New application of agricultural enzyme and method for controlling soil pathogenic bacteria biological pollution

Abstract

The application discloses a new use of an agricultural enzyme and a method for controlling and inhibiting soil pathogenic bacteria biological pollution, wherein the agricultural enzyme is used as a soil repair agent to prevent or treat soil pathogenic bacteria biological pollution, and the soil pathogenic bacteria are at least one of Klebsiella pneumoniae, Acidovorax xylosoxidans, Streptomyces orientatis, Vibrio cholerae, Riemerella anatipestifer, Clostridium botulinum, Serratia marcescans, Rhodococcus globerulus and Aeromonas salmonicida. The agricultural enzyme is applied to the pathogenic bacteria biological pollution soil to be treated in different ways. The application directly kills the pathogenic bacteria or regulates the soil microbial community through a quorum sensing gene and a virulence factor gene, so that the abundance of the pathogenic bacteria in the soil community is reduced, and the soil pathogenic bacteria biological pollution is reduced. The application is an economic, green, convenient, efficient agricultural soil environment repair technology.

Description

Technical Field

[0001] This invention belongs to the field of prevention and control of soil pathogenic microbial pollution, specifically involving a method for controlling soil pathogenic microbial pollution. This method indirectly antagonizes soil pathogens by directly killing them or reducing quorum sensing genes and virulence factor genes, thereby alleviating soil pathogenic microbial pollution caused by the application of organic fertilizers. Background Technology

[0002] Pathogenic bacterial infections can cause diseases in humans and animals, and are a major cause of global health losses, becoming the second leading cause of death worldwide, second only to ischemic heart disease. Soil, as the most important habitat for microorganisms, contains pathogens including, but not limited to, Klebsiella pneumoniae, Xylose-oxidizing achromobacterium, Streptomyces orientalis, Vibrio cholerae, Riemerella anatipestifer, Clostridium botulinum, Serratia marcescens, Rhodococcus spp., and Aeromonas salmonicidae. Soil pathogens can spread to humans and animals through the digestive tract, respiratory tract, and skin / mucous membranes. Furthermore, pathogens can enhance their overall resistance and survival rate by using quorum sensing, a social behavior that increases resistance to foreign substances such as fertilizers, pesticides, and fungicides. Quorum sensing can also regulate horizontal gene transfer, promoting the amplification of virulence factors from pathogens in non-pathogenic bacteria, potentially leading to non-pathogenic bacteria carrying pathogenic factors and becoming pathogenic. Studies show that with global warming, the proportion of soil pathogens is increasing, and soil pathogenic biological pollution is becoming increasingly serious. Therefore, the prevention and control of soil pathogenic microbial pollution is of utmost urgency.

[0003] The main sources of soil pathogens include untreated human and animal excrement as fertilizer, landfilling of domestic waste, direct irrigation with industrial or hospital wastewater containing pathogens, and improper disposal of dead livestock carcasses. These sources allow large quantities of pathogenic and infectious bacteria to enter and proliferate in the soil, leading to soil-borne biological pollution. Based on this principle of soil-borne biological pollution, current methods for preventing or controlling it can be divided into chemical and biological methods. Chemical methods involve adding chemical substances such as fertilizers and organic acids to the soil. The basic principle is to kill or antagonize soil pathogens by altering soil fertility, pH, or salinity. For example, Chinese invention application numbers CN201811207698, CN201910194557, and CN202010283287 disclose a fertilizer for increasing soil fertility that inhibits the growth and proliferation of soil pathogens by improving soil permeability and regulating soil pH; an organic acid produced by anaerobic fermentation of organic material applied to tobacco fields that can directly kill some pathogens in the soil; and a protein fertilizer formula that achieves the purpose of killing or antagonizing soil pathogens by improving the dynamic balance of the rhizosphere microbial community.

[0004] Biological methods involve adding animals such as earthworms and microorganisms, including but not limited to fungi and bacteria, to the soil to antagonize or kill soil pathogens. For example, Chinese invention application CN201610780440 discloses a straw composting agent made by compound fermentation of Streptomyces cirrhosa and Streptomyces niger, which can significantly reduce soil pathogenic microorganisms; CN201910854981 discloses a method that uses Bacillus to inhibit the growth of soil pathogens and improve the soil micro-ecological environment; CN202011478399 provides a biological disinfection method for vegetable greenhouse soil using yeast and actinomycetes in a multi-stage facility, which kills soil pathogens by regulating the micro-environment through the formation of a dynamic balance of soil water, fertilizer, air and heat.

