Application of a bacteriophage in the remediation of organochlorine pesticide contaminated saline-alkali soil

Abstract

Application of a bacteriophage in the remediation of organochlorine pesticide contaminated saline-alkali soil. The bacteriophage of Bacillus cereus was inoculated into the organochlorine pesticide contaminated saline-alkali soil in the form of suspension. The titer of the bacteriophage in the soil was 10 6 -10 9 PFU / g. By inoculating the bacteriophage φYSZBA2 and the host Bacillus cereus into the saline-alkali contaminated soil, the degradation of organochlorine pesticides was accelerated by the synergistic effect of the bacteriophage and the host bacteria, and the degradation rate of typical organochlorine pesticides such as hexachlorocyclohexane and nitrochlorobenzene in the saline-alkali soil was significantly improved. The bacteriophage has good tolerance to high salt and wide pH environment, which can overcome the technical bottleneck of traditional degradation bacteria, such as difficult colonization and decreased activity under saline-alkali stress. At the same time, the inhibition of saline-alkali stress on crop growth was relieved by optimizing the structure of rhizosphere microbial community, the residual amount of organochlorine pesticides in the edible part of crops was reduced, and the health of soil micro-ecology was maintained.

Description

Technical Field

[0001] This invention belongs to the field of bioremediation technology for contaminated soil, specifically relating to the field of microbial remediation technology for soil contaminated by organochlorine pesticides, and particularly to the application of a bacteriophage in the remediation of organochlorine pesticide contaminated soil in saline-alkali land. Background Technology

[0002] In recent years, due to the long-term and large-scale application of organochlorine pesticides, the difficulty of persistent environmental degradation, and the lack of remediation technologies for historically contaminated sites, farmland soil-crop systems in many countries worldwide have become high-risk hotspots for the accumulation of organochlorine pesticide residues. Organochlorine pesticides (such as hexachlorocyclohexane, DDT, pentachloronitrobenzene, and chlordane) are characterized by high hydrophobicity, strong bioaccumulation, and extremely low degradation rates. They can remain in the soil for decades and continuously enter the farmland ecosystem through surface runoff, wet and dry deposition, and farmland irrigation. Especially under the combined effects of multiple factors such as adsorption by soil organic matter, hindered anaerobic dechlorination, and limited microbial degradation activity, organochlorine pesticides and their toxic metabolic intermediates continuously accumulate in the rhizosphere soil and within crops. This not only severely inhibits the structure and function of soil microbial communities and disrupts the nutrient cycling and self-purification capacity of the farmland ecosystem, but also poses potential risks to human health, including endocrine disruption, carcinogenicity, and neurotoxicity, through the food chain. Therefore, it is both necessary and urgent to develop efficient and targeted microbial degradation technologies for farmland soil contaminated by organochlorine pesticides under adverse conditions such as saline-alkali stress.

[0003] Salt and alkali stress is one of the major abiotic stressors limiting crop growth and agricultural productivity in global farmland ecosystems. my country has vast areas of saline-alkali land along its coast and inland, with high concentrations of soluble salts (such as Na₂SO₄) in the soil. + Cl -High salinity and high pH environments can trigger osmotic stress, ion toxicity, and oxidative damage, leading to inhibited seed germination, suppressed root development, and reduced photosynthetic efficiency, ultimately resulting in severe yield reduction or even crop failure. Furthermore, saline-alkali stress significantly inhibits the activity and diversity of native soil microorganisms, disrupts the rhizosphere microecological balance, and further weakens soil nutrient cycling capacity and crop resistance. Previous studies have shown that under saline-alkali stress, screening for salt-tolerant functional bacteria and their specific bacteriophages, and synergistically reintroducing both into contaminated soil, can effectively alleviate the inhibitory effect of salinity on plant growth. On the one hand, salt-tolerant functional bacteria can enhance plant salt tolerance by producing plant hormones (such as indoleacetic acid), secreting extracellular polysaccharides, and regulating ion homeostasis; on the other hand, specific bacteriophages indirectly promote plant resistance to saline-alkali stress by infecting and regulating the metabolic activity of host bacteria and optimizing the rhizosphere microbial community structure. Therefore, isolating and applying bacteriophage-bacteria systems adapted to saline-alkali environments has important theoretical and practical value for improving the stress resistance of crops in saline-alkali farmland, ensuring crop yield, and promoting sustainable agricultural development.

[0004] Bacteriophages (or bacteriophages for short) are a class of viruses that specifically infect and lyse living bacteria. They are widely distributed in soil, water, air, and on the surface or intestinal tract of humans and animals. It is estimated that their total number can reach 10^ ... 31 Order of magnitude. Phage therapy refers to a bioremediation method that involves isolating, screening, purifying, and enriching host bacteria-specific bacteriophages, selecting multivalent bacteriophages with high titers, short lysis cycles, and strong environmental tolerance, and then adding specific bacteriophage suspensions to contaminated soil-plant systems to target and inactivate pathogenic bacteria.

