Method for eliminating virulence genes of pathogenic bacteria in soil

Abstract

The present application provides a method for eliminating virulence genes of pathogenic bacteria in soil, comprising: obtaining soil to be repaired and cultured soil; performing microbial community detection on the cultured soil to obtain community diversity, relative abundance of Proteobacteria community, and relative abundance of Acidobacteria community; when the community diversity, relative abundance of Proteobacteria community, and relative abundance of Acidobacteria community of the cultured soil meet preset conditions, placing biochar in the cultured soil for incubation to obtain a microbial-loaded biochar material; placing the microbial-loaded biochar material in the soil to be repaired for incubation to obtain repaired soil. This method for eliminating virulence genes of pathogenic bacteria in soil is simple and convenient, can effectively eliminate virulence genes of pathogenic bacteria in the soil, will not affect the viability of bacteria in the soil, and has no effect on the absolute abundance of bacteria in the soil.

Description

Technical Field

[0001] The present application relates to the technical field of soil remediation, and in particular to a method for eliminating virulence genes of pathogenic bacteria in soil. Background Art

[0002] Pathogenic bacteria in the environment and their migration and spread can cause some zoonotic diseases, which in turn threaten human health and ecosystem safety. According to statistics, about 61% of human pathogens are zoonotic in nature. Zoonotic bacterial pathogens (i.e., pathogens related to humans and animals) can be transmitted to humans through any contact point with domestic or wild animals. In this process, they can indirectly increase their pathogenicity and induce diseases by producing virulence factors (encoded by corresponding virulence genes). The virulence genes carried by pathogenic bacteria are a new type of pollutant with microbial properties, which has potential health and ecological risks.

[0003] Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Salmonella enterica are four typical zoonotic pathogens widely distributed in soil and water environments. According to the prevention and control standards established by the World Health Organization (WHO) in 2017, Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli are classified as critical groups, and Salmonella enterica belongs to the high-priority group. As common foodborne pathogens, they pose a threat to human and animal health. Although infection studies in the clinical field have provided us with most of the information about virulence genes, there are few reports on virulence gene ablation technologies.

[0004] Since the research on virulence genes of pathogenic bacteria in soil is still in its infancy, many mechanisms are not clear and there are only a few technologies to eliminate them. How to achieve the effect of eliminating virulence genes of soil pathogenic bacteria still needs to be explored. Therefore, there is an urgent need for a simple and effective method to eliminate virulence genes of pathogenic bacteria in soil. Summary of the Invention

[0005] In view of this, the purpose of this application is to propose a method for eliminating virulence genes of pathogenic bacteria in soil.

[0006] Based on the above-mentioned purpose, the present application provides a method for eliminating virulence genes of pathogenic bacteria in soil, comprising: obtaining soil to be repaired and culture soil; performing microbial community detection on the culture soil to obtain community diversity, relative abundance of Proteobacteria community and relative abundance of Acidobacteria community; when the community diversity, relative abundance of Proteobacteria community and relative abundance of Acidobacteria community of the culture soil meet preset conditions, placing biochar in the culture soil for incubation to obtain a microorganism-loaded biochar material; placing the microorganism-loaded biochar material in the soil to be repaired for repair to obtain the repaired soil.

[0007] Furthermore, the preset conditions include: the community diversity is greater than 9.5, the relative abundance of the Proteobacteria community is 40% to 50%, the relative abundance of the Acidobacteria community is 15% to 25%, and the sum of the relative abundance of the Proteobacteria community and the relative abundance of the Acidobacteria community is greater than 60%.

[0008] Furthermore, the relative abundance of the Proteobacteria community is 46%, and the relative abundance of the Acidobacteria community is 20%.

[0009] Furthermore, placing the biochar in the culture soil for incubation to obtain the microorganism-loaded biochar material includes: sieving the biochar and placing it in a mesh bag, burying the mesh bag in the culture soil and incubating it for a first preset time, and then removing the mesh bag from the culture soil to obtain the microorganism-loaded biochar material.