[0005] The aforementioned methods utilize chemical compound fertilizers such as protein fertilizers and organic materials, or biological fungicides such as fungi and bacteria, to improve the soil microbial community and antagonize or kill soil pathogens. However, these methods all have limitations, such as being uncommon and only applicable to specific soil types; and being complex to produce, requiring the addition of multiple fungi or bacteria. Chinese invention application CN2021110451426 discloses an agricultural enzyme that has shown good effects in controlling soil resistance gene pollution, and its effects are correlated with actinomycetes in the soil. Since resistance genes are deoxyribonucleotides, which are chemical substances, the soil resistance gene pollution described in this application is essentially soil chemical pollution rather than soil biological pollution. Summary of the Invention

[0006] This application provides an application of agricultural enzymes as antagonists to kill or antagonize soil pathogens and a method for controlling soil pathogen biopollution. The method utilizes agricultural enzymes to directly kill pathogens or regulates the soil microbial community through quorum sensing genes and virulence factor genes, thereby reducing the abundance of pathogens in the soil community and reducing soil pathogen biopollution. This is an economical, green, convenient and efficient agricultural soil environment remediation technology.

[0007] The application of an agricultural enzyme as a soil remediation agent in the prevention or treatment of soil pathogenic bacterial biopollution, wherein the soil pathogenic bacteria are at least one of Klebsiella pneumoniae, Achromobacter xylosoxidans, Streptomyces orientalis, Vibrio cholerae, Riemerella anatipestifer, Clostridium botulinum, Serratia marcescens, Rhodococcus fascians, and Aeromonas salmonicida.

[0008] Optionally, the soil pathogenic bacteria are at least one of Klebsiella pneumoniae, Xylose-oxidizing achromobacterium, Vibrio cholerae, Riemerella anatipestifer, Clostridium botulinum, Serratia marcescens, and Aeromonas salmonidae.

[0009] Optionally, the soil pathogen is *Achromobacter xylose-oxidizing* or *Vibrio cholerae*. High concentrations of *Achromobacter xylose-oxidizing* resulted in a 25% reduction, while high concentrations of *Vibrio cholerae* resulted in a 52% reduction.

[0010] Optionally, the agricultural enzyme is a liquid fermented product obtained by fermenting fruit and vegetable residues.

[0011] The present invention also provides a method for inhibiting soil pathogenic microbial biofouling, comprising:

[0012] Agricultural enzymes can be diluted and applied directly to the soil contaminated with pathogenic bacteria, or fertilizer can be applied first and then applied to the soil contaminated with pathogenic bacteria, or agricultural enzymes can be mixed with fertilizer and then applied to the soil contaminated with pathogenic bacteria.

[0013] The agricultural enzyme is a liquid fermented product obtained through the fermentation of fruit and vegetable residues;

[0014] The pathogenic bacteria are at least one of the following: Klebsiella pneumoniae, Xylose-oxidizing achromobacterium, Streptomyces orientalis, Vibrio cholerae, Riemerella anatipestifer, Clostridium botulinum, Serratia marcescens, Rhodococcus spp., and Aeromonas salmonicidae.

[0015] Optionally, the soil pathogenic bacteria are at least one of Klebsiella pneumoniae, Xylose-oxidizing achromobacterium, Vibrio cholerae, Riemerella anatipestifer, Clostridium botulinum, Serratia marcescens, and Aeromonas salmonidae.

[0016] Furthermore, the soil pathogen is *Achromobacter xylose oxidizing* or *Vibrio cholerae*.

[0017] Optionally, the soil remediation agent is a liquid fermentation stock solution diluted 10 to 20 times;

[0018] The preparation process of the liquid fermentation stock solution is as follows:

[0019] Mix fruit and vegetable residues, brown sugar, water, and baking powder in a certain proportion, seal and ferment. The liquid obtained by filtering the fermentation product is the liquid fermentation product.

[0020] Optionally, the ratio of the fruit and vegetable residues, brown sugar, water and baking powder is as follows (by weight): 4-6 parts fruit and vegetable residues, 0.5-2.5 parts brown sugar, 15-25 parts water, and 0.5-2.5 parts baking powder.

[0021] Optionally, the fruit and vegetable residues include, but are not limited to, fruit pits, peels, discarded vegetable leaves, rotten fruit and vegetables, etc.

[0022] Optionally, the filter screen required for filtration should be no less than 10 mesh to ensure separation of residue and liquid fermentation product, and the resulting liquid fermentation product should not contain obvious solids.

[0023] Optionally, the fermentation includes an early aerobic fermentation and a later anaerobic fermentation. The early aerobic fermentation time is 10-15 days, and the later anaerobic fermentation time is 35-65 days, with a total fermentation time of 45-80 days and a fermentation temperature of 20-40℃. During the early aerobic fermentation, the lid needs to be opened to release gas and stirred once every 3-5 days.

[0024] Furthermore, the proportions are in parts by weight: 5 parts fruit and vegetable residues, 2 parts brown sugar, 20 parts water, and 2 parts baking powder.

