Bacillus strain jz-5 and application thereof

Abstract

The application discloses a bacillus strain JZ-5 and application, and belongs to the technical field of microorganisms, the strain is separated from the plough layer soil, has been preserved in the China typical culture preservation center, and the preservation number is CCTCC NO: M 2025651. The bacillus strain JZ-5 not only has the characteristics of high efficiency, broad spectrum and rapid degradation of pyrethroid insecticides, but also has strong salt tolerance, and is suitable for the remediation of saline-alkali soil and other salinization soil; and the strain can produce indole-3-acetic acid at high yield, can effectively promote the root system development and plant growth of crops, realizes the triple effects of degradation detoxification, salt tolerance and stress resistance and growth promotion and yield increase. The production process of the microbial inoculant produced based on the strain is simple, low in cost, convenient to use and high in environmental safety, is suitable for large-area popularization and application, and has important significance for guaranteeing the quality and safety of agricultural products, repairing contaminated soil, improving saline-alkali soil, protecting the ecological environment and promoting the green development of agriculture.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Bacillus strain JZ-5 and its applications. Background Technology

[0002] Pyrethroid insecticides are a class of broad-spectrum, highly effective insecticides synthesized artificially using natural pyrethrin as the parent structure. Since the 1970s, they have been widely used in agriculture, forestry, animal husbandry, storage, and sanitary pest control due to their advantages such as high insecticidal activity, rapid knockdown speed, low dosage, and relatively low acute toxicity to mammals. Among them, cypermethrin, deltamethrin, and fenvalerate have become the most widely used pyrethroid pesticide varieties. With long-term and large-scale application, pyrethroid insecticides have become one of the most common pesticide residues in farmland ecosystems. Due to the presence of ester bonds, halogenated benzene rings, and cyclopropane carboxylic acids in their molecules, they have high hydrophobicity and chemical stability, easily adsorbing into soil organic matter and sediments. They are widely detected in soil, water bodies, sediments, and agricultural products, and can migrate and accumulate through the food chain, adversely affecting aquatic organisms, non-target insects, and soil microbial communities. At the same time, they increase the risk of pesticide residue exceeding the standard in agricultural products, posing a potential threat to ecological and environmental safety and food safety.

[0003] Currently, methods for controlling pyrethroid pollution mainly include chemical degradation, photocatalysis, adsorption, and microbial remediation. Among these, microbial remediation technology utilizes microbial metabolism or its produced esterases, carboxylesterases, and other degrading enzymes to catalyze the hydrolysis of ester bonds in pyrethroid molecules and further metabolize intermediate products. It boasts advantages such as environmental friendliness, low cost, no secondary pollution, and in-situ remediation capabilities, and is considered one of the most promising technical routes for controlling pyrethroid pesticide pollution. Microorganisms reported to possess pyrethroid degradation capabilities include *Sphingomonas* spp. (…). Sphingobium ), genus *Pseudomonas* Ochrobactrum ), Bacillus spp. Bacillus ), Pseudomonas spp. Pseudomonas (e.g., can degrade one or more pyrethroid pesticides.)

[0004] However, existing pyrethroid-degrading bacteria still have certain limitations. For example, most strains can only efficiently degrade a single or a few types of pyrethroid pesticides, resulting in a narrow degradation spectrum. Some strains have low degradation rates, insufficient tolerance to high concentrations of pesticides, and poor colonization and environmental adaptability in complex soil environments. Adverse conditions such as high salinity, drought, and poor nutrition can further inhibit the growth and degradation activity of the strains. In addition, there are currently no reports of pyrethroid-degrading bacteria that also have crop growth-promoting functions. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a Bacillus strain JZ-5 that simultaneously possesses the triple functions of degradation and detoxification, salt tolerance and stress resistance, and growth promotion and yield increase; the second purpose is to provide applications for this Bacillus strain JZ-5.

[0006] Technical solution: The Bacillus strain described in this invention ( Bacillus sp. JZ-5, accession number CCTCCNO: M 2025651.

[0007] Preservation Notes: The Bacillus strain JZ-5 in this invention is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2025651, deposited on March 31, 2025. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, 430072, China.

[0008] The microbial preparation of the present invention contains Bacillus strain JZ-5.

[0009] Preferably, the microbial preparation further contains a biologically acceptable carrier.