[0010] Furthermore, the mesh number of the sieve is 200, the depth of the mesh bag buried in the culture soil is 5 cm to 20 cm, and the first preset time is 10 days to 15 days.

[0011] Furthermore, the placing of the microorganism-loaded biochar material in the soil to be repaired for repair to obtain the repaired soil includes: adding the microorganism-loaded biochar material to the soil to be repaired according to a preset addition amount and mixing, and repairing the soil for a second preset time to obtain the repaired soil.

[0012] Furthermore, the preset addition amount is 1% of the weight of the soil to be repaired, and the second preset time is 4 to 5 weeks.

[0013] Furthermore, the method for preparing the biochar comprises: subjecting the biochar raw material to a pyrolysis reaction under anoxic or anaerobic conditions, and obtaining the biochar after cooling and drying.

[0014] Furthermore, the biomass charcoal raw material includes plant-derived raw material or animal-derived raw material, the temperature of the pyrolysis reaction is 400° C. to 500° C., and the time of the pyrolysis reaction is 3 h to 5 h.

[0015] Furthermore, the culture soil is one or more of coniferous forest soil, coniferous-broadleaved mixed forest soil, deciduous broadleaved forest soil or evergreen broadleaved forest soil.

[0016] As can be seen from the above, the method provided by the present application for eliminating virulence genes of pathogenic bacteria in soil is to first obtain soil to be repaired and culture soil, and then perform microbial community detection on the culture soil to obtain community diversity, relative abundance of Proteobacteria community and relative abundance of Acidobacteria community; when the community diversity, relative abundance of Proteobacteria community and relative abundance of Acidobacteria community of the culture soil meet the preset conditions, biochar is placed in the culture soil for incubation to obtain a microorganism-loaded biochar material; finally, the microorganism-loaded biochar material is placed in the soil to be repaired for repair, After the repaired soil is obtained, microorganisms are used to load biochar materials to inhibit pathogenic bacteria. Experimental tests have shown that the reduction rate of virulence genes of pathogenic bacteria in the soil is over 60%, and microorganisms can better colonize in the soil. The absolute abundance of bacteria in the soil after repair is not significantly different from that before repair, and the survival ability of bacteria in the soil is not affected. The method of reducing virulence genes of pathogenic bacteria in the soil is simple and convenient, can effectively reduce virulence genes of pathogenic bacteria in the soil, will not affect the survival ability of bacteria in the soil, and has no effect on the absolute abundance of bacteria in the soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in this application or related technologies, the following briefly introduces the drawings required for use in the embodiments or related technical descriptions. Obviously, the drawings described below are merely embodiments of this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0018] Figure 1 Schematic diagram of a process for eliminating virulence genes of pathogenic bacteria in soil according to an embodiment of the present application;

[0019] Figure 2 This is a test chart of the absolute abundance of bacteria in the examples, comparative examples and control soils of this application. DETAILED DESCRIPTION

[0020] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the present disclosure is further described in detail below with reference to specific embodiments.

[0021] It should be noted that, unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods described, unless otherwise specified, are all conventional methods.

[0022] Pathogenic bacteria in the environment and their migration and spread can cause some zoonotic diseases, which in turn threaten human health and ecosystem safety. According to statistics, about 61% of human pathogens are zoonotic in nature. Zoonotic bacterial pathogens (i.e., pathogens related to humans and animals) can be transmitted to humans through any contact point with domestic or wild animals. In this process, they can indirectly increase their pathogenicity and induce diseases by producing virulence factors (encoded by corresponding virulence genes). The virulence genes carried by pathogenic bacteria are a new type of pollutant with microbial properties, which has potential health and ecological risks.

[0023] Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Salmonella enterica are four typical zoonotic pathogens widely distributed in soil and water environments. According to the prevention and control standards established by the World Health Organization (WHO) in 2017, Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli are classified as critical groups, and Salmonella enterica belongs to the high-priority group. As common foodborne pathogens, they pose a threat to human and animal health. Although infection studies in the clinical field have provided us with most of the information about virulence genes, there are few reports on virulence gene ablation technologies.