[0005] A review of relevant literature and patent searches revealed no publicly available publications or applications regarding a single bacteriophage strain and its application in the remediation of organochlorine pesticide-contaminated soil in saline-alkali land. The closest existing methods to this invention include: bacteriophage-assisted degradation technology for organochlorine pesticide-contaminated soil, screening and application of salt-tolerant and growth-promoting microbial strains in saline-alkali land, and targeted inactivation applications of bacteriophage therapy in other environments or diseases. Current research primarily focuses on using bacteriophages or functional bacteria alone to degrade organochlorine pesticides or improve saline-alkali land. However, there are almost no remediation technologies that utilize the same bacteriophage to simultaneously degrade organochlorine pesticide pollution and alleviate the stress of salinity on plants under saline-alkali conditions. Application No.: CN202510134046.0 provides the application of a bacteriophage composition in enhancing the degradation of nitrochlorobenzene in soil. This invention utilizes a bacteriophage composition to enhance the degradation effect of nitrochlorobenzene in soil. Application No.: CN202610198712.1 provides the application of Enterobacter AD8 in improving saline-alkali soil and enhancing the salt-alkali stress resistance of soybeans. This invention screens strain AD8 from saline-alkali soil, which possesses phosphorus-solubilizing and indoleacetic acid-producing characteristics, and can reduce soil pH and soluble salt content, thereby enhancing the salt-alkali stress resistance of soybeans. Application No.: CN202511720606.7 provides a Lindian alkaline halophilic bacterium LH-SA-1 and its saline-alkali soil remediation agent. This strain is resistant to... Due to the high salinity and alkalinity environment, the soil salinity and alkali content is significantly reduced and the soil enzyme activity is improved; Application No.: CN202310755792.2 provides a microbial composition and method for improving saline-alkali land. This invention uses Bacillus tasakae and Bacillus subtilis to form a bacterial agent, which effectively improves the pH and salinity of saline-alkali land; Application No.: CN202410978417.9 provides a bacteriophage and its application in compensating for soil nitrogen fixation. This Klebsiella pneumoniae bacteriophage improves soil nitrogen fixation efficiency through bacteriophage transplantation; Application No.: CN201510008569.7 provides a technical method for preventing and controlling tobacco bacterial wilt using bacteriophage therapy. This method involves injecting a bacteriophage suspension into tobacco stems to prevent and control bacterial wilt. In summary, existing technologies have not yet addressed the application of bacteriophage φYSZBA2 in the degradation and remediation of organochlorine pesticide-contaminated soils in saline-alkali land, nor have they achieved the synergistic effect of bacteriophage-mediated pesticide degradation and plant stress resistance promotion under saline-alkali stress.

[0006] The main shortcomings of existing technologies are: most existing research on the remediation of soil contaminated by organochlorine pesticides adopts physicochemical methods (such as thermal desorption, chemical oxidation, leaching, etc.) or microbial enhancement methods using single degrading strains. These methods often suffer from high costs, high risks of secondary pollution, difficulties in colonization of degrading bacteria under saline-alkali stress, and decreased activity. There is a serious lack of development of synergistic degradation technologies for organochlorine pesticides in the special habitat of saline-alkali land. Existing saline-alkali land improvement technologies mainly reduce soil salinity and improve soil structure by applying salt-tolerant growth-promoting bacteria, organic fertilizers, or soil conditioners. However, these methods focus on improving crop salt tolerance and do not combine them with the efficient degradation of organochlorine pesticide pollution, let alone achieve the synchronous synergy between pesticide removal and plant stress resistance promotion under saline-alkali stress. On the other hand, the current applications of bacteriophage technology in environmental remediation mainly focus on the specific inactivation of pathogenic bacteria (such as Pseudomonas aeruginosa and Ralstonia solanacearum) or compensating for soil nitrogen fixation. There are almost no reports on the direct application of bacteriophages to the degradation and remediation of organochlorine pesticide-contaminated soils (especially under saline-alkali stress). Furthermore, assessments of the functional stability, diversity, and ecological safety of the microbial community in the soil-crop system after phage inoculation are relatively scarce. Therefore, developing a bacteriophage bioremediation technology capable of simultaneously degrading organochlorine pesticide pollution and mitigating the inhibitory effects of salinity on plant growth under saline-alkali stress has significant practical implications and broad application prospects.

[0007] The main reasons for these deficiencies are as follows: In recent years, the academic community has gradually recognized that saline-alkali farmland soils are "hotspots" and important "sinks" for the accumulation of persistent organochlorine pesticide residues. The combined effects of saline-alkali stress and organochlorine pesticide pollution not only significantly inhibit soil microbial activity and nutrient cycling, but also threaten agricultural product safety and human health through the food chain. Existing remediation research mostly focuses on chemical oxidation, thermal desorption, or enhanced remediation of organochlorine pesticides under non-saline-alkali environments using single degrading bacteria, or on improving salt-tolerant growth-promoting bacteria in saline-alkali land to enhance crop stress resistance. Research on biosynergistic remediation technologies under coupled saline-alkali stress and organochlorine pesticide pollution is severely lacking, especially in the absence of technical solutions that utilize bacteriophages to target and regulate functional bacteria to simultaneously achieve organochlorine pesticide degradation and plant salt-alkali stress resistance promotion. Even less attention has been paid to the bacteriophage-host bacteria interaction mechanism under organochlorine pesticide pollution stress in saline-alkali land and its contribution to the restoration of soil-crop system ecological functions. Therefore, there is an urgent need to develop efficient, safe, and targeted bacteriophage remediation technologies for organochlorine pesticide-contaminated soils in saline-alkali land. Summary of the Invention