[0010] Preferably, the biologically acceptable carrier includes: culture medium, enrichment agent, wetting agent, penetrant, dispersant, emulsifier, stabilizer, adhesive, filler, surfactant, controlled release agent, synergist, binder, spreader, anti-drift agent, safener, or spraying aid.

[0011] The application of Bacillus strain JZ-5 or microbial preparations described in this invention in the degradation of pyrethroid compounds.

[0012] Preferably, the pyrethroid compound is selected from any one or more of cypermethrin, deltamethrin, fenvalerate, bifenthrin, permethrin, deltamethrin, flufenoxuron, and flufenoxuron.

[0013] The application of the Bacillus strain JZ-5 or microbial preparation described in this invention in agricultural production.

[0014] Preferably, the application is for remediating pyrethroid compound pollution in agricultural production.

[0015] Preferably, the application is an application that promotes crop growth.

[0016] Preferably, the application is performed in a high-salt environment; the high-salt environment is defined as NaCl concentration not exceeding 100 g / L.

[0017] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. The Bacillus strain JZ-5 is a newly isolated strain derived from topsoil, possessing the characteristics of highly efficient, broad-spectrum, and rapid degradation of pyrethroid insecticides. It can degrade more than 99% of 0.2 mM cypermethrin within 48 hours; 2. This strain has extremely strong salt tolerance, maintaining growth in a high-salt environment containing 75 g / L NaCl and maintaining a certain degradation activity at 100 g / L NaCl, making it suitable for the remediation of saline-alkali land and other saline-alkali soils; 3. This strain can produce high levels of indole-3-acetic acid, with a peak concentration reaching 12.6438 mg / L, effectively promoting crop root development and plant growth, achieving the triple effects of degradation and detoxification, salt tolerance and stress resistance, and growth promotion and yield increase; 4. The inoculant produced based on this strain has a simple production process, low cost, convenient use, and high environmental safety, making it suitable for large-scale promotion and application. It is of great significance for ensuring the quality and safety of agricultural products, remediating polluted soils, improving saline-alkali lands, protecting the ecological environment, and promoting green agricultural development. Attached Figure Description

[0018] Figure 1 The image shows the morphological characteristics of Bacillus strain JZ-5 grown on LB solid culture plates. In the image, A is a representative image of the colony and B is a representative image of the transmission electron microscope.

[0019] Figure 2 Phylogenetic tree analysis of Bacillus strain JZ-5;

[0020] Figure 3 Representative images of Bacillus strain JZ-5 grown on LB solid culture plates containing different concentrations of NaCl;

[0021] Figure 4 Time-degradation curve of Bacillus strain JZ-5 against pyrethroid insecticides;

[0022] Figure 5 The graph shows the effects of environmental factors on the degradation of cypermethrin by Bacillus strain JZ-5. In the graph, A shows the effect of different temperatures, B shows the effect of different pH values, and C shows the effect of different NaCl concentrations.

[0023] Figure 6 The figures show the analytical results of the degradation of cypermethrin by Bacillus strain JZ-5, where A is the result of liquid chromatography and B is the result of mass spectrometry. Detailed Implementation

[0024] The technical solution of the present invention will be further described below.

[0025] Example 1: Screening and identification of Bacillus strain JZ-5

[0026] 1. Screening of strain JZ-5

[0027] In September 2024, soil samples were collected from the 0-20 cm topsoil layer of a tomato greenhouse in Chuzhou City, Anhui Province. The residual amount of cypermethrin was 0.46 mg / kg, the pH was 6.8, the organic matter was 25.4 g / kg, and the total nitrogen was 1.8 g / kg.

[0028] 10.0 g of the previously collected soil sample was weighed and added to a 250 mL Erlenmeyer flask containing 100 mL of MM medium. Simultaneously, a cypermethrin (cis-cypermethrin, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., C110030)-methanol solution was added, with a final cypermethrin concentration of 0.2 mM. The sample was cultured in the dark at 30℃ and 150 rpm for 7 days. Samples were taken every 48 h to determine the residual amount. Specifically, an equal volume of ethyl acetate was added to the sample for extraction, and the extraction was performed twice. The organic phases were combined, dried under nitrogen, and then diluted to 0.5 mL with methanol. The sample was filtered through a 0.22 μm organic filter membrane and then analyzed by high performance liquid chromatography (HPLC). The HPLC conditions were as follows: a Thermo Fisher Scientific Dionex UltiMate 3000SD liquid chromatography system was used, with a 250 mm × 4.6 mm C18 reversed-phase column, a column temperature of 40 °C, a mobile phase of 90% (v / v) methanol aqueous solution containing 0.5% (v / v) acetic acid, a flow rate of 1.0 mL / min, and detection wavelengths of 230 nm and 280 nm.