[0024] Since the research on the virulence genome of pathogenic bacteria in soil is still in its infancy, many mechanisms are not clear and there are only a few technologies for its elimination. How to achieve the effect of eliminating the virulence genes of soil pathogens still needs to be explored. Therefore, there is an urgent need for a method that can simply and effectively eliminate the virulence genes of pathogens in soil.

[0025] Related technologies use specific microbial agents to repair specific crop types or pollutant types, such as rhizobia agents in the rhizosphere of legumes. The purpose is to gain a survival advantage in competition with indigenous microbial communities through interactions with plants. For example, certain Bacillus can reduce the content of pollutants through adsorption, degradation and other mechanisms, and gain an advantage in interspecies competition with other bacteria. However, there are many types of virulence genes in the soil, and the soil environment is often very complex. It is difficult for a single or specific microorganism to gain a survival advantage and colonize, so the effect of eliminating the virulence genes of pathogenic bacteria in the soil is poor.

[0026] Below, through specific embodiments and combined Figures 1 to 2 To describe the technical solution of this application in detail.

[0027] In some embodiments of the present application, a method for eliminating virulence genes of pathogenic bacteria in soil is provided, such as Figure 1 As shown, the following steps are included:

[0028] S1. Obtain soil to be repaired and cultivate soil.

[0029] The soil to be repaired refers to soil with a relatively high proportion of pathogenic bacteria such as Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli or Salmonella enterica, for example, the relative abundance of pathogenic bacteria is greater than 60%, and there is no specific limitation; the culture soil is, for example, one or more of coniferous forest soil, coniferous and broad-leaved mixed forest soil, deciduous broad-leaved forest soil or evergreen broad-leaved forest soil, and there is no specific limitation. The culture soil can be taken from the soil in the forest that is less than 30 cm away from the trees. Obtaining the culture soil provides a basis for the subsequent preparation of microbial-loaded biochar materials.

[0030] S2. Performing microbial community detection on the culture soil to obtain community diversity, relative abundance of Proteobacteria community, and relative abundance of Acidobacteria community.

[0031] Microbial community detection was performed by 16s-rRNA amplicon sequencing and analyzed using Qiime2 software. The community diversity, relative abundance of Proteobacteria community and relative abundance of Acidobacteria community were measured to provide a basis for screening culture soil.

[0032] S3. When the community diversity, the relative abundance of the Proteobacteria community, and the relative abundance of the Acidobacteria community of the culture soil meet preset conditions, placing biochar in the culture soil for incubation to obtain a microbial-loaded biochar material.

[0033] The results showed that the higher the diversity of soil microbial communities, the better their ability to eliminate virulence genes. The relative abundance of Proteobacteria and Acidobacteria communities represent the health of the cultivated soil. The higher the relative abundance, the more conducive it is to the elimination of virulence genes.

[0034] The preset conditions include a community diversity greater than 9.5, such as 9.6, 9.7 or 9.8, a relative abundance of the Proteobacteria community of 40% to 50%, such as 40%, 45% or 50%, a relative abundance of the Acidobacteria community of 15% to 25%, such as 15%, 20% or 25%, and a sum of the relative abundance of the Proteobacteria community and the relative abundance of the Acidobacteria community greater than 60%, such as 60%, 65%, 70%, 75% or 80%, etc., without specific limitation; after testing, using microorganisms in cultured soil that meet the preset conditions to repair the soil can effectively reduce the virulence genes of pathogens, and the total reduction rate reaches more than 35%.

[0035] Biochar is placed in a culture soil that meets the preset conditions for incubation to obtain a microbial-loaded biochar material. As a good microbial carrier, biochar can provide more survival sites and nutrient supply for microorganisms, especially soil microorganisms. Its ability to adsorb pollutants can also reduce the stress of pollutants in the environment on microorganisms, thereby enabling microorganisms to better colonize in the soil, enhancing their survival ability and competition with the original pathogenic bacteria, thereby achieving a better effect of reducing virulence genes.

[0036] S4. Placing the microorganism-loaded biochar material into the soil to be repaired to obtain repaired soil.