[0025] Optionally, the baking powder contains lactic acid bacteria; any commercially available baking powder containing lactic acid bacteria is acceptable, for example, the commonly available Angel Yeast baking powder can be selected.

[0026] The pH of the liquid fermented product was 3.5-5, and it had a slightly acidic ester aroma. The dominant bacterial group in the liquid fermented product was Lactobacillus, with an abundance of no less than 90% of the sequenced product.

[0027] Optionally, the soil remediation agent is applied at a rate of 10-20 ml per square meter of soil or 10-20 ml per plant root; applied once every 3-6 months.

[0028] Optionally, the fertilizer is one of pig manure organic fertilizer, chicken manure organic fertilizer, cow manure organic fertilizer, and silkworm manure organic fertilizer.

[0029] This application discovers that antagonists made from agricultural enzymes can effectively reduce the abundance of pathogens in the soil and reduce quorum sensing genes and virulence factor genes. This application also discovers that the antagonists have good effects in killing or antagonizing soil pathogens. The antagonists are inexpensive, easy to apply, and have obvious effects, and have broad and sustainable application prospects.

[0030] This invention discovers that: on the one hand, agricultural enzymes can directly kill the pathogens; on the other hand, agricultural enzymes can regulate pathogens by reducing quorum sensing genes and virulence factor genes.

[0031] Compared with existing technologies for antagonizing soil pathogenic bacteria biocontamination, this application has at least one of the following superior effects:

[0032] (1) Fruit and vegetable residues are widely available and inexpensive, making them a high-quality and readily available raw material for agricultural enzymes;

[0033] (2) The liquid fermentation product formed by aerobic and anaerobic fermentation of fruit and vegetable residues has the characteristics of high organic matter, high probiotics, high mineral nutrients and high humic acid. It can not only treat fruit and vegetable residues to reduce the pressure of garbage disposal, but also serve as a good substitute for chemical fertilizers in the soil.

[0034] (3) The above-mentioned agricultural enzyme is simple to prepare, has a simple process, is convenient to transport and store, and has no operational threshold. It is an agricultural enzyme with low process cost.

[0035] (4) The pathogenic bacteria antagonist prepared by diluting the above-mentioned agricultural enzymes has a good inhibitory effect on pathogenic bacteria in the soil; on the one hand, it can directly kill the pathogenic bacteria; on the other hand, it can regulate pathogenic bacteria by reducing quorum sensing genes and virulence factor genes.

[0036] (5) Compared with methods such as inhibiting antibiotic resistance genes, the above-mentioned method of directly killing the pathogens can prevent the proliferation and spread of pathogens from the source, which is direct and efficient in controlling soil pathogen pollution. Secondly, by reducing the two genes directly related to the proliferation of pathogens, namely quorum sensing genes and virulence factor genes, the information exchange between soil pathogens can be minimized, thereby destroying their self-protection mechanism and preventing the proliferation of pathogens during the spread process.

[0037] (6) The method of this application has a very positive effect on increasing the yield of grain crops in my country and reducing soil pathogenic bacteria pollution. Therefore, the agricultural enzyme has a wide application prospect and promotion area. Attached Figure Description

[0038] Figure 1 This is a graph showing the changes in the total abundance of the nine pathogenic bacteria in the soil in Example 2.

[0039] Figure 2 This is a graph showing the abundance changes of Klebsiella pneumoniae, Xylose-oxidizing achromobacterium, and Streptomyces orientalis in Example 3.

[0040] Figure 3 This is a graph showing the abundance changes of Vibrio cholerae, Riemerella anatipestifer, and Clostridium botulinum in Example 4.

[0041] Figure 4 This is a graph showing the abundance changes of Serratia marcescens, Rhodococcus fasciatus, and Aeromonas salmonidae in Example 5.

[0042] Figure 5 The graph shows the abundance changes of the three quorum sensing synthetic genes (cqsA, TraI, AvsI) in Example 6.

[0043] Figure 6 This is a graph showing the abundance changes of the three quorum sensing receptor genes (fusK, CinR, BisR) in Example 7.

[0044] Figure 7 This is a graph showing the abundance changes of six types of virulence factor genes (Adherence, Immune modulation, Effector delivery system, Nutritious / Metabolic factor, Stress survival, Others) in Example 8.

[0045] Figure 8 This is a diagram showing the sequencing results of the microbial community of the liquid fermentation product prepared in Example 1. Detailed Implementation

[0046] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0048] This application discovers that antagonists prepared by diluting agricultural enzymes can effectively inhibit soil pathogens. The method for preparing agricultural enzymes includes:

[0049] (1) By weight, collect 5 parts of fruit and vegetable residues, add 2 parts of brown sugar, 20 parts of water and 2 parts of baking powder, stir to obtain a slurry and pour it into a food fermentation tank; the fruit and vegetable residues include, but are not limited to, fruit pits, fruit peels, discarded vegetable leaves, rotten fruits and vegetables, etc.