[0029] The external standard method was used for the quantification of cypermethrin. Specifically, cypermethrin solutions with concentrations of 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, and 0.4 mM were prepared. After HPLC detection, concentration-peak area standard curves were plotted, and the cypermethrin content in the samples was calculated based on the standard curves. The degradation rate was further calculated based on the initial cypermethrin content in the samples.

[0030] When the residual amount of cypermethrin was less than 50%, it was transferred to fresh MM medium containing 0.2 mM cypermethrin at 5% (v / v) and enriched 4 times consecutively.

[0031] Take the 5th generation enrichment solution and process it for 10... -1 ~10 -6 After serial dilution, the bacteria were spread onto MM solid culture plates containing 0.2 mM cypermethrin and incubated at 30°C for 72 h. Single colonies that formed degradation zones were picked and streaked onto fresh LB solid culture plates for purification. This process was repeated three times to obtain a strain that could effectively degrade cypermethrin, which was named JZ-5.

[0032] The MM medium formula is as follows: 1.0 g / L (NH4)2SO4, 0.5 g / L KH2PO4·2H2O, 0.2 g / L MgSO4·7H2O, 1.5 g / L K2HPO4·3H2O, 1.0 g / L NaCl, and the remainder is water.

[0033] The aforementioned JZ-5 strain was inoculated onto LB solid culture plates and incubated at 30°C for 24 h. Its colony morphology is as follows: Figure 1 As shown in Figure A, the colonies of this strain on LB solid culture plates are round with irregular edges, yellowish-brown, opaque, and have a rough, dry surface.

[0034] Further observation using transmission electron microscopy at an accelerating voltage of 80 kV yielded the following results: Figure 1 As shown in B, strain JZ-5 is rod-shaped, approximately (0.6~0.8) μm × (2.0~4.0) μm in size, with peritrichous flagella, a relatively thick cell wall, and typical characteristics of Gram-positive bacteria; the cytoplasm has a uniform electron density and contains a small amount of electron-transparent granular inclusions.

[0035] 2. Identification of strain JZ-5

[0036] 2.1 Physiological and biochemical identification

[0037] Strain JZ-5 was inoculated onto LB solid culture plates and incubated at 30°C for 24 h. Fresh single colonies were then picked and a bacterial suspension with a McFarland turbidity of 0.5 was prepared using API 50CHB / E (purchased from bioMérieux, 50430). This suspension was then inoculated onto API 50 CH strips (purchased from bioMérieux, 50300) and incubated at 30°C for 48 h. The carbohydrate utilization reaction results were recorded, and the strain was identified.

[0038] Further identification was conducted with reference to Bergey's Manual® of Systematic Bacteriology (David R. Boone, Richard W. Castenholz, George M. Garrity. Springer, New York, NY:DOI:10.1007 / 978-0-387-21609-6.).

[0039] The results showed that JZ-5 is a strict aerobic bacterium; it was positive for catalase test, oxidase test, VP test, gelatin liquefaction test, starch hydrolysis test and nitrate reduction test; it was negative for urease test, indole test and H2S production test; and it can produce acid using glucose, sucrose and mannitol.

[0040] 2.2 Molecular Identification

[0041] Genomic DNA was extracted from strain JZ-5 using a bacterial genomic DNA extraction kit (DP302, Tiangen Biotech Co., Ltd.). The 16S rDNA fragment of strain JZ-5 was amplified using universal bacterial primers 27F / 1492R and a 2× Phanta Max MasterMix kit (P515, Nanjing Novizan Biotechnology Co., Ltd.). The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, for a total of 30 cycles; final extension at 72℃ for 5 min. The obtained PCR products were bidirectionally sequenced by Beijing Qingke Biotechnology Co., Ltd.; the resulting sequences were assembled and compared and analyzed in the EzBioCloud database.