[0037] The microorganism-loaded biochar material is placed in the soil to be repaired for repair to obtain the repaired soil, and the repaired soil is tested for virulence genes. The detection method uses, for example, a pathogenic bacteria virulence gene chip (VFG-Chip). The chip targets 96 virulence genes of five major functions of typical human and animal pathogens in the environment (Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli and Salmonella enterica). Based on a high-throughput real-time quantitative PCR (polymerase chain reaction) platform, it can detect the virulence group of 42 samples at one time within 2 hours, and the amplification efficiency of primers greater than 91% is 90% to 110%, with the characteristics of high efficiency, rapidity and accuracy.

[0038] The reduction rate of pathogenic virulence genes in the remediated soil was 60.26%-87.11%, especially for the virulence genes of Klebsiella pneumoniae and Salmonella enterica, with a significant reduction rate of 73.52%-88.30% and 76.39%-91.56%, respectively. The microorganisms loaded with biochar have a stable community structure and rich functions. When applied to the soil to be remediated, they trigger interspecies competition among microorganisms, thereby inhibiting the proliferation of pathogenic bacteria and virulence genes in the contaminated soil community and achieving the purpose of reducing virulence genes. This method can replace the traditional method of preparing specific microbial agents. The method of burying and incubating in culture soil replaces the matrix culture process of traditional microbial agents, reducing preparation costs. In addition, the introduction of microorganisms in the biochar-loaded culture soil not only enables the microorganisms to better colonize and exert their effects in the soil, but also does not affect the survival ability of bacteria in the soil. Experimental tests show that the absolute abundance of bacteria in the remediated soil is not significantly different from that before remediation.

[0039] The method for eliminating virulence genes of pathogenic bacteria in soil is simple and convenient, can effectively eliminate virulence genes of pathogenic bacteria in soil, will not affect the viability of bacteria in soil, and has no effect on the absolute abundance of bacteria in soil.

[0040] In some embodiments, the relative abundance of the Proteobacteria community is 46%, and the relative abundance of the Acidobacteria community is 20%.

[0041] Setting the relative abundance of Proteobacteria community to 46%, the relative abundance of Acidobacteria community to 20%, and the sum of the relative abundance of Proteobacteria community and Acidobacteria community to 66% can achieve the best effect on the reduction of virulence genes of pathogenic bacteria in the soil. After testing, the total reduction rate reached 87.11%.

[0042] In some embodiments, the step of placing the biochar in the culture soil for incubation to obtain the microorganism-loaded biochar material comprises:

[0043] S301, sieving the biochar and placing it into a mesh bag, burying the mesh bag in the culture soil and incubating for a first preset time, and then taking out the mesh bag from the culture soil to obtain the microorganism-loaded biochar material.

[0044] The biochar is screened, and the mesh number of the screen is, for example, 200, to avoid the mesh number being too small, so that the biochar is not easy to load microorganisms, and to avoid the mesh number being too large, so that the relative area of ​​the biochar is too small and it cannot load more microorganisms; the sieved biochar is placed in a mesh bag, which is, for example, a nylon mesh bag, which is corrosion-resistant and has good chemical stability, and can also be a polyethylene mesh bag, a polypropylene mesh bag, etc., without specific limitation; the mesh bag is buried in the culture soil and incubated for a first preset time, and the depth of the mesh bag buried in the culture soil is 5 cm to 2 0cm, for example, 5cm, 10cm, 15cm or 20cm, etc., to avoid a depth that is too shallow and easily disturbed, and also to avoid a depth that is too deep and has a low microbial content. The first preset time is 10 to 15 days, for example, 10 days, 11 days, 12 days, 13 days, 14 days or 15 days; after incubation, the mesh bag is taken out from the culture soil to obtain the microbial-loaded biochar material, and the obtained microbial-loaded biochar material can be stored at a low temperature for standby use, for example, at 0°C-4°C to maintain activity.

[0045] In some embodiments, step S4 includes:

[0046] S401, adding the microorganism-loaded biochar material into the soil to be repaired according to a preset addition amount and mixing them, and obtaining the repaired soil after repairing for a second preset time.