[0008] Technical Problem Solved: To address the problems of existing technologies in the remediation of organochlorine pesticide-contaminated soils in saline-alkali land, such as difficulties in colonization of degrading bacteria, inhibited activity, and the lack of synergistic remediation schemes that combine organochlorine pesticide degradation with the mitigation of saline-alkali stress, this invention provides the application of a bacteriophage φYSZBA2 in the remediation of organochlorine pesticide-contaminated soils in saline-alkali land. This bacteriophage was deposited at the China Center for Type Culture Collection on August 1, 2018, with accession number CCTCC NO:M 2018518, classified and named Bacillus cereus phage φYSZBA2, and the deposit address is Wuhan University, Wuhan City, Hubei Province, China. This invention involves reintroducing bacteriophage φYSZBA2, isolated from saline-alkali land, into saline-alkali soil contaminated with organochlorine pesticides. Utilizing its specific infection and lysis of host bacteria carrying organochlorine pesticide degradation genes, it significantly improves the degradation rate of typical organochlorine pesticides such as hexachlorocyclohexane (HCH) and nitrochlorobenzene in saline-alkali soil. Furthermore, this bacteriophage exhibits good tolerance to high-salt (≤5% NaCl) and wide pH (6.0~10.0) environments, effectively overcoming the technical bottlenecks of traditional degrading bacteria facing colonization difficulties and decreased activity under saline-alkali stress. By optimizing the rhizosphere microbial community structure, it alleviates the inhibitory effect of saline-alkali stress on crop growth, reduces the residue of organochlorine pesticides in the edible parts of crops, and maintains soil microecological health. With its low cost, ease of use, and environmental friendliness, this invention provides an efficient and safe solution for the bioremediation of organochlorine pesticide contaminated farmland in coastal and inland saline-alkali areas of my country.

[0009] Technical Solution: Application of Bacillus cereus phage φYSZBA2 in the remediation of organochlorine pesticide contaminated soil in saline-alkali land.

[0010] The aforementioned organochlorine pesticides include at least one of hexachlorocyclohexane (HCH) and nitrochlorobenzene.

[0011] The pH of the aforementioned saline-alkali soil is 8.0–10.0, and the soluble salt content is 0.3%–5.0%.

[0012] The aforementioned bacteriophage φYSZBA2 was reintroduced into saline-alkali soil contaminated with organochlorine pesticides in the form of a bacteriophage suspension. After reintroduction, the bacteriophage titer in the soil was 10. 6 ~10 9 PFU / g.

[0013] The above applications also include inoculating soil with Bacillus cereus, wherein the concentration of the inoculum of Bacillus cereus is 10. 7 ~10 8 CFU / g.

[0014] Application of Bacillus cereus phage φYSZBA2 in the preparation of microbial agents for the remediation of organochlorine pesticide-contaminated soil in saline-alkali land.

[0015] A method for remediating organochlorine pesticide-contaminated soil in saline-alkali land includes applying bacteriophage φYSZBA2 in the form of a bacteriophage suspension to the organochlorine pesticide-contaminated saline-alkali land soil.

[0016] The above method also includes applying Bacillus cereus to the soil, wherein the concentration of the Bacillus cereus bacterial solution is 10. 7 ~10 8 CFU / g.

[0017] The working principle of this invention is as follows: 1. Bacillus cereus phage φYSZBA2 is a known virulent phage isolated from saline-alkali soil. It can specifically infect and lyse specific Bacillus host bacteria, which carry functional genes related to the degradation of organochlorine pesticides and are important microorganisms driving the transformation of organochlorine pesticides in soil; 2. The high-salt and high-pH environment in saline-alkali soil inhibits the activity of native microorganisms, resulting in extremely low natural degradation rates of organochlorine pesticides. However, phage φYSZBA2 has strong tolerance to saline-alkali stress and can be isolated in high-salt (≤5%) soils. 1. Under conditions of NaCl and a wide pH range (6.0~10.0), it maintains high infectivity and lysis capacity; 2. Bacillus cereus phage φYSZBA2 recognizes and adsorbs specific binding sites on the surface of the host bacterial cell membrane in the environment, then injects its own DNA into the host bacteria, utilizes the host metabolic system to rapidly complete nucleic acid replication and protein synthesis, assembles progeny phages, and finally releases lysozymes to lyse the host bacteria, releasing a large number of progeny phages; 3. After the host bacteria are lysed, the intracellular enzyme system involved in the degradation of organochlorine pesticides (such as dechlorinase, oxygenase, etc.) is released into the soil environment, which can continue to catalyze the degradation reaction of organochlorine pesticides, thereby achieving a synergistic effect of "lysis desiccant-enzyme-enzyme degradation"; 4. The introduction of Bacillus cereus phage φYSZBA2 can directionally reduce the excessive proliferation of host bacteria in saline-alkali soils, optimize the rhizosphere microbial community structure, and reduce the metabolic intermediates of organochlorine pesticides. 6. It inhibits the toxicity of beneficial microorganisms such as nitrogen-fixing bacteria and phosphate-solubilizing bacteria, thereby restoring the soil nutrient cycle function; 7. While lysing host bacteria, Bacillus cereus phage φYSZBA2 does not significantly affect other non-target microorganisms in the soil (such as salt-tolerant nitrogen-fixing bacteria and plant growth-promoting rhizosphere bacteria), exhibiting good host specificity and maintaining the diversity and stability of the soil microecology; 8. Bacillus cereus phage φYSZBA2 "originates from saline-alkali soil and returns to saline-alkali soil," without any genetic engineering modification, and will not introduce exogenous genetic material into the environment after application, exhibiting good ecological safety; 9. After Bacillus cereus phage φYSZBA2 is reintroduced into saline-alkali soil contaminated with organochlorine pesticides, it can migrate to the aboveground parts through plant root absorption and transpiration, reducing the residue of organochlorine pesticides in the edible parts of crops, while alleviating the inhibitory effect of saline-alkali stress on crop growth and improving crop yield and quality.