[0042] The results showed that strain JZ-5 was related to Bacillus ( Bacillus They are the closest relatives, among whom are... Bacillus paranthracis Mn5 T , Bacillus nitratireducens 4049 T and Bacillus cereus The similarity to bacteria such as ATCC 145791T is 100%. Based on the colony morphology, physiological and biochemical characteristics, and phylogenetic analysis of the 16S rRNA gene, strain JZ-5 is classified as a Bacillus strain.

[0043] Example 2: Determination of the ability of Bacillus strain JZ-5 to produce indole-3-acetic acid

[0044] Single colonies of JZ-5 were picked and inoculated into 4 mL of LB liquid medium. After culturing at 30℃ and 150 rpm for 24 h, activated JZ-5 was obtained. The activated JZ-5 was then inoculated into 100 mL of LB liquid medium containing 500 mg / L L-tryptophan at a 2% (v / v) inoculation rate and cultured at 30℃ and 150 rpm. Samples were taken at 24 h, 48 h, and 72 h. The fermentation broth samples were centrifuged at 12,000 rpm for 10 min, and the supernatant was collected and mixed with an equal volume of Salkowski chromogenic solution. After reacting at room temperature in the dark for 30 min, the absorbance was measured at 530 nm. The Salkowski chromogenic solution was prepared by mixing 1 mL of 0.5 mol / L FeCl3 aqueous solution with 50 mL of 35% (v / v) perchloric acid solution.

[0045] Indole-3-acetic acid (IAA) solutions with concentrations of 0, 1, 5, 10, 15, and 20 mg / L were mixed with an equal volume of Salkowski colorimetric solution and reacted at room temperature in the dark for 30 min. The absorbance was measured at 530 nm, a standard curve was plotted, and the IAA concentration in the supernatant of the fermentation broth sampled at each of the aforementioned time points was calculated.

[0046] The results showed that the IAA production of JZ-5 reached its peak at 48 h, at 12.6438 mg / L; it decreased slightly at 72 h, to 10.8925 mg / L.

[0047] Example 3: Salt tolerance test of Bacillus strain JZ-5

[0048] LB agar plates containing 25, 50, 75, and 100 g / L NaCl, as well as LB agar plates without NaCl, were prepared. Activated JZ-5 was prepared into OD using sterile physiological saline. 600 5 μL of a bacterial suspension with a concentration of 0.1 was streaked onto plates of different NaCl concentrations, with three replicates for each concentration. After incubation at 30°C for 48 h, colony growth was observed and images were collected and recorded.

[0049] The results are as follows Figure 3 As shown, JZ-5 can form visible colonies with full morphology and maintain growth in the range of NaCl below 75 g / L; no colonies are formed under high salt stress of 100 g / L NaCl; indicating that JZ-5 has extremely strong salt tolerance, which enables JZ-5 to effectively colonize and play a role in saline-alkali soils and other saline soils.

[0050] Example 4: Determination of pyrethroid degradation performance of Bacillus strain JZ-5

[0051] 1. Degradation performance determination under laboratory conditions

[0052] 1.1 Degradation of pyrethroid insecticides by Bacillus strain JZ-5

[0053] The activated JZ-5 was determined according to its final turbidity OD. 600An inoculum of 0.15 g was inoculated into 100 mL of MM medium containing 0.2 mM cypermethrin, deltamethrin (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., D114244), and fenvalerate (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., F109864). The medium was incubated at 30°C and 150 rpm using a shaker. A sample was taken at the start of the experiment (0 h), followed by 5 mL samples every 8 h for 48 h. The samples were extracted twice with an equal volume of ethyl acetate. The organic phases were combined, dried under nitrogen, and then diluted to 0.5 mL with methanol. The mixture was filtered through a 0.22 μm organic filter membrane and analyzed by high-performance liquid chromatography (HPLC). The HPLC conditions were as follows: a Thermo Fisher Scientific Dionex UltiMate 3000SD liquid chromatography system was used, with a 250 mm × 4.6 mm C18 reversed-phase column, a column temperature of 40 °C, a mobile phase of 90% (v / v) methanol aqueous solution containing 0.5% (v / v) acetic acid, a flow rate of 1.0 mL / min, and detection wavelengths of 230 nm and 280 nm.

[0054] The external standard method was used to quantify pyrethroid insecticides. Specifically, solutions of cypermethrin, deltamethrin, and fenvalerate with concentrations of 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, and 0.4 mM were prepared. After HPLC detection, concentration-peak area standard curves were plotted, and the pyrethroid insecticide content in the aforementioned samples was calculated based on the standard curves.