[0050] (2) Place the fermentation bucket in a cool, ventilated place, avoiding direct sunlight, seal it, affix a label, and begin fermentation;

[0051] (3) During the first 10 to 15 days of fermentation, it is aerobic fermentation. The lid needs to be opened every 3 to 5 days to release gas and stir once to ensure that the fruit and vegetable residues are completely covered by liquid. After 15 days of fermentation, the lid is not opened again and anaerobic fermentation is carried out. The temperature of the entire fermentation process is 20 to 40℃.

[0052] (4) After 45 to 80 days of fermentation, the fermentation tank is opened and the liquid is dark yellow to yellowish-brown with a slightly acidic ester aroma and wine aroma.

[0053] (5) Filter the residue with a filter screen of not less than 10 mesh. The resulting filtrate is the liquid fermentation product, i.e. agricultural enzyme, and its pH is acidic between 3.5 and 5. During filtration, it should be ensured that the residue and liquid fermentation product are separated, and the resulting liquid fermentation product should not contain obvious solids.

[0054] The enzymes of different concentrations mentioned above can be applied directly to soil contaminated with pathogenic bacteria, or the enzymes can be mixed evenly with pig manure and then applied to soil contaminated with pathogenic bacteria, or the enzymes can be applied after the pig manure is applied.

[0055] The dosage of the antagonist was 10-20 ml / m 2 Apply 10-20 ml to the soil or the roots of each plant, once every 3-6 months.

[0056] The following specific examples further illustrate this:

[0057] Example 1: Preparation of agricultural enzymes:

[0058] Table 1 shows the weight ratio of raw materials for enzyme stock solution:

[0059] Table 1

[0060]

[0061] According to the raw material ratio in Table 1, the operating steps are as follows:

[0062] (1) By weight, collect 5 parts of fruit and vegetable residues, add 2 parts of brown sugar, 20 parts of water, and 2 parts of baking powder (commercially available Angel yeast baking powder). After thorough stirring, the mixture is poured into a food fermentation tank.

[0063] (2) Place the fermentation bucket in a cool, ventilated place, avoiding direct sunlight, seal it, affix a label, and begin fermentation;

[0064] (3) During the first 15 days of fermentation, it is aerobic fermentation. The lid needs to be opened every 3 days to release gas and stir once to ensure that the fruit and vegetable residues are completely covered by liquid. After the first 15 days of fermentation, the lid is not opened and anaerobic fermentation is carried out. The temperature of the entire fermentation process is 20-40℃.

[0065] (4) After about 60 days of fermentation, open the fermentation tank. The liquid will be dark yellow to yellowish-brown and have a slightly acidic ester aroma and wine aroma.

[0066] (5) Filter the residue using a filter screen with a mesh size of at least 10 mesh. The resulting filtrate is the liquid fermentation product, i.e., the enzyme stock solution, with a pH between 3.5 and 5, indicating acidity. The dominant bacterial group in the liquid fermentation product is Lactobacillus, with an abundance of at least 90% of the sequenced product. The sequencing results are as follows: Figure 8 As shown, enzyme 1 corresponds to 1# in Table 1, enzyme 2 corresponds to 2# in Table 1, and enzyme 3 corresponds to 3# in Table 1.

[0067] In the following examples, the high-concentration enzymes used were enzyme stock solutions diluted 10 times, and the low-concentration enzymes were enzyme stock solutions diluted 20 times.

[0068] In the following examples, the antagonism of soil pathogens by agricultural enzymes was demonstrated using a soil microcosm experiment.

[0069] Soil microcosm experiment: The microcosm experiment was conducted in a plastic box with dimensions of 35cm (length), 24cm (width) and 11cm (height).

[0070] 0.25 kg of pig manure organic fertilizer (from an organic fertilizer company in Xiaoshan, Hangzhou, Zhejiang) was mixed with 3.75 kg of soil (soil collected from farmland of Zhejiang University, paddy soil, with basic properties: pH 7.3, organic matter 19.4 g / kg, available nitrogen 74.4 mg / kg, available phosphorus 52.6 mg / kg, available potassium 122.0 mg / kg). Agricultural enzymes diluted at different ratios were sprayed onto the soil every two months. Every three months, 100 g of pig manure organic fertilizer was added to the soil as a nutrient supplement. Appropriate amounts of water were sprayed every three days to maintain suitable moisture content. The experimental temperature was room temperature (22℃±2℃). The control group received only pig manure organic fertilizer, the low-concentration enzyme group received both pig manure organic fertilizer and low-concentration enzyme, and the high-concentration enzyme group received both pig manure organic fertilizer and high-concentration enzyme.