[0018] Beneficial Effects: This invention can efficiently degrade organochlorine pesticides. The Bacillus cereus phage φYSZBA2 can specifically infect host Bacillus species carrying genes for organochlorine pesticide degradation, significantly improving the degradation rate and mineralization degree of typical organochlorine pesticides such as hexachlorocyclohexane (HCH) and chloronitrobenzene in saline-alkali soils. It exhibits strong tolerance to saline-alkali stress and adaptability. Isolated from coastal saline-alkali soils, this phage demonstrates good tolerance to high-salt (≤5% NaCl) and wide pH (6.0~10.0) environments, maintaining high infectivity and lytic capacity even under saline-alkali stress conditions, overcoming the technical bottlenecks of traditional degrading bacteria's difficulty in colonization and decreased activity in saline-alkali soils. It alleviates saline-alkali inhibition and reduces pesticide residues in crops. After reinoculation, the phage can migrate to the aboveground parts of crops through root absorption and transpiration, effectively reducing the amount of organochlorine pesticide residues in edible parts; simultaneously, by optimizing the rhizosphere microbial community structure, it indirectly enhances the crop's resistance to saline-alkali stress, promotes crop growth, and improves yield and quality. Low cost, easy to use, and environmentally friendly. The Bacillus cereus phage φYSZBA2 originates from saline-alkali soil, has not undergone any genetic engineering modification, and poses no risk of introducing foreign genetic material when reintroduced into the soil. The preparation process is simple, easy to store and transport, and convenient to operate, effectively preventing secondary pollution and facilitating large-scale application. It maintains soil microecological health. This phage has strong host specificity and does not significantly interfere with non-target microorganisms (such as halophilic nitrogen-fixing bacteria and phosphate-solubilizing bacteria). After application, the functional diversity and stability of the soil microbial community are positively promoted, aligning with the concept of green and sustainable remediation. This invention provides a highly efficient, safe, and easy-to-implement new method for the bioremediation of organochlorine pesticide-contaminated farmland in coastal and inland saline-alkali areas of my country, with broad application prospects. Attached Figure Description

[0019] Figure 1 The values ​​represent the OD values ​​of Bacillus cereus under different salt concentrations.

[0020] Figure 2 Image of plaque morphology of Bacillus cereus phage φYSZBA2 on a plate.

[0021] Figure 3 Transmission electron microscopy image of Bacillus cereus phage φYSZBA2.

[0022] Figure 4 To demonstrate the effectiveness of this invention in degrading HCH in coastal saline-alkali farmland soil in Nantong City, Jiangsu Province, under salt-alkali stress, this diagram verifies the degradation effect of HCH on HCH-contaminated soil.

[0023] Figure 5 To utilize the technical solution of this invention, the residual amount of hexachlorocyclohexane (HCH) in rice grains from coastal saline-alkali farmland in Nantong City, Jiangsu Province, under saline-alkali stress was determined.

[0024] Figure 6 To utilize the technical solution of this invention, the biomass of the aboveground parts of rice paddies in coastal saline-alkali land of Nantong City, Jiangsu Province, under salt-alkali stress and HCH-contaminated soil was measured.

[0025] Figure 7 To utilize the technical solution of this invention, the degradation rate of hexachlorocyclohexane (HCH) was compared in saline-alkali stressed soil and ordinary farmland soil by applying bacterial and bacteriophage suspensions. Detailed Implementation

[0026] The following specific embodiments do not limit the technical solutions of the present invention in any way. All technical solutions obtained by means of equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

[0027] The aforementioned Bacillus cereus phage φYSZBA2, with accession number CCTCC NO M 2018518, is classified as Bacillus cereus phage. Electron microscopy images show a typical icosahedral head and a retractable tail sheath. The head diameter is approximately 85–95 nm, and the tail length is approximately 110–120 nm. Phage plaques formed on double-layer agar plates are circular and transparent, with clear edges and no halo, and a diameter of approximately 2–4 mm.

[0028] The host bacteria mentioned above is Bacillus cereus. This strain was isolated from organochlorine pesticide-contaminated soil in saline-alkali land. It carries the key genes C2001290, C2001804 and C2009263 for the metabolism of p-chloroaniline and has the ability to degrade organochlorine pesticides such as hexachlorocyclohexane and DDT.

[0029] The aforementioned organochlorine pesticides include at least one of hexachlorocyclohexane (HCH) and nitrochlorobenzene, and the concentration of p-chloroaniline in the contaminated soil ranges from 3 to 600 mg / kg.

[0030] The aforementioned saline-alkali soil refers to coastal saline-alkali land, with a soil pH of 8.0–10.0 and a soluble salt content of 0.3%–5.0%.

[0031] In the above specific implementation scheme, the phage φYSZBA2 suspension is prepared at 10 6 ~10 9 The PFU / g titer was reinjected into saline-alkali soil contaminated with organochlorine pesticides, and simultaneously inoculated with Bacillus cereus (bacterial concentration of 10). 7 ~10 8 The two (CFU / g) work synergistically to achieve a degradation rate of 75%–95% for the tested organochlorine pesticides within 30 days. At the same time, the plant height, biomass, and yield of crops (rice and wheat) increase by 20%–40%, and the residual amount of organochlorine pesticides in the edible parts of crops is reduced to below the national food safety standard limit.

[0032] Example 1

[0033] The soil used for the potted plants was collected from saline-alkali farmland, and the test crop was a salt-tolerant rice variety (Yan Dao 12). This area has long been contaminated with hexachlorocyclohexane (HCH), and soil samples were collected from five topsoil layers (0–20 cm). Basic soil physicochemical properties: sand 52.6%, silt 38.2%, clay 9.2%, pH 8.7, soluble salt content 0.75%, total nitrogen 0.85 g·kg⁻¹. -1 Total phosphorus 0.68 g·kg -1 Total potassium 22.15 g·kg -1 Organic matter 12.6 g·kg -1 CEC 15.3 cmol·kg -1 The initial concentration of hexachlorocyclohexane (HCH) was 0.68 mg·kg⁻¹. -1 .