[0047] The microorganism-loaded biochar material is added to the soil to be repaired according to a preset addition amount and mixed. The preset addition amount is 1% of the weight of the soil to be repaired, that is, 100g of the soil to be repaired corresponds to 1g of the microorganism-loaded biochar material. The repaired soil is obtained after the second preset repair time. The second preset time is 4 to 5 weeks, for example, 4 weeks, 4.5 weeks or 5 weeks, etc., and is not specifically limited.

[0048] In some embodiments, the method for preparing biochar comprises:

[0049] S300, subjecting the biochar raw material to pyrolysis reaction under anoxic or anaerobic conditions, and obtaining the biochar after cooling and drying.

[0050] Biomass charcoal raw materials include plant-derived raw materials or animal-derived raw materials. Plant-derived raw materials include one or more of straw, rice husks, wheat bran or bamboo chips. Animal-derived raw materials include one or more of pig manure or chicken bones, and are not specifically limited. The biomass charcoal raw materials are subjected to pyrolysis reaction under anoxic or anaerobic conditions, for example, pyrolysis reaction is carried out under nitrogen protection. The temperature of the pyrolysis reaction is 400°C to 500°C, for example, 400°C, 450°C or 500°C, etc., to avoid the temperature being too low, which makes the pores of the biochar too small, and to avoid the temperature being too high, which makes the yield of the biochar low. The pyrolysis reaction time is 3h to 5h, for example, 3h, 4h or 5h, etc., and is not specifically limited.

[0051] Comparative Example 1

[0052] The first culture soil was obtained and divided into five parts. The microbial community was detected in each part of the first culture soil (16s-rRNA amplicon sequencing was used for analysis using Qiime2 software). The average value of the community diversity (Shannon index) was 9.61, the average value of the relative abundance of the Proteobacteria community was 42.6%, the average value of the relative abundance of the Acidobacteria community was 21.7%, and the average value of the sum of the relative abundance of the Proteobacteria community and the relative abundance of the Acidobacteria community was 64.3%. The microorganisms in each part of the first culture soil were extracted using PBS buffer.

[0053] The soil to be remediated was obtained and divided into five parts. The microorganisms in each part of the first cultured soil were added to one part of the soil to be remediated and mixed. The soil was remediated at 60% of the field capacity for 4 weeks to obtain the remediated soil (D1-1, D1-2, D1-3, D1-4 and D1-5). The total reduction rate of pathogenic bacteria virulence genes in the remediated soil was detected (calculated based on the relative abundance of the 16s gene, detected using the VFG-Chip pathogenic bacteria virulence gene chip). The average total reduction rate was 35.24%.

[0054] Comparative Example 2

[0055] A second culture soil was obtained and divided into five parts. The microbial community was detected in each part of the second culture soil (16s-rRNA amplicon sequencing, analyzed using Qiime2 software). The average value of the community diversity (Shannon index) was 9.7, the average value of the relative abundance of the Proteobacteria community was 48.93%, the average value of the relative abundance of the Acidobacteria community was 17.82%, and the average value of the sum of the relative abundance of the Proteobacteria community and the relative abundance of the Acidobacteria community was 66.75%. The microorganisms in each part of the second culture soil were extracted using PBS buffer.

[0056] The soil to be remediated was obtained and divided into five parts. The microorganisms from each part of the second cultured soil were added to one part of the soil to be remediated and mixed. The soil was remediated at 60% of the field capacity for 4 weeks to obtain the remediated soil (D2-1, D2-2, D2-3, D2-4 and D2-5). The total reduction rate of pathogenic bacteria virulence genes in the remediated soil was detected (calculated based on the relative abundance of the 16s gene, detected using the VFG-Chip pathogenic bacteria virulence gene chip). The average total reduction rate was 49.25%.