[0071] Example 2: Experiment on the microcosm of soil microorganisms to kill or antagonize total pathogenic bacteria in soil using agricultural enzymes.

[0072] In this embodiment, the soil microcosm project began at the end of September 2022, and included a high-concentration enzyme group, a low-concentration enzyme group, and a control group:

[0073] High-concentration enzyme group: The No. 3 agricultural enzyme stock solution and pig manure organic fertilizer diluted 10 times in Example 1 were applied once in early October and once in late November.

[0074] Low-concentration enzyme group: The No. 1 agricultural enzyme stock solution and pig manure organic fertilizer diluted 20 times in Example 1 were applied once in early October and once in late November.

[0075] Control group: Only pig manure organic fertilizer was applied.

[0076] Finally, soil samples were taken and sequenced in early December, with all other conditions being the same as described above for the soil microcosm experiment.

[0077] The abundance of total pathogenic bacteria in the three soil groups is as follows: Figure 1 As shown, the control group had a concentration as high as 3.35 × 10⁻⁶. -4 ±3.48×10 -5 The total abundance of pathogenic bacteria in the soil (rpkm) decreased to 2.79 × 10⁻⁶ in both the low-concentration and high-concentration enzyme groups. -4 ±1.95×10 -5 (rpkm) and 2.04×10 -4 ±1.41×10 -5 (rpkm), in which the total abundance of pathogenic bacteria in the low concentration enzyme group was reduced by 16.68% compared with the control group, and the total abundance of pathogenic bacteria in the high concentration enzyme group was reduced by 39.15% compared with the control group.

[0078] Example 3: Experiment on soil microcosm of killing or antagonizing Klebsiella pneumoniae, Xylose-oxidizing Achromobacterium xylose and Streptomyces orientalis with agricultural enzymes.

[0079] In this embodiment, the soil microcosm project began at the end of September 2022, and included a high-concentration enzyme group, a low-concentration enzyme group, and a control group:

[0080] High-concentration enzyme group: The No. 3 agricultural enzyme stock solution and pig manure organic fertilizer diluted 10 times in Example 1 were applied once in early October and once in late November.

[0081] Low-concentration enzyme group: The No. 3 agricultural enzyme stock solution and pig manure organic fertilizer diluted 20 times in Example 1 were applied once in early October and once in late November.

[0082] Control group: Only pig manure organic fertilizer was applied.

[0083] Finally, soil samples were taken and sequenced in early December.

[0084] The abundance of Klebsiella pneumoniae, Achromobacter xylose oxidase, and Streptomyces orientalis in the three soil samples is as follows: Figure 2As shown, the abundance of Klebsiella pneumoniae, Achromobacter xylose oxidase, and Streptomyces orientalis in the control group was as high as 2.08 × 10⁻⁶. -4 ±2.12×10 -5 (rpkm), 3.33×10 -5 ±7.93×10 -7 (rpkm) and 3.26×10 -5 ±7.17×10 -6 (rpkm), after applying low concentrations of enzymes, the abundance of Klebsiella pneumoniae, Achromobacter xylose oxidizing bacteria, and Streptomyces orientalis decreased to 1.78 × 10⁻⁶. -4 ±9.25×10 -6 (rpkm), 2.99×10 -5 ±2.30×10 -6 (rpkm) and 2.37×10 -5 ±1.28×10 -6 (rpkm), which reduced the abundance of Klebsiella pneumoniae, Achromobacter xylose oxidase, and Streptomyces orientalis to 1.24 × 10⁻⁶, respectively. -4 ±8.15×10 -6 (rpkm), 2.49×10 -5 ±8.16×10 -7 (rpkm) and 2.25×10 -5 ±2.26×10 -6 (rpkm), which were reduced by 40.39%, 25.15%, and 31.05%, respectively.

[0085] Example 4: Experiment on soil microcosm of killing and antagonizing Vibrio cholerae, Riemerella anatipestifer, and Clostridium botulinum with agricultural enzymes.

[0086] In this embodiment, the soil microcosm project began at the end of September 2022, and included a high-concentration enzyme group, a low-concentration enzyme group, and a control group:

[0087] High-concentration enzyme group: The No. 3 agricultural enzyme stock solution and pig manure organic fertilizer diluted 10 times in Example 1 were applied once in early October and once in late November.

[0088] Low-concentration enzyme group: The No. 3 agricultural enzyme stock solution and pig manure organic fertilizer diluted 20 times in Example 1 were applied once in early October and once in late November.

[0089] Control group: Only pig manure organic fertilizer was applied.

[0090] Finally, soil samples were taken and sequenced in early December.