[0034] The experiment consisted of 6 treatments, with 3 replicates per treatment. Three rice plants were planted in each pot, and the water level was maintained at 1-2 cm. The temperature was 27±2℃: (1) Blank control (CK): No bacteriophage or host bacteria were added; (2) Bacteriophage alone (P): 300 mL of bacteriophage suspension was inoculated; (3) Host bacteria alone (B): 300 mL of host bacteria suspension was inoculated; (4) Bacteriophage + host bacteria (VP): Equal amounts of the two suspensions were inoculated simultaneously; (5) Inactivated bacteriophage control (IP): 300 mL of bacteriophage suspension inactivated by heating at 65℃ for 30 min was inoculated; (6) Ordinary farmland soil control (NS): Non-saline-alkali soil (pH 6.5, soluble salt 0.12%) was used as the substrate, and the same treatment as VP was applied. Rice samples were collected and measured after 90 days of growth.

[0035] The test results showed that: ① The degradation effect of HCH in the soil: the residue in the CK group was 0.59±0.04 mg·kg -1 The degradation rate was 13.2%; the P group had a dosage of 0.55 ± 0.03 mg / kg. -1 The degradation rate was 19.1%, which was not significantly different from the control (P > 0.05), proving that the phage alone has no degradation function; Group B: 0.38 ± 0.03 mg·kg -1 The degradation rate was 44.1%, significantly higher than that of the control (CK) (P < 0.05), indicating that the host bacteria had a certain degradation capacity; the residue in the VP group was only 0.12 ± 0.02 mg·kg. -1 The degradation rate was 82.4%, significantly better than group B (P < 0.01), indicating that bacteriophages significantly enhanced the degradation efficiency of the host bacteria; IP group: 0.58 ± 0.04 mg·kg -1The degradation rate was 14.7%, which was no different from the control (CK), excluding interference from inactivated products. ② DDT residue in rice grains: CK grain residue was 0.23 ± 0.03 mg·kg⁻¹. -1 The levels in group P were 0.21±0.02 and in group IP were 0.22±0.03, both far exceeding the national standard (GB 2763-2026 limit of 0.05 mg·kg). -1 Group B had a concentration of 0.15±0.02, which still did not meet the standard; while Group VP had a concentration of only 0.04±0.01, which was below the national standard. This indicates that the combined treatment can effectively block the migration of HCH to edible parts. ③ Alleviating the inhibition of rice growth by salt-alkali stress: Under salt-alkali conditions, the CK rice plant height was 52.3±3.1 cm, and the chlorophyll content was 1.85±0.12 mg·g. -1 FW growth was inhibited. There were no significant differences between groups P and IP and the control (CK). Group B plant height was 58.7±2.5 cm, and chlorophyll content was 2.31±0.11, showing significant improvement (P<0.05). Group VP plant height reached 64.2±2.2 cm, an increase of 22.8% compared to CK; aboveground dry weight was 28.7±1.4 g / pot. -1 The root dry weight increased by 54.3%; the root dry weight was 5.2 ± 0.3 g per pot. -1 The chlorophyll content increased by 62.5%; chlorophyll content was 2.78 ± 0.09 mg·g. -1 The degradation rate of FW increased by 50.3%, and all indicators were significantly better than CK (P < 0.01). This indicates that the phage and host synergy can effectively alleviate saline-alkali stress. ④ The specificity advantage of phage in saline-alkali environment: When VP treatment was compared with ordinary farmland soil (NS), the degradation rate of ordinary soil was 54.4%, while the degradation rate of saline-alkali soil reached 82.4%, and the effect in saline-alkali environment was significantly better than that in ordinary soil (P < 0.01). ⑤ Degradation half-life and intermediate product toxicity: According to first-order kinetics, the half-life of HCH in CK was about 433 days, while the half-life of VP group was only 32.7 days, shortened by 92.4%, which greatly accelerated degradation. GC-MS detection of intermediate products (γ-pentachlorocyclohexene, etc.) showed that the relative content in CK was 2.3%, which posed a risk of secondary pollution; the content in VP group was only 0.4%, which was 82.6% lower than the control, indicating that the combined treatment promoted the deep mineralization of intermediate products and significantly reduced the risk of toxicity.

[0036] This invention utilizes the synergistic effect of bacteriophage φYSZBA2 and the host Bacillus cereus to achieve a degradation rate of 82.4% for HCH in potted rice grown in saline-alkali soil. This is significantly superior to bacteriophage alone (19.1%), host bacteria alone (44.1%), and the inactivated control (14.7%). Rice grain residue was reduced to 0.04 mg·kg⁻¹. -1It meets national standards; it significantly alleviates saline-alkali stress and promotes growth; the degradation half-life is reduced from 433 days to 32.7 days, and the toxicity of intermediate products is reduced by 82.6%; its effect in saline-alkali environments is better than that in ordinary soils, proving its unexpected applicability. This example fully verifies the effectiveness and unique advantages of this bacteriophage in the remediation of organochlorine pesticide pollution in saline-alkali land.