[0057] Comparative Example 3

[0058] The third incubation soil was obtained and divided into five parts. The microbial community was detected in each part of the third incubation soil (16s-rRNA amplicon sequencing was used for analysis using Qiime2 software). The average value of the community diversity (Shannon index) was 9.74, the average value of the relative abundance of the Proteobacteria community was 46.21%, the average value of the relative abundance of the Acidobacteria community was 19.58%, and the average value of the sum of the relative abundance of the Proteobacteria community and the relative abundance of the Acidobacteria community was 65.79%. The microorganisms in each part of the third incubation soil were extracted using PBS buffer.

[0059] The soil to be remediated was obtained and divided into five parts. The microorganisms from each part of the third cultured soil were added to one part of the soil to be remediated and mixed. The soil was remediated at 60% of the field capacity for 4 weeks to obtain the remediated soil (D3-1, D3-2, D3-3, D3-4 and D3-5). The total reduction rate of pathogenic bacteria virulence genes in the remediated soil was detected (calculated based on the relative abundance of the 16s gene, detected using the VFG-Chip pathogenic bacteria virulence gene chip). The average total reduction rate was 47.81%.

[0060] Comparative Example 4

[0061] The fourth incubation soil was obtained and divided into five parts. The microbial community was detected in each part of the fourth incubation soil (16s-rRNA amplicon sequencing was used for analysis using Qiime2 software). The average value of the community diversity (Shannon index) was 9.25, the average value of the relative abundance of the Proteobacteria community was 16.21%, the average value of the relative abundance of the Acidobacteria community was 28.79%, and the average value of the sum of the relative abundance of the Proteobacteria community and the relative abundance of the Acidobacteria community was 45%. The microorganisms in each part of the fourth incubation soil were extracted using PBS buffer.

[0062] The soil to be remediated was obtained and divided into five parts. The microorganisms from each fourth culture soil were added to one part of the soil to be remediated and mixed. The soil was remediated at 60% of the field capacity for 4 weeks to obtain the remediated soil (D4-1, D4-2, D4-3, D4-4 and D4-5). The total reduction rate of pathogenic bacteria virulence genes in the remediated soil was detected (calculated based on the relative abundance of the 16s gene and detected using the VFG-Chip pathogenic bacteria virulence gene chip). The average total reduction rate was 6.47%.

[0063] Comparative Example 5

[0064] The fifth incubation soil was obtained and divided into five parts. The microbial community was detected in each part of the fifth incubation soil (16s-rRNA amplicon sequencing, analyzed using Qiime2 software). The average value of the community diversity (Shannon index) was 8.94, the average value of the relative abundance of the Proteobacteria community was 36.06%, the average value of the relative abundance of the Acidobacteria community was 9.2%, and the average value of the sum of the relative abundance of the Proteobacteria community and the relative abundance of the Acidobacteria community was 45.26%. The microorganisms in each part of the fifth incubation soil were extracted using PBS buffer.

[0065] The soil to be remediated was obtained and divided into five parts. The microorganisms from each fifth culture soil were added to one part of the soil to be remediated and mixed. The soil was remediated at 60% of the field capacity for 4 weeks to obtain the remediated soil (D5-1, D5-2, D5-3, D5-4 and D5-5). The total reduction rate of pathogenic bacteria virulence genes in the remediated soil was detected (calculated based on the relative abundance of the 16s gene, detected using the VFG-Chip pathogenic bacteria virulence gene chip). The average total reduction rate was 45.26%.

[0066] Table 1 Comparative Examples 1-5 Test Results

[0067]

[0068]

[0069] The cultured soil tested was, for example, taken from the coniferous and broad-leaved mixed forest soil of Tiantong Mountain National Forest Park, Yinzhou District, Ningbo City, Zhejiang Province. The cultured soils of Comparative Examples 1 to 5 were respectively subjected to microbial community detection. The experimental results are shown in Table 1. The microorganisms in the cultured soils were extracted and mixed into the soil to be repaired for repair. The virulence genes of the repaired soil were tested and the total reduction rate was calculated. The experimental results are shown in Table 1. The cultured soils of Comparative Examples 1 to 3 had a significantly better effect on reducing the virulence genes of pathogens than Comparative Examples 4 and 5, indicating that the community diversity was greater than 9.5, the relative abundance of the Proteobacteria community was 40% to 50%, the relative abundance of the Acidobacteria community was 15% to 25%, and the sum of the relative abundance of the Proteobacteria community and the relative abundance of the Acidobacteria community was greater than 60%. The cultured soil can effectively reduce the virulence genes.