[0091] The abundance of Vibrio cholerae, Riemerella anatipestifer, and Clostridium botulinum in the three soil samples is as follows: Figure 3 As shown, the abundance of Vibrio cholerae, Riemerella anatipestifer, and Clostridium botulinum in the control group was as high as 2.72 × 10⁻⁶. -5 ±1.35×10 -6 (rpkm), 1.13×10 -5 ±1.44×10 -6 (rpkm) and 7.09×10 -6 ±1.02×10 -6 (rpkm), after applying low concentrations of enzymes, the abundance of Vibrio cholerae, Riemerella anatipestifer, and Clostridium botulinum decreased to 2.20 × 10⁻⁶. -5 ±3.42×10 -6 (rpkm), 8.02×10 -6 ±9.96×10 -7 (rpkm) and 5.67×10 -6 ±6.28×10 -7 (rpkm), which reduced the abundance of pathogens by 19.00%, 29.22%, and 20.03%, respectively; after applying high concentrations of enzymes, the abundance of the three pathogens decreased to 1.30 × 10⁻⁶. -5 ±1.81×10 -6 (rpkm), 5.61×10 -6 ±4.05×10 -7 (rpkm) and 4.56×10 -6 ±5.73×10 -8 (rpkm), which were reduced by 52.98%, 50.44% and 35.73% respectively.

[0092] Example 5: Soil microcosm experiment on the killing or antagonism of Serratia marcescens, Rhodococcus spp. and Aeromonas salmonidae by agricultural enzymes.

[0093] In this embodiment, the soil microcosm project began at the end of September 2022, and included a high-concentration enzyme group, a low-concentration enzyme group, and a control group:

[0094] High-concentration enzyme group: The No. 3 agricultural enzyme stock solution and pig manure organic fertilizer diluted 10 times in Example 1 were applied once in early October and once in late November.

[0095] Low-concentration enzyme group: The No. 3 agricultural enzyme stock solution and pig manure organic fertilizer diluted 20 times in Example 1 were applied once in early October and once in late November.

[0096] Control group: Only pig manure organic fertilizer was applied.

[0097] Finally, soil samples were taken and sequenced in early December.

[0098] The abundance of Serratia marcescens, Rhodococcus spp., and Aeromonas salmonidae in the three soil groups is as follows: Figure 4 As shown, the abundances of *Serratia marcescens*, *Rhodococcus solani*, and *Aeromonas salmonicida* in the control group were 5.93 × 10⁻⁶. -6 ±3.21×10 -7 (rpkm), 5.31×10 -6 ±4.59×10 -7 (rpkm) and 3.99×10 -6 ±1.08×10 -6 (rpkm), after applying low concentrations of enzymes, the abundances of Rhodococcus faecalis, Listeria monocytogenes, and Aeromonas salmonidae decreased to 4.90 × 10⁻⁶. -6 ±8.60×10 -7 (rpkm), 4.04×10 -6 ±5.07×10 -7 (rpkm) and 2.43×10 -6 ±2.42×10 -7 (rpkm), which reduced the abundance of pathogens by 17.31%, 23.81%, and 38.96%, respectively; after applying high concentrations of enzymes, the abundance of the three pathogens decreased to 3.59 × 10⁻⁶. -6 ±2.56×10 -7 (rpkm), 3.37×10 -6 ±1.22×10 -7 (rpkm) and 2.12×10 -6 ±1.81×10 -7 (rpkm), which were reduced by 39.40%, 36.43%, and 46.82% respectively.

[0099] Example 6: Soil microcosm experiment to reduce soil quorum sensing synthesis genes (cqsA, TraI, AvsI) using agricultural enzymes.

[0100] In this embodiment, the soil microcosm project began at the end of September 2022, and included a high-concentration enzyme group, a low-concentration enzyme group, and a control group:

[0101] High-concentration enzyme group: The No. 3 agricultural enzyme stock solution and pig manure organic fertilizer diluted 10 times in Example 1 were applied once in early October and once in late November.

[0102] Low-concentration enzyme group: The No. 3 agricultural enzyme stock solution and pig manure organic fertilizer diluted 20 times in Example 1 were applied once in early October and once in late November.

[0103] Control group: Only pig manure organic fertilizer was applied.

[0104] Finally, soil samples were taken and sequenced in early December.