[0055] The degradation rate was further calculated based on the initial content of pyrethroid insecticides in the aforementioned samples.

[0056] The results are as follows Figure 4 As shown, at a final concentration of 0.2 mM, JZ-5 can degrade 99.2% of cypermethrin, 96.8% of deltamethrin, and 90.2% of fenvalerate within 48 h.

[0057] 1.2 Effects of Environmental Factors on the Degradation Performance of Cypermethrin by Bacillus strain JZ-5

[0058] Single colonies of JZ-5 were inoculated into LB liquid medium and incubated at 30°C and 180 rpm until OD500 was reached. 600 =1.0, centrifuged at 6000 rpm for 10 min to collect bacterial cells, washed twice with MM medium and resuspended in MM medium, OD 600 =1.0, used as seed solution for subsequent use.

[0059] 1.2.1 Effect of Temperature

[0060] Take six 50 mL Erlenmeyer flasks and fill each with 20 mL of MM medium containing 0.2 mM cypermethrin. Calculate the final turbidity (OD) according to the concentration. 600 Inoculate the aforementioned seed culture at an inoculum size of 0.15, mix thoroughly, and immediately take 10 mL of bacterial culture. Centrifuge at 12000 rpm for 5 min, and collect the supernatant as a 0 h control. Subsequently, incubate at 16, 20, 25, 30, 37, and 42℃ on a shaker at 150 rpm for 48 h, with three replicates for each treatment temperature. After incubation, take 10 mL of sample, centrifuge using the same method, collect the supernatant, fully extract according to the aforementioned method, and determine the cypermethrin concentration and calculate the degradation rate by HPLC.

[0061] The results are as follows Figure 5 As shown in A, JZ-5 exhibits the best degradation effect at 30℃, with a degradation rate of 99.1% after 48 h, and a degradation rate of >80% in the range of 25-37℃; when the temperature drops to 16℃, the degradation rate is only 47.2%, and at 42℃, the degradation rate is 61.4%.

[0062] 1.2.2 Effect of pH

[0063] Take six 50 mL Erlenmeyer flasks and fill each with 20 mL of MM medium containing 0.2 mM cypermethrin, adjusted to pH 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 respectively using 5M HCl or 5M NaOH. Calculate the final turbidity (OD) according to the OD value. 600 Inoculate the aforementioned seed culture at an inoculum size of 0.15, mix thoroughly, and immediately take 10 mL of bacterial culture. Centrifuge at 12000 rpm for 5 min, and collect the supernatant as a 0 h control. Subsequently, incubate at 30℃ and 150 rpm for 48 h, with three replicates for each pH treatment. After the incubation period, take 10 mL of sample, centrifuge using the same method, collect the supernatant, and after thorough extraction, determine the cypermethrin concentration and calculate the degradation rate according to the aforementioned HPLC method.

[0064] The results are as follows Figure 5 As shown in B, JZ-5 has a degradation rate of >99% at pH 7.0, and degradation rates of 80.5% and 82.3% at pH 6.0 and 8.0, respectively; the degradation rate drops to 63.3% at pH 5.0, and the degradation rate is 73.7% at pH 9.0.

[0065] 1.2.3 Effect of NaCl concentration

[0066] Take six 50 mL Erlenmeyer flasks and fill each with 20 mL of 0.2 mM cypermethrin-containing medium based on NaCl-free MM medium, then add NaCl to each flask at final concentrations of 0, 25, 50, 75, 100, and 125 g / L, respectively. Analyze the final turbidity (OD) according to... 600Inoculate the seed culture obtained above with an inoculum of 0.15 g / L. After thorough mixing, immediately take 10 mL of the bacterial culture, centrifuge at 12000 rpm for 5 min, and collect the supernatant as a 0 h control. Subsequently, incubate at 30℃ and 150 rpm for 48 h, with three replicates for each NaCl concentration treatment. After the incubation period, take 10 mL of the sample, centrifuge using the same method, collect the supernatant, fully extract according to the aforementioned method, and determine the cypermethrin concentration and degradation rate by HPLC.

[0067] The results are as follows Figure 5 As shown in C, the degradation rate of JZ-5 remained above 80% in the range of 0-50 g / L NaCl; it decreased to 72.2% at 75 g / L; and it still maintained 4.6% degradation activity at 100 g / L, proving that JZ-5 still has a certain degradation ability under extremely high salt concentrations.