[0070] Example 1

[0071] The first culture soil of comparative example 1 was obtained for microbial community detection. The test showed that the community diversity was 9.61, the relative abundance of the Proteobacterium community was 42.6%, the relative abundance of the Acidobacteria community was 21.7%, and the sum of the relative abundance of the Proteobacteria community and the relative abundance of the Acidobacteria community was 64.3%, which met the preset conditions.

[0072] The biochar was sieved with a mesh size of 200 to remove biochar with smaller particle size. The sieved biochar was put into a nylon mesh bag with a mesh size of 200. The nylon mesh bag was buried in the first culture soil to a depth of 10 cm and incubated for 10 days. The nylon mesh bag was removed from the first culture soil to obtain a microbial-loaded biochar material.

[0073] The weight of the first culture soil was weighed, and 1% of the weight of the microorganism-loaded biochar material was added to the first culture soil and mixed. The soil was repaired for 4 weeks at 60% of the field capacity to obtain the repaired soil.

[0074] Example 2

[0075] The second culture soil of Comparative Example 2 was obtained for microbial community detection. The community diversity was 9.7, the relative abundance of the Proteobacterium community was 48.93%, the relative abundance of the Acidobacteria community was 17.82%, and the sum of the relative abundance of the Proteobacteria community and the relative abundance of the Acidobacteria community was 66.75%, which met the preset conditions.

[0076] The biochar was sieved with a mesh size of 200 to remove biochar with smaller particle size. The sieved biochar was put into a nylon mesh bag with a mesh size of 200. The nylon mesh bag was buried in the second culture soil to a depth of 10 cm. After incubation for 10 days, the nylon mesh bag was removed from the second culture soil to obtain a microbial-loaded biochar material.

[0077] The second culture soil was weighed, and 1% of the weight of the microbial-loaded biochar material was added to the second culture soil and mixed. The soil was repaired at 60% of the field capacity for 4 weeks to obtain the repaired soil.

[0078] Example 3

[0079] The third culture soil of comparative example 3 was obtained for microbial community detection. After testing, the community diversity was 9.74, the relative abundance of the Proteobacterium community was 46.21%, the relative abundance of the Acidobacteria community was 19.58%, and the sum of the relative abundance of the Proteobacteria community and the relative abundance of the Acidobacteria community was 65.79%, which met the preset conditions.

[0080] The biochar was sieved with a mesh size of 200 to remove biochar with smaller particle size. The sieved biochar was put into a nylon mesh bag with a mesh size of 200. The nylon mesh bag was buried in the third culture soil to a depth of 10 cm. After incubation for 10 days, the nylon mesh bag was removed from the third culture soil to obtain a microbial-loaded biochar material.

[0081] The third culture soil was weighed, and 1% of the weight of the microorganism-loaded biochar material was added to the third culture soil and mixed. The soil was repaired for 4 weeks at 60% of the field capacity to obtain the repaired soil.

[0082] Table 2 Comparative Examples and Examples Reduction Rate Test Table

[0083]

[0084] The virulence genes of the soil repaired by Examples 1 to 3 and Comparative Examples 1 to 3 were tested, and the reduction rate and total reduction rate of the virulence genes of various pathogens were calculated. The experimental results are shown in Table 2. The reduction effect of virulence genes in Examples 1 to 3 was significantly better than that in Comparative Examples 1 to 3. From the results, it can be seen that a certain virulence gene elimination effect can be achieved by simply adding microorganisms. After applying microbial-loaded biochar using the technology of this embodiment, the effect is significantly improved compared to the effect of applying microorganisms alone, and a total of 60.26%-87.11% of the virulence genes of soil pathogens can be reduced. In particular, the virulence genes of the main pathogens in the soil: Klebsiella pneumoniae and Salmonella enterica have a good reduction effect, with the effects reaching 73.52%-88.30% and 76.39%-91.56%, respectively. Although the sum of the relative abundance of the Proteobacteria community and the relative abundance of the Acidobacteria community in Example 3 is not the highest, the various indicators have achieved the best reduction effect.