[0105] The abundance of quorum sensing synthetic genes (cqsA, TraI, AvsI) in the three soil samples is as follows: Figure 5 As shown, the abundances of cqsA, TraI, and AvsI in the control group were 4.79 × 10⁻⁶. -6 ±8.79×10 -7 (rpkm), 9.30×10 -7 ±4.75×10 -7 (rpkm) and 8.72×10 -7 ±3.41×10 -7 (rpkm), after applying low concentrations of enzymes, the abundance of the three genes decreased to 4.54 × 10⁻⁶. -6 ±5.52×10 -7 (rpkm), 6.41×10 -7 ±2.58×10 -7 (rpkm) and 6.95×10 -7 ±3.08×10 -7 (rpkm), decreased by 5.29%, 31.08%, and 20.37%, respectively; after applying high concentrations of enzymes, the abundance of the three genes decreased to 4.07 × 10⁻⁶. -6 ±3.28×10 -7 (rpkm), 4.56×10 -7 ±7.18×10 -8 (rpkm) and 5.05×10 -7 ±3.24×10 -7 (rpkm) decreased by 15.08%, 50.98%, and 42.16%, respectively; the total abundance of the three quorum sensing synthesis genes in the low-concentration and high-concentration enzyme groups decreased by 10.92% and 23.73%, respectively.

[0106] Example 7: Soil microcosm experiment to reduce soil quorum sensor receptor genes (fusK, CinR, BisR) using agricultural enzymes.

[0107] In this embodiment, the soil microcosm project began at the end of September 2022, and included a high-concentration enzyme group, a low-concentration enzyme group, and a control group:

[0108] High-concentration enzyme group: The No. 3 agricultural enzyme stock solution and pig manure organic fertilizer diluted 10 times in Example 1 were applied once in early October and once in late November.

[0109] Low-concentration enzyme group: The No. 3 agricultural enzyme stock solution and pig manure organic fertilizer diluted 20 times in Example 1 were applied once in early October and once in late November.

[0110] Control group: Only pig manure organic fertilizer was applied.

[0111] Finally, soil samples were taken and sequenced in early December.

[0112] The abundance of quorum sensor receptor genes (fusK, CinR, BisR) in the three soil samples is as follows: Figure 6 As shown, the abundances of fusK, CinR, and BisR in the control group were 1.55 × 10⁻⁶. -5 ±2.89×10 -6 (rpkm), 7.64×10 -7 ±3.92×10 -8 (rpkm) and 1.59×10 -7 ±1.13×10 -7 (rpkm), after applying low concentrations of enzymes, the abundance of the three genes decreased to 1.14 × 10⁻⁶. -5 ±1.24×10 -7 (rpkm), 5.36×10 -6 ±1.63×10 -7 (rpkm) and 0 decreased by 26.59%, 29.82%, and 100.00%, respectively; after applying high concentrations of enzymes, the abundance of the three genes decreased to 1.02 × 10⁻⁶. -5 ±1.26×10 -6 (rpkm), 4.20×10 -7 ±6.08×10 -8 (rpkm) and 0 decreased by 34.25%, 44.94% and 100.00%, respectively; the total abundance of the three quorum sensing receptor genes in the low-concentration and high-concentration enzyme groups decreased by 27.45% and 35.38%, respectively.

[0113] Example 8: Soil microcosm experiment on the reduction of six types of toxicity factor genes (Adherence, Immune modulation, Effector delivery system, Nutritious / Metabolic factor, Stress survival, Others) by agricultural enzymes.

[0114] In this embodiment, the soil microcosm project began at the end of September 2022, and included a high-concentration enzyme group, a low-concentration enzyme group, and a control group:

[0115] High-concentration enzyme group: The No. 3 agricultural enzyme stock solution and pig manure organic fertilizer diluted 10 times in Example 1 were applied once in early October and once in late November.

[0116] Low-concentration enzyme group: The No. 3 agricultural enzyme stock solution and pig manure organic fertilizer diluted 20 times in Example 1 were applied once in early October and once in late November.

[0117] Control group: Only pig manure organic fertilizer was applied.

[0118] Finally, soil samples were taken and sequenced in early December.

[0119] Abundance of toxicity factor genes in 3 groups of soils as follows Figure 7 As shown, the abundances of the six virulence factor genes in the control group were 0.411±0.040 (copies / cell), 0.267±0.015 (copies / cell), 0.123±0.027 (copies / cell), 0.178±0.026 (copies / cell), 0.083±0.009 (copies / cell), and 0.187±0.016 (copies / cell), respectively. After applying low-concentration enzymes, the abundances of the six virulence factor genes decreased to 0.400±0.017 (copies / cell), 0.259±0.019 (copies / cell), 0.108±0.023 (copies / cell), 0.165±0.023 (copies / cell), 0.077±0.009 (copies / cell), and 0.178±0.016 (copies / cell), respectively. The abundance of the six virulence factor genes decreased to 0.012 (copies / cell), representing reductions of 2.76%, 3.03%, 12.15%, 7.54%, 4.67%, and 5.01, respectively. After applying high concentrations of enzymes, the abundance of these genes decreased to 0.376±0.024 (copies / cell), 0.222±0.014 (copies / cell), 0.076±0.008 (copies / cell), and 0.1, respectively. The abundances of the six virulence factor genes were 47±0.004 (copies / cell), 0.074±0.001 (copies / cell), and 0.154±0.012 (copies / cell), representing decreases of 8.51%, 17.08%, 38.29%, 17.18%, 11.63%, 17.37%, and 16.03%, respectively. The total abundance of the six virulence factor genes in the low-concentration and high-concentration enzyme groups decreased by 5.01% and 16.03%, respectively.