[0068] 1.2.4 Influence of Exogenous Carbon and Nitrogen Sources

[0069] Take 12 50 mL Erlenmeyer flasks and fill each with 20 mL of MM medium containing 0.2 mM cypermethrin, supplemented with either glucose, sucrose, maltose, lactose, mannitol, glycerol, sodium acetate, urea, yeast extract (purchased from Beijing Solarbio Science & Technology Co., Ltd., Y8020), tryptone (purchased from Beijing Solarbio Science & Technology Co., Ltd., LP0042B), or beef extract (purchased from Beijing Solarbio Science & Technology Co., Ltd., G8270) at a final concentration of 1 g / L. Use 20 mL of MM medium containing 0.2 mM cypermethrin without supplementation as a control. Analyze the final turbidity (OD)... 600 Inoculate the seed culture obtained above with an inoculum of 0.15 g / L. After thorough mixing, immediately take 10 mL of the bacterial culture, centrifuge at 12,000 rpm for 5 min, and collect the supernatant as a 0 h control. Subsequently, incubate at 30℃ and 150 rpm for 24 h, with 3 replicates per treatment. After the incubation period, take 10 mL of the sample, centrifuge using the same method, collect the supernatant, fully extract according to the aforementioned method, and determine the cypermethrin concentration and degradation rate by HPLC.

[0070] The results are shown in Table 1. The addition of glucose, sucrose, and maltose significantly promoted degradation, increasing the degradation rate by 15.3%, 12.7%, and 10.4% respectively compared to the control group without any additives. Mannitol and glycerol had no significant effect. Sodium acetate inhibited degradation, decreasing the degradation rate by 8.6%. Yeast extract, peptone, and beef extract significantly promoted degradation, increasing the degradation rate by 8.2%, 6.5%, and 12.1% respectively. Urea had no significant promoting effect.

[0071] Table 1. Effects of exogenous carbon and nitrogen sources on the degradation of cypermethrin by JZ-5

[0072]

[0073] Where ns indicates no significant difference compared to the no-additive control group, and ** indicates a significant difference compared to the no-additive control group. P <0.01.

[0074] 1.3 Identification of metabolites of cypermethrin degradation by Bacillus strain JZ-5

[0075] The seed culture of JZ-5 was determined according to its final turbidity OD. 600 =0.15% inoculum was inoculated into 100 mL MM medium containing 0.2 mM cypermethrin and cultured at 30 °C and 160 rpm on a shaker. Every 4 h, 10 mL samples were taken, and the resulting samples were extracted with an equal volume of dichloromethane. After drying under nitrogen, the extract was dissolved in 200 μL of methanol and filtered through a 0.22 μm organic filter. LC-MS identification was performed when the substrate residue decreased to approximately 50% as determined by HPLC. LC-MS analysis of metabolites was conducted using an AB SCIEX Triple TOF5600 micrometer. + Identification was performed using a UPLC mass spectrometry system. The column was an ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm). The mobile phase was acetonitrile (B) and a 5 mM ammonium acetate aqueous solution at pH 7.0 (A). The flow rate was 0.3 mL / min, and the gradient elution program was: 0–2 min 5% B, 2–10 min ramp to 95% B, 10–13 min hold, and 13–13.5 min equilibration back to 5% B. Mass spectrometry was performed in positive and negative ion switching mode (ESI). + / ESI - Acquisition was performed using capillary voltage +5.5 / -4.5 kV, source temperature 550℃, curtain gas 35 psi, nebulizer gas and auxiliary gas both 55 psi, first-stage full scan range m / z 50-600, and IDA mode automatically triggered second-stage MS / MS (CE35±15 eV).

[0076] Liquid phase analysis results as follows Figure 6 As shown in Figure A, JZ-5 completely degraded cypermethrin within 48 h, converting it into the key intermediate 3-phenoxybenzoic acid (3-PBA); the mass spectrometry analysis results of the product are as follows. Figure 6 As shown in B, 3-PBA in ESI - The following are [MH] - The sample was detected at m / z 213.0559, with secondary fragments at m / z 93.0340 and 169.0653. Comparison with the standard confirmed the metabolic pathway. Therefore, the key metabolite for the degradation of cypermethrin by strain JZ-5 is 3-PBA.