[0085] In addition, the absolute abundance of bacteria was tested on the soils repaired in Examples 1 to 3, Comparative Examples 1 to 3, and the control soil. The control soil was the soil to be repaired without any treatment. The experimental results are as follows: Figure 2 As shown, the absolute abundance of bacteria in the soil after repair in Comparative Examples 1 to 3 was significantly reduced, indicating that the survival ability of bacteria in the soil was affected. However, the absolute abundance of bacteria in the soil after repair in Examples 1 to 3 was not significantly different from that before repair, which did not affect the survival ability of bacteria in the soil. The colonization ability of microorganisms can be improved, thereby achieving a better effect of reducing virulence genes.

[0086] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples. Within the scope of the present disclosure, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in detail for the sake of simplicity.

[0087] In addition, when details are set forth to describe exemplary embodiments of the present disclosure, it will be apparent to those skilled in the art that the present disclosure embodiments can be implemented without or with variations in these details. Therefore, these descriptions should be considered illustrative rather than restrictive.

[0088] While the disclosure has been described in conjunction with embodiments thereof, many alternatives, modifications and variations of these embodiments will be apparent to those skilled in the art in light of the foregoing description.

[0089] The embodiments of the present disclosure are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present disclosure should be included in the scope of protection of the present disclosure.

Claims

1. A method for eliminating virulence genes of pathogenic bacteria in soil, characterized in that: include: Obtaining soil to be remediated and cultivated soil, wherein the soil to be remediated refers to soil with a high proportion of pathogenic bacteria such as Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, or Salmonella enterica; Conducting microbial community testing on the cultured soil to obtain community diversity, relative abundance of Proteobacteria community, and relative abundance of Acidobacteria community; When the community diversity, the relative abundance of the Proteobacteria community and the relative abundance of the Acidobacteria community of the culture soil meet preset conditions, the biochar is placed in the culture soil for incubation to obtain a microbial-loaded biochar material; the preset conditions include: the community diversity is greater than 9.5, the relative abundance of the Proteobacteria community is 46%, and the relative abundance of the Acidobacteria community is 20%; the placing of the biochar in the culture soil for incubation to obtain the microbial-loaded biochar material includes: sieving the biochar and placing it in a mesh bag, burying the mesh bag in the culture soil and incubating it for a first preset time, and then taking out the mesh bag from the culture soil to obtain the microbial-loaded biochar material; the mesh number of the sieve is 200, the depth of the mesh bag buried in the culture soil is 5 cm to 20 cm, and the first preset time is 10 days to 15 days; Placing the microorganism-loaded biochar material in the soil to be repaired for repair to obtain the repaired soil, comprising: adding the microorganism-loaded biochar material to the soil to be repaired according to a preset addition amount and mixing, and repairing the soil after a second preset time to obtain the repaired soil; the preset addition amount is 1% of the weight of the soil to be repaired, and the second preset time is 4 to 5 weeks.

2. The method for eliminating virulence genes of pathogenic bacteria in soil according to claim 1, characterized in that: The method for preparing biochar comprises: The biomass charcoal raw material is subjected to pyrolysis reaction under anoxic or anaerobic conditions, and the biomass charcoal is obtained after cooling and drying.

3. The method for eliminating virulence genes of pathogenic bacteria in soil according to claim 2, characterized in that: The biomass charcoal raw material includes plant-derived raw material or animal-derived raw material. The temperature of the pyrolysis reaction is 400° C. to 500° C., and the time of the pyrolysis reaction is 3 hours to 5 hours.

4. The method for eliminating virulence genes of pathogenic bacteria in soil according to claim 1, characterized in that: The culture soil is one or more of coniferous forest soil, coniferous-broadleaved mixed forest soil, deciduous broadleaved forest soil or evergreen broadleaved forest soil.

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