[0120] In summary, the agricultural enzyme of this application can effectively reduce soil pathogenic microbial pollution caused by pig manure organic fertilizer. In particular, the high-concentration enzyme shows excellent killing effects on Klebsiella pneumoniae, Achromobacter xylose oxidizing bacteria, Streptomyces orientalis, Vibrio cholerae, Riemerella anatipestifer, Clostridium botulinum, Serratia marcescens, Rhodococcus spp., and Aeromonas salmonidae, significantly reducing their abundance. Furthermore, the agricultural enzyme of this application can antagonize soil pathogens and reduce their abundance by reducing quorum sensing-related genes and virulence factor-related genes. By directly killing or indirectly antagonizing soil pathogens, the agricultural enzyme of this application can reduce their abundance, thereby reducing the risk of human and animal infection with pathogens.

[0121] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. The application of agricultural enzymes as soil remediation agents in the prevention or treatment of soil pathogenic microbial pollution, characterized in that, The soil pathogen is Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella Pneumoniae ), xylose-oxidizing achromobacterium ( Achromobacter xylosoxidans ), Vibrio cholerae ( Vibrio cholerae ), Riemerella anatipestifer ( Riemerella anatipestifer ), botulinum toxin ( Clostridium botulinum Serratia marcescens ( ), Serratia marcescens ) and Aeromonas salmonidae ( Aeromonas salmonicida At least one of the following: the agricultural enzyme is a liquid fermented product obtained by fermenting fruit and vegetable residues; the dominant bacterial group of the liquid fermented product is lactobacillus, and its abundance is not less than 90% of the sequenced bacteria; the agricultural enzyme can kill the pathogenic bacteria and can also regulate the pathogenic bacteria by reducing quorum sensing genes and virulence factor genes.

2. The application according to claim 1, characterized in that, The soil pathogens are Xylose-oxidizing Achromobacterium or Vibrio cholerae.

3. A method for inhibiting soil pathogenic microbial pollution, characterized in that, include: Agricultural enzymes can be diluted and applied directly to the soil contaminated with pathogenic bacteria, or fertilizer can be applied first and then applied to the soil contaminated with pathogenic bacteria, or agricultural enzymes can be mixed with fertilizer and then applied to the soil contaminated with pathogenic bacteria. The agricultural enzyme is a liquid fermented product obtained by fermenting fruit and vegetable residues; the dominant bacterial group of the liquid fermented product is lactobacillus, and its abundance is not less than 90% of the sequenced bacteria; the agricultural enzyme can kill the pathogenic bacteria and can also regulate the pathogenic bacteria by reducing quorum sensing genes and virulence factor genes. The pathogenic bacteria are at least one of Klebsiella pneumoniae, Xylose-oxidizing achromobacterium, Vibrio cholerae, Riemerella anatipestifer, Clostridium botulinum, Serratia marcescens, and Aeromonas salmonidae. The fertilizer is one of the following: pig manure organic fertilizer, chicken manure organic fertilizer, cow manure organic fertilizer, and silkworm manure organic fertilizer.

4. The method according to claim 3, characterized in that, The soil remediation agent is a liquid fermentation stock solution diluted 10 to 20 times; The preparation process of the liquid fermentation stock solution is as follows: Mix fruit and vegetable residues, brown sugar, water, and baking powder in a certain proportion, seal and ferment. The liquid obtained by filtering the fermentation product is the liquid fermentation product.

5. The method according to claim 4, characterized in that, The ratio of the fruit and vegetable residues, brown sugar, water and baking powder is as follows (by weight): 4-6 parts fruit and vegetable residues, 0.5-2.5 parts brown sugar, 15-25 parts water, and 0.5-2.5 parts baking powder. The fermentation includes an early aerobic fermentation and a later anaerobic fermentation. The early aerobic fermentation lasts for 10 to 15 days, and the later anaerobic fermentation lasts for 35 to 65 days, with a total fermentation time of 45 to 80 days. The fermentation temperature is 20 to 40°C. During the early aerobic fermentation, the lid needs to be opened to release gas and stirred once every 3 to 5 days.

6. The method according to claim 3, characterized in that, The soil remediation agent is applied at a rate of 10-20 ml per square meter of soil or 10-20 ml per plant root; it is applied once every 3-6 months.

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