[0077] 2. Degradation performance determination in soil environment

[0078] Soil samples were taken from a typical vegetable garden in the suburbs of Nanjing, Jiangsu Province, where tomatoes had been grown for three consecutive years. The soil sample was silty loam, with sand, silt, and clay content of 42%, 38%, and 20%, respectively. The soil pH was 7.0, organic matter content was 2.1%, field capacity was 28%, and the background content of cypermethrin was 0.02 mg / kg. After air-drying, the soil sample was sieved through a 2 mm sieve to remove stones and plant debris. The moisture content was adjusted to 60% of the field capacity. The resulting soil sample was used for subsequent experiments.

[0079] Cypermethrin was dissolved in chromatographically pure methanol to prepare a 1000 mg / L stock solution. 2.5 mL of this stock solution was added dropwise to 100 g of diatomaceous earth dried at 120℃ for 2 h. The solution was then placed in a fume hood to evaporate the solvent, ensuring complete adsorption of the cypermethrin. The diatomaceous earth containing the cypermethrin was then thoroughly mixed with 400 g of the previously obtained soil sample in a sterile stainless steel dish. The final theoretical concentration of cypermethrin in the soil was 5 mg / kg (dry weight).

[0080] Weigh 500 g of contaminated soil into a 500 mL sterile plastic cup (9 cm in diameter, 12 cm in height), and set up the following 3 treatment groups: T1: Inoculate with 5 mL of JZ-5 bacterial agent (equivalent to 1×10⁻⁶). 7 T2: Inoculated with 5 mL of sterile MM medium; T3: No treatment. T2 was the blank control, and T3 was the chemical control. Each treatment group had three replicates. The JZ-5 inoculum was: cultured Bacillus strain JZ-5, centrifuged and washed, resuspended in sterile MM medium at a concentration of 10. 10 CFU / mL bacterial suspension.

[0081] During treatment, the soil moisture content was maintained at 60% field capacity. The cup mouths were covered with breathable sealing film and incubated in a 30℃ constant temperature incubator in the dark for 7 days. Samples were taken at 0, 1, 3, 5, and 7 days. Three points were randomly selected from the 0-5 cm surface layer using a sterile soil auger each time. After mixing, 20g of the sample was taken and immediately placed at -20℃ to terminate microbial activity.

[0082] Add 40 mL of methanol to 20 g of the sampled wet soil, sonicate at 200 W and 40 kHz for 30 min, shake at 200 rpm for 30 min, centrifuge at 8000 rpm for 5 min, collect the supernatant and filter it using a 0.22 µm organic filter membrane; repeat the above operation on the residue once more, combine the two supernatants, blow dry with nitrogen, and determine the concentration of cypermethrin and calculate the degradation rate by HPLC according to the above method.

[0083] Table 2 Degradation dynamics of cypermethrin by JZ-5 in soil (mean ± SD, n=3)

[0084]

[0085] The results are shown in Table 2. In T1 (inoculated with JZ-5), the degradation rate was 53.8% on day 1, 83.6% on day 3, 92.4% on day 5, and 94.9% on day 7. In T2 (blank control), the degradation rate was only naturally decreased by 5.1%, 7.9%, 9.7%, and 11.2% during the same period. There was no significant difference between T3 (chemical control) and T2. P >0.05). This indicates that JZ-5 can rapidly and stably degrade cypermethrin in soil, and no "soil inhibition" phenomenon was observed.

[0086] qPCR absolute quantification was further performed using specific primers. Using genomic DNA from JZ-5 pure culture as a template, the V4-V5 region of the 16S rRNA gene was amplified using specific primers JZ5-F: 5'-TCAACCGTGGAGGGTCATTG-3' (SEQ ID NO: 1) and JZ5-R: 5'-CAGTGTCAGTTACAGACCAG-3' (SEQ ID NO: 2). The PCR amplification products were purified, and their concentrations were measured and converted to copy numbers. Subsequently, PCR amplification products with different copy numbers were detected using a SYBR Green Pro Taq HS premixed qPCR kit (purchased from Hunan Aike Rui Biotechnology Co., Ltd., AG11701) at 10-fold serial dilutions. A copy number-Ct standard curve was established based on the detection results.

[0087] Total DNA was extracted from the aforementioned soil samples using a soil genomic DNA extraction kit (purchased from Tiangen Biotech Co., Ltd., DP336). After determining the DNA concentration and purity, qPCR detection was performed using the aforementioned JZ5-F