[0037] Example 2

[0038] The soil used for the potted plants was collected from saline-alkali farmland, and the test crop was the salt-tolerant rice variety Yandao 12. This area has long been contaminated with nitrochlorobenzene; soil samples were collected from the topsoil layer (0–20 cm) at five sampling points. Basic soil physicochemical properties: sand 52.6%, silt 38.2%, clay 9.2%, pH 8.9, soluble salt content 0.83%, total nitrogen 0.93 g·kg⁻¹. -1 Total phosphorus 0.63 g·kg -1 Total potassium 21.34 g·kg -1 Organic matter 13.2 g·kg -1 CEC 16.1 cmol·kg -1 The initial contamination concentration of nitrochlorobenzene was 0.58 mg / kg. -1 .

[0039] The experiment consisted of 6 treatments, with 3 replicates per treatment. Three rice plants were planted in each pot, and the water level was maintained at 1-2 cm. The temperature was 27±2℃: (1) Blank control (CK): No bacteriophage or host bacteria were added; (2) Bacteriophage alone (P): 300 mL of bacteriophage suspension was inoculated; (3) Host bacteria alone (B): 300 mL of host bacteria suspension was inoculated; (4) Bacteriophage + host bacteria (VP): Equal amounts of the two suspensions were inoculated simultaneously; (5) Inactivated bacteriophage control (IP): 300 mL of bacteriophage suspension inactivated by heating at 65℃ for 30 min was inoculated; (6) Ordinary farmland soil control (NS): Non-saline-alkali soil (pH 6.5, soluble salt 0.12%) was used as the substrate, and the same treatment as VP was applied. Rice samples were collected and measured after 90 days of growth.

[0040] The test results showed that: ① The degradation effect of nitrochlorobenzene in the soil: the residual amount in the CK group was 0.44±0.03 mg·kg -1 The degradation rate was 15.4%; the P group had a dosage of 0.42 ± 0.03 mg·kg⁻¹. -1 The degradation rate was 19.2%, which was not significantly different from the control (P > 0.05), proving that the phage alone has no degradation function; Group B: 0.31 ± 0.02 mg·kg -1 The degradation rate was 40.4%, significantly higher than that of the control (CK) (P < 0.05), indicating that the host bacteria had a certain degradation capacity; the residue in the VP group was only 0.08 ± 0.01 mg·kg. -1The degradation rate was 84.6%, significantly better than group B (P<0.01), indicating that bacteriophages significantly enhanced the degradation efficiency of the host bacteria; IP group: 0.43±0.03 mg·kg -1 The degradation rate was 17.3%, which was no different from the control (CK), excluding interference from inactivated products. ② Nitrochlorobenzene residue in rice grains: Referring to relevant food safety standards, the residue in CK grains was 0.18 ± 0.02 mg / kg. -1 The levels in group P (0.17±0.02) and group IP (0.18±0.02) both far exceeded the limits; group B (0.12±0.01) still did not meet the standard; while group VP (0.03±0.01) was below the safe limit. This indicates that the combined treatment can effectively block the migration of nitrochlorobenzene to the edible parts. ③ Alleviating the inhibition of rice growth by salt-alkali stress: Under salt-alkali conditions, the CK rice plant height was 51.8±3.0 cm, and the chlorophyll content was 1.82±0.11 mg·g. -1 FW growth was inhibited. There were no significant differences between groups P and IP and CK. Group B plant height was 57.9±2.6 cm, and chlorophyll content was 2.28±0.10, showing significant improvement (P<0.05). Group VP plant height reached 63.5±2.3 cm, an increase of 22.6% compared to CK; aboveground dry weight was 28.1±1.5 g / pot. -1 The root dry weight increased by 53.6%; the root dry weight was 5.1 ± 0.3 g per pot. -1 The chlorophyll content increased by 61.9%; chlorophyll content was 2.75 ± 0.10 mg·g. -1 FW increased by 51.1%, and all indicators were significantly better than CK (P<0.01). This indicates that the phage and host synergy can effectively alleviate saline-alkali stress. ④ Specificity advantage of phage in saline-alkali environment: Comparing VP treatment in ordinary farmland soil (NS), the degradation rate in ordinary soil was 50.0%, while the degradation rate in saline-alkali soil reached 84.6%, showing a significantly better effect in saline-alkali environment than in ordinary soil (P<0.01). Those skilled in the art would have no motivation to use a phage known only for inactivating pathogenic bacteria for pesticide degradation in saline-alkali land, and the enhanced effect of this invention under saline-alkali conditions is unpredictable. ⑤ Degradation half-life and intermediate product toxicity: According to first-order kinetics, the half-life of nitrochlorobenzene in CK was about 362 days (the half-life of p-nitrochlorobenzene in atmospheric photochemical oxidation is 62 days, and the half-life in soil is significantly prolonged), while the half-life of VP group was only 28.5 days, shortened by 92.1%, greatly accelerating degradation. GC-MS analysis of intermediate products (such as p-chloroaniline) showed that the relative content in the control group was 1.9%, indicating a risk of secondary contamination; while the content in the VP group was only 0.3%, a reduction of 84.2% compared to the control, indicating that the combined treatment promoted the deep mineralization of intermediate products and significantly reduced the risk of toxicity.

[0041] Example 3

[0042] The soil used for the potted plants was collected from saline-alkali farmland, and the test crop was a salt-tolerant wheat variety (Yanmai 1902). This area has long been contaminated with hexachlorocyclohexane (HCH), and soil samples were collected from five topsoil layers (0–20 cm). Basic soil physicochemical properties: sand 51.3%, silt 36.1%, clay 12.6%, pH 8.9, soluble salt content 0.75%, total nitrogen 0.79 g·kg⁻¹. -1 Total phosphorus 0.71 g·kg -1 Total potassium 21.35 g·kg -1 Organic matter 11.8 g·kg -1 CEC 14.9 cmol·kg -1 The initial concentration of hexachlorocyclohexane (HCH) was 0.65 mg·kg⁻¹. -1 .

[0043] The experiment consisted of 6 treatments, with 3 replicates per treatment. Three wheat plants were planted in each pot, and the soil moisture content was maintained at 70% of field capacity. The temperature was 22±2℃. (1) Blank control (CK): no bacteriophage or host bacteria were added. (2) Bacteriophage alone (P): 300 mL of bacteriophage suspension was inoculated. (3) Host bacteria alone (B): 300 mL of host bacteria suspension was inoculated. (4) Bacteriophage + host bacteria (VP): equal amounts of the two suspensions were inoculated simultaneously. (5) Inactivated bacteriophage control (IP): 300 mL of bacteriophage suspension inactivated by heating at 65℃ for 30 min was inoculated. (6) Ordinary farmland soil control (NS): non-saline-alkali soil (pH 6.5, soluble salt 0.12%) was used as the substrate, and the same treatment as VP was applied. Wheat samples were collected and measured after 90 days of growth.

[0044] The test results showed that: ① The degradation effect of HCH in the soil: the residue in the CK group was 0.58±0.04 mg·kg -1 The degradation rate was 14.7%; the P group had a dosage of 0.55 ± 0.03 mg·kg⁻¹. -1 The degradation rate was 19.1%, which was not significantly different from the control (P>0.05), proving that the phage alone has no degradation function; Group B: 0.39±0.03 mg·kg -1 The degradation rate was 42.6%, significantly higher than that of the control (CK) (P<0.05), indicating that the host bacteria had a certain degradation capacity; the residue in the VP group was only 0.13±0.02 mg·kg. -1 The degradation rate was 80.9%, significantly better than group B (P<0.01), indicating that bacteriophages significantly enhanced the degradation efficiency of the host bacteria; IP group: 0.57±0.04 mg·kg -1 The degradation rate was 16.2%, which was no different from the control (CK), excluding interference from inactivated products. ② DDT residues in wheat grains: Referencing GB 2763-2026 standard (maximum residue limit of DDT in rice: 0.05 mg / kg)... -1(Wheat, referring to similar standards), CK grain residue was 0.21 ± 0.03 mg·kg. -1 The concentrations in group P (0.19±0.02) and group IP (0.20±0.03) far exceeded the national standard; group B (0.14±0.02) still did not meet the standard; while group VP (0.04±0.01) fell below the national standard. This indicates that the combined treatment can effectively block the migration of HCH to edible parts. ③ Alleviating the inhibition of wheat growth by salt-alkali stress: Under salt-alkali conditions, the CK wheat plant height was 48.6±2.8 cm, and the chlorophyll concentration was 1.78±0.10 mg·g. -1 FW growth was inhibited. There were no significant differences between groups P and IP and the control (CK). Group B plant height was 54.3±2.4 cm, and chlorophyll content was 2.22±0.09, showing significant improvement (P<0.05). Group VP plant height reached 59.8±2.1 cm, an increase of 23.0% compared to CK; aboveground dry weight was 25.6±1.3 g / pot. -1 The yield increased by 52.4%; the root dry weight was 4.7 ± 0.3 g per pot. -1 Increased by 61.0%; chlorophyll 2.69±0.08 mg·g -1 The degradation rate of FW was increased by 51.1%, and all indicators were significantly better than CK (P<0.01). This indicates that the phage and host synergy can effectively alleviate saline-alkali stress. ④ The specificity advantage of phage in saline-alkali environment: When VP treatment was compared with ordinary farmland soil (NS), the degradation rate of ordinary soil was 52.9%, while the degradation rate of saline-alkali soil reached 80.9%, and the effect in saline-alkali environment was significantly better than that in ordinary soil (P<0.01). Those skilled in the art would have no motivation to use a phage known only for inactivating pathogenic bacteria for pesticide degradation in saline-alkali land, and the enhancement effect of this invention under saline-alkali conditions is unpredictable. ⑤ Degradation half-life and intermediate product toxicity: According to first-order kinetics, the half-life of HCH in CK was about 425 days, while the half-life of VP group was only 34.2 days, shortened by 92.0%, which greatly accelerated degradation. GC-MS analysis of intermediate products (γ-pentachlorocyclohexene, etc.) showed that the relative content in the CK group was 2.1%, posing a risk of secondary pollution; while the content in the VP group was only 0.4%, a decrease of 81.0% compared with the control, indicating that the combined treatment promoted the deep mineralization of intermediate products and significantly reduced the risk of toxicity.

[0045] Example 4

[0046] The soil used for the potted plants was collected from saline-alkali farmland, and the tested crop was a salt-tolerant wheat variety (Yanmai 1902). This area has long been contaminated with nitrochlorobenzene; soil samples were collected from the topsoil layer (0–20 cm) at five sampling points. Basic soil physicochemical properties: sand 53.1%, silt 35.5%, clay 11.4%, pH 8.8, soluble salt content 0.85%, total nitrogen 0.77 g·kg⁻¹. -1 Total phosphorus 0.65 g·kg -1 Total potassium 21.98 g·kg-1 Organic matter 11.9 g·kg -1 CEC 14.9 cmol·kg -1 The initial contamination concentration of nitrochlorobenzene was 0.52 mg·kg⁻¹. -1 .

[0047] The experiment consisted of 6 treatments, with 3 replicates per treatment. Three wheat plants were planted in each pot, and the soil moisture content was maintained at 70% of field capacity. The temperature was 22±2℃. (1) Blank control (CK): no bacteriophage or host bacteria were added. (2) Bacteriophage alone (P): 300 mL of bacteriophage suspension was inoculated. (3) Host bacteria alone (B): 300 mL of host bacteria suspension was inoculated. (4) Bacteriophage + host bacteria (VP): equal amounts of the two suspensions were inoculated simultaneously. (5) Inactivated bacteriophage control (IP): 300 mL of bacteriophage suspension inactivated by heating at 65℃ for 30 min was inoculated. (6) Ordinary farmland soil control (NS): non-saline-alkali soil (pH 6.5, soluble salt 0.12%) was used as the substrate, and the same treatment as VP was applied. Wheat samples were collected and measured after 90 days of growth.

[0048] The test results showed that: ① The degradation effect of nitrochlorobenzene in the soil: the residual amount in the CK group was 0.43±0.03 mg·kg -1 The degradation rate was 17.3%; the P group had a dosage of 0.41 ± 0.03 mg·kg⁻¹. -1 The degradation rate was 21.2%, which was not significantly different from the control (P>0.05), proving that the phage alone has no degradation function; Group B 0.30±0.02 mg·kg -1 The degradation rate was 42.3%, significantly higher than that of the control (CK) (P<0.05), indicating that the host bacteria had a certain degradation capacity; the residue in the VP group was only 0.09±0.01 mg·kg. -1 The degradation rate was 82.7%, significantly better than group B (P<0.01), indicating that bacteriophages significantly enhanced the degradation efficiency of the host bacteria; IP group: 0.42±0.03 mg·kg -1 The degradation rate was 19.2%, which was no different from the control (CK), excluding interference from inactivated products. ② Nitrochlorobenzene residue in wheat grains: Referring to relevant food safety standards, the residue in CK grains was 0.17 ± 0.02 mg / kg. -1 The concentrations in group P (0.16±0.02) and group IP (0.17±0.02) were both far exceeding the limits; group B (0.11±0.01) still did not meet the standard; while group VP (0.03±0.01) was below the safe limit. This indicates that the combined treatment can effectively block the migration of nitrochlorobenzene to the edible parts. ③ Alleviating the inhibitory effect of salt-alkali stress on wheat growth: Under salt-alkali conditions, the CK wheat plant height was 47.9±2.7 cm, and the chlorophyll concentration was 1.75±0.09 mg·g. -1FW growth was inhibited. There were no significant differences between groups P and IP and CK. Group B plant height was 53.8±2.3 cm, and chlorophyll content was 2.18±0.09, showing significant improvement (P<0.05). Group VP plant height reached 59.2±2.0 cm, 23.6% higher than CK; aboveground dry weight was 25.2±1.2 g / pot. -1 The root dry weight increased by 53.7%; the root dry weight was 4.6 ± 0.2 g per pot. -1 Increased by 61.4%; chlorophyll 2.65±0.08 mg·g -1 The degradation rate of FW was increased by 51.4%, and all indicators were significantly better than CK (P<0.01). This indicates that the phage and host synergy can effectively alleviate saline-alkali stress. ④ The specificity advantage of phage in saline-alkali environment: When VP treatment was compared with ordinary farmland soil (NS), the degradation rate of ordinary soil was 48.1%, while the degradation rate of saline-alkali soil reached 82.7%, and the effect in saline-alkali environment was significantly better than that in ordinary soil (P<0.01). Those skilled in the art would have no motivation to use a phage known only for inactivating pathogenic bacteria for pesticide degradation in saline-alkali land, and the enhancement effect of this invention under saline-alkali conditions is unpredictable. ⑤ Degradation half-life and intermediate product toxicity: According to first-order kinetics, the half-life of nitrochlorobenzene in CK was about 355 days, while the half-life of VP group was only 27.8 days, shortened by 92.2%, greatly accelerating degradation. GC-MS analysis of intermediate products (such as p-chloroaniline) showed that the relative content in the control group was 1.8%, indicating a risk of secondary pollution; while the content in the VP group was only 0.3%, a reduction of 83.3% compared to the control, indicating that the combined treatment promoted the deep mineralization of intermediate products and significantly reduced the risk of toxicity.

Claims

1. Application of Bacillus cereus phage φYSZBA2 in the remediation of organochlorine pesticide contaminated soil in saline-alkali land.

2. The application according to claim 1, characterized in that... The organochlorine pesticides include one of hexachlorocyclohexane (HCH) and nitrochlorobenzene.

3. The application according to claim 1, characterized in that... The pH of the saline-alkali soil is 8.0–10.0, and the soluble salt content is 0.3%–5.0%.

4. The application according to claim 1, characterized in that... The bacteriophage φYSZBA2 was reintroduced into saline-alkali soil contaminated with organochlorine pesticides in the form of a bacteriophage suspension. After reintroduction, the bacteriophage titer in the soil was 10. 6 ~10 9 PFU / g.

5. The application according to claim 4, characterized in that... It also includes inoculating the soil with Bacillus cereus, wherein the concentration of the inoculum of Bacillus cereus is 10. 7 ~10 8 CFU / g.

6. Application of Bacillus cereus phage φYSZBA2 in the preparation of microbial agents for the remediation of organochlorine pesticide-contaminated soil in saline-alkali land.

7. The application according to claim 6, characterized in that... The repair agent also contains Bacillus cereus.

8. A method for remediating organochlorine pesticide-contaminated soil in saline-alkali land, comprising applying bacteriophage φYSZBA2 in the form of a bacteriophage suspension to the organochlorine pesticide-contaminated saline-alkali land soil.

9. The method according to claim 8, characterized in that... It also includes applying Bacillus cereus to the soil, wherein the concentration of the Bacillus cereus solution is 10. 7 ~10 8 CFU / g.

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