strB during the model fertilization experiment, (a) the absolute abundance of strB in the DT treatment group; (b) the absolute abundance of strB in the AT treatment group; (c) the absolute abundance of strB in the DA treatment group; (d) the relative abundance of strB in the DT treatment group; (e) the relative abundance of strB in the AT treatment group; and (f) the relative abundance of strB in the DA treatment group.

[0039] Figure 10 To restore the abundance of ermA during the model fertilization experiment, (a) the absolute abundance of ermA in the DT treatment group; (b) the absolute abundance of ermA in the AT treatment group; (c) the absolute abundance of ermA in the DA treatment group; (d) the relative abundance of ermA in the DT treatment group; (e) the relative abundance of ermA in the AT treatment group; and (f) the relative abundance of ermA in the DA treatment group.

[0040] Figure 11To restore the abundance of intl1 during the model fertilization experiment, (a) the absolute abundance of intl1 in the DT treatment group; (b) the absolute abundance of intl1 in the AT treatment group; (c) the absolute abundance of intl1 in the DA treatment group; (d) the relative abundance of intl1 in the DT treatment group; (e) the relative abundance of intl1 in the AT treatment group; and (f) the relative abundance of intl1 in the DA treatment group.

[0041] Figure 12 To restore the abundance of Tn916 / 1545 during the model fertilization experiment, the following data were collected: (a) absolute abundance of Tn916 / 1545 in the DT treatment group; (b) absolute abundance of Tn916 / 1545 in the AT treatment group; (c) absolute abundance of Tn916 / 1545 in the DA treatment group; (d) relative abundance of Tn916 / 1545 in the DT treatment group; (e) relative abundance of Tn916 / 1545 in the AT treatment group; and (f) relative abundance of Tn916 / 1545 in the DA treatment group.

[0042] Figure 13 The figures show the characteristics of culturable bacteria in soil under different treatments; (a), (b), (c), (d), and (e) in the figure represent the number of culturable bacteria in soil on days 0, 15, 30, 45, and 60 of the recovery model fertilization experiment, respectively.

[0043] Figure 14The figures show the variation characteristics of soil bacterial community α diversity index under different treatments; (a), (b), (c), and (d) represent the ACE, Chao1, Shannon, and Simpson indices of soil bacteria, respectively; CK100 represents the 100% CK soil microbial community recovery suspension group, DT100 represents the 100% DT soil suspension group, DT90 represents the 90% DT soil suspension + 10% CK soil microbial community recovery suspension group, DT70 represents the 70% DT soil suspension + 30% CK soil microbial community recovery suspension group, DT50 represents the 50% DT soil suspension + 50% CK soil microbial community recovery suspension group, and AT100 represents... The groups are: 100% AT soil suspension group, AT90 group (90% AT soil suspension + 10% CK soil microbial regeneration suspension group), AT70 group (70% AT soil suspension + 30% CK soil microbial regeneration suspension group), AT50 group (50% AT soil suspension + 50% CK soil microbial regeneration suspension group), DA100 group (100% DA soil suspension group), DA90 group (90% DA soil suspension + 10% CK soil microbial regeneration suspension group), DA70 group (70% DA soil suspension + 30% CK soil microbial regeneration suspension group), and DA50 group (50% DA soil suspension + 50% CK soil microbial regeneration suspension group). * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference (P > 0.05).

[0044] Figure 15 The β-diversity characteristics of soil bacterial communities under different treatments: (a) β-diversity of soil bacterial communities in the CK group and the DT treatment group; (b) β-diversity of soil bacterial communities in the CK group and the AT treatment group; (c) β-diversity of soil bacterial communities in the CK group and the DA treatment group. Detailed Implementation

[0045] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific drawings and embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0046] The selected soil samples showed significantly higher levels of microbial biomass carbon, basal respiration rate, and various enzyme activities compared to conventional agricultural soils. The tested soils were farmland soils from the teaching and research base of the College of Agriculture, South China Agricultural University, with sampling points located at 23° 9'27.83"N, 113° 21' 28.13"E. Topsoil (0–20 cm) was collected from the farmland within the base, and a mixed sample was prepared according to the 5-point sampling method.

[0047] Example 1

[0048] Preparation of biochar: Moso bamboo was selected to prepare biochar. First, it was screened through a 200-mesh sieve to ensure suitable particle size. Then, the moso bamboo was pyrolyzed at 600℃ for 2.5 h and prepared in a tube furnace under nitrogen atmosphere.

[0049] Preparation of microbial community restorer (CK): Normal soil suspension and biochar are mixed in a ratio of 1:3 to prepare microbial community restorer (CK).

[0050] Construction of a soil microbial diversity perturbation model: Soil was cultured in sterile incubators, and soil parameters were measured under four treatment groups: control (CK), drought at 40% WHC (DT), 16 mg / kg DOX antibiotic (AT), and its synergistic stress (DA). Each incubator contained 1 kg of sterile dry soil, and all treatments were incubated in a 23°C artificial climate chamber in the dark for 60 days. Changes in the number of culturable bacteria and soil microbial structure were measured to ensure the successful establishment of the perturbation model.

[0051] Construction of a soil microbial diversity restoration model: The prepared microbial restoration agent (CK) was mixed into the stressed soil (DT / AT / DA groups) at proportions (10%-50%), as shown in Table 1. After 60 days of incubation, the number of culturable bacteria and the soil microbial structure were measured to ensure the restoration of microbial diversity.

[0052] Table 1. Grouping information for soil microbial diversity restoration models

[0053] Soil microbial community function testing after restoration: Soil cultured with microbial restoration agent was mixed with PBS at a 1:1 (V:V) ratio and shaken for 30 min. Soil impurities were filtered through a full-width microporous homogenizing bag (Huankai, China) to obtain different soil microbial community restoration suspensions. The bacterial count of the normal soil suspension was determined to be 10⁻⁶ using the plate culture method. 6 CFU / mL. Thirteen treatment groups were obtained by adding 22.5% of the solution to 1 kg dry weight of sterilized soil, with four replicates per group. After adjusting the soil moisture content to 80% WHC, 40 g of pig manure was added to 1 kg of soil at a 4% addition rate. The mixture was thoroughly mixed and then incubated in a climate chamber at 23°C in the dark for 60 days. Changes in the abundance of ARGs and MGEs, soil bacterial count and community structure, and soil physicochemical properties were detected.

[0054] Table 2. Grouping information for soil microbial community function testing after restoration

[0055] The results showed that after the soil microbial diversity perturbation model was constructed, the number of culturable bacteria decreased by 33.41%, 29.06%, and 41.85% in DT, AT, and DA compared with the control group. The α-diversity index of soil bacterial communities in each treatment group was significantly reduced on day 30 (P<0.05), and the β-diversity of each soil bacterial community changed significantly. Drought and antibiotic stress treatments both significantly inhibited soil bacterial quantity and diversity, but synergistic treatments showed a stronger inhibitory effect (e.g., minimum bacterial quantity, minimum carbon source use efficiency) and promoted significant homogenization of soil bacterial communities, confirming a significant synergistic effect between the two treatments. Figures 1 to 3 ).

[0056] In the preliminary experiments for constructing the soil microbial diversity restoration model, the mixing ratio of biochar and soil bacterial suspension in the microbial restoration agent used in this invention was explored. First, biochar and soil bacterial suspension were mixed into stressed soil at mass ratios of 1:1, 1:2, 1:3, 1:4, and 1:5. At ratios of 1:4 and 1:5, it was observed that increasing the ratio led to a decrease in bacterial diversity and richness. Further increases in the biochar ratio could inhibit microbial growth due to drastic changes in soil physicochemical properties (such as altered pore structure or excessive nutrient adsorption) or the release of toxic substances. Excessive biochar addition could lead to a decrease in bacterial OTUs and exacerbate soil moisture loss or salt accumulation, indirectly inhibiting microorganisms. High proportions of bacterial suspension could cause biochar particle aggregation or sedimentation, affecting uniform application. Conversely, excessively high concentrations of soil bacterial suspension could inhibit the activity of functional bacteria in the soil due to nutrient competition or the accumulation of metabolic products.

[0057] Therefore, taking all factors into consideration, this invention selects biochar and soil bacterial suspension mixed at a mass ratio of 1:3 and added to stressed soil for subsequent experiments.

[0058] After constructing a soil microbial diversity restoration model, we found that adding a high proportion (30%, 50%) of microbial community restorer effectively restored bacterial quantity and community diversity (α and β diversity) in drought- or antibiotic-stressed soils. Within each treatment group, a general trend was observed where the α diversity index increased with the increase in the proportion of microbial community restorer suspension. This indicates that adding a certain proportion of microbial community restorer to disturbed soil bacterial communities can have a restorative effect, and the higher the proportion, the more significant the restoration effect. The dominant bacterial phyla in the soil of each experimental group were Actinobacteria, Pseudomonas, Bacillus, Chlorconiosis, and Acidobacteria. The relative abundance of Actinobacteria and Bacillus showed a gradually decreasing trend in each experimental group; while Pseudomonas and Bacteroidetes showed a gradually increasing trend. At day 60, compared with the CK group, the relative abundance of Actinobacteria was higher in each treatment group. From day 0 to day 60, the relative abundance of high-risk drug-resistant genera such as Nocardia spp. and Sinomenium spp. gradually decreased in all experimental groups; while the relative abundance of low-risk drug-resistant genera such as Sphingomonas spp. and Lysobacterium spp. gradually increased in all experimental groups. The groups with 30% of the bacterial colony recovery agent (DT70, AT70, DA70) showed the most significant improvement in carbon source utilization, reaching 83.45%, 50.85%, and 54.73%, respectively, which was better than the 50% or 90% addition ratios (e.g., DA50 only 27.62%), indicating that 30% may be a better recovery ratio. Figures 4 to 5 ).

[0059] Adding soil microbial regeneration suspension (especially at high concentrations of 30% and 50%) significantly inhibited the spread of pig manure-derived ARGs (such as sul1, strB, and intl1), with a maximum control rate of 83.42% (strB, AT 50 group). The addition of pig manure led to a significant decrease in soil bacterial α-diversity (ACE, Chao1, Shannon, Simpson indices), indicating that pig manure microbiota has an invasive effect on native soil microbiota. β-diversity analysis showed that the soil microbial structure tended to stabilize after 30 days after the addition of pig manure. Restoring soil microbial diversity (through the addition of soil microbial regeneration suspension) effectively altered the microbial structure and function, thereby inhibiting the spread of most pig manure-derived ARGs. Its effect was influenced by both the restoration ratio and the type of stress. Figure 6 ).

[0060] Example 2: Variation characteristics of ARGs and MGEs abundance

[0061] 1. Abundance variation of tetG

[0062] On day 30, the absolute abundance of tetG in the AT90 and AT70 groups was similar and significantly lower than that in the other groups. However, by day 60, the absolute abundance of tetG in these two groups was the highest among all groups, significantly higher than that in CK100 and AT100 (P<0.05). From day 0 to day 60, the absolute abundance of tetG in the DA50 group was significantly higher than that in the other groups (P<0.05). The relative abundance of tetG in each experimental group first decreased on day 30, then gradually increased, reaching its highest value on day 60, which was significantly higher than that on day 45.

[0063] 2. Abundance variation of sul1

[0064] The absolute abundance of sul1 in each treatment group was low on day 0, gradually increasing until day 60, when it was significantly higher than on day 0 (P < 0.05). On day 60, the absolute abundance of sul1 in the DT90 group was significantly higher than that in the DT70 and DT50 groups (P < 0.05), while there were no significant differences among the other groups (P > 0.05). From day 15 to day 60, the absolute abundance of sul1 in the AT100 group was significantly higher than that in the other groups (P < 0.05), with the order being AT100 > AT70 > AT90 > AT50 among the other treatment groups. On day 60, compared with AT100, the inhibition rates of AT90, AT70, and AT50 on sul1 were 40.87%, 33.66%, and 56.36%, respectively. On day 60, compared with DT100, the control rates of DA90, DA70 and DA50 for sul1 were 50.69%, 36.14% and 38.39%, respectively.

[0065] 3. Abundance variation of strB

[0066] The absolute abundance of strB in each treatment group was low on day 0, gradually increasing until day 60, when it was significantly higher than on day 0 (P < 0.05). On day 30, the absolute abundance of strB in the DT70 and DT50 groups was significantly lower than that in the DT100 and DT90 groups (P < 0.05); on day 60, the absolute abundance of strB in the AT50 group was significantly lower than that in the other groups (P < 0.05), and the absolute abundance of strB in each treatment group was AT100 > AT70 > AT90 > AT50; on day 60, compared with AT100, the blocking rates of AT90, AT70, and AT50 for strB were 67.56%, 66.93%, and 83.42%, respectively. The absolute abundance of strB in each treatment group was DA100 > DA50 > DA70 > DA90; on day 60, the blocking rates of strB by DA90, DA70 and DA50 were 76.88%, 56.11% and 53.78% respectively compared with DA100.

[0067] From day 15 to day 60, the relative abundance of strB in the DT70 and DT50 groups was lower than that in the other groups, and was significantly lower than that in the DT90 group at day 15 (P < 0.05), significantly lower than that in the CK100 and DT100 groups at day 45 (P < 0.05), and significantly lower than that in the DT100 group at day 60 (P < 0.05).

[0068] 4. Abundance changes of ermA

[0069] The absolute abundance of ermA in each treatment group showed a slow increasing trend from day 0 to day 60, but there was no significant difference (P > 0.05). The relative abundance of ermA in each experimental group showed a slow decreasing trend from day 0 to day 60. By day 60, the relative abundance of ermA in each experimental group was significantly lower than that on day 0 (P < 0.05). On day 15, the absolute abundance of ermA in the CK100 and AT100 groups was significantly higher than that in the AT90 and AT50 groups (P < 0.05). On day 45, the absolute abundance of ermA in the AT100 group was significantly higher than that in the AT50 group (P < 0.05).

[0070] 5. Abundance variation of intl1

[0071] The absolute abundance of intl1 in each treatment group was low on day 0, gradually increasing until day 60, when the absolute abundance of intl1 in all groups was significantly higher than on day 0 (P < 0.05). From day 15 to day 60, the absolute abundance of intl1 in the AT100 group was significantly higher than that in the other groups (P < 0.05), and the absolute abundance of intl1 in each treatment group was AT100 > AT70 > AT90 > AT50. On day 60, compared with AT100, the blocking rates of intl1 by AT90, AT70, and AT50 were 47.73%, 47.05%, and 55.78%, respectively. On day 60, the absolute abundance of intl1 in the DA100 group was significantly higher than that in the CK100, DA90, and DA70 groups (P < 0.05). The absolute abundance of intl1 in each treatment group was DA100 > DA50 > DA70 > DA90. On day 60, compared with DA100, the blocking rates of intl1 by DA90, DA70, and DA50 were 53.93%, 30.36%, and 17.90%, respectively.

[0072] The relative abundance of intl1 in each experimental group did not change significantly from day 0 to day 60. On day 45, the relative abundance of intl1 in the DT70 group was significantly lower than that in the DT100 group (P < 0.05); on day 15, the relative abundance of intl1 in the DA90, DA70, and DA50 groups was significantly lower than that in the DA100 group (P < 0.05); on day 30, the relative abundance of intl1 in the DA90 and DA50 groups was significantly lower than that in the DA100 group (P < 0.05); on day 45, the relative abundance of intl1 in the DA90 and DA50 groups was significantly lower than that in the other groups (P < 0.05); and on day 60, the relative abundance of intl1 in the DA90 group was significantly lower than that in the DA100 group (P < 0.05).

[0073] 6. Abundance variation of Tn916 / 1545

[0074] On day 30, the absolute abundance of Tn916 / 1545 in the DT70 group was significantly lower than that in the other groups (P < 0.05). On day 60, the absolute abundance of Tn916 / 1545 in the DT100 and DT50 groups was significantly lower than that in the DT90 group (P < 0.05). From day 30 to day 60, the absolute abundance of Tn916 / 1545 in the AT50 group was significantly lower than that in the AT100 group.

[0075] The relative abundance of Tn916 / 1545 in all experimental groups on day 0 was significantly higher than that on days 15 to 60 (P < 0.05). After a sharp drop from day 0 to day 15, the relative abundance of Tn916 / 1545 in all experimental groups remained relatively stable until day 60.

[0076] 7. Effects of different treatments on the number of culturable bacteria in soil

[0077] From day 0 to day 60, the number of culturable bacteria in each experimental group ranged from 3.33 × 10⁻⁶. 5 ~ 1.35×10 7 The CFU / g ratio shows a gradually increasing trend.

Claims

1. A soil microbial community restorer, characterized by, The soil microbial community recovery agent is obtained by mixing a local normal soil suspension and bamboo charcoal in a certain proportion, and the mass ratio of the local normal soil suspension to the bamboo charcoal is 1:

3.

2. The soil bacterial community rejuvenator of claim 1, wherein, The bamboo charcoal is prepared by the following method: the bamboo is first screened through a 200-mesh sieve, then pyrolyzed at high temperature, and prepared in a nitrogen environment.

3. The use of the soil microbial community recovery agent of claim 2 in improving the microbial community diversity in soil.

4. Use according to claim 3, characterized in that, The improvement of the microbial community diversity in soil refers to: (1) increasing the number of beneficial microorganisms in soil under drought and / or antibiotic stress; (2) restoring the alpha diversity and / or beta diversity of microorganisms in soil under drought and / or antibiotic stress.

5. The use of the soil microbial community recovery agent of claim 2 in inhibiting the spread of ARGs in pig manure source contaminated soil.

6. Use according to claim 4 or 5, characterized in that, The soil microbial community recovery agent is mixed into the soil under drought and / or antibiotic stress at a mass ratio of 10-50%, and then the recovered soil is obtained, which is made into a suspension to obtain a normal soil suspension, which can be used for soil under drought and / or antibiotic stress.

7. The use of the normal soil suspension of claim 6 in inhibiting the spread of ARGs in pig manure source contaminated soil.

8. Use according to claim 7, characterized in that, The inhibition of the spread of ARGs in pig manure source contaminated soil refers to: (1) reducing the abundance of antibiotic resistance genes tetG, sul1, strB, ermA, intl1, Tn916 / 1545 in pig manure source contaminated soil; (2) increasing the number of cultivable bacteria in pig manure source contaminated soil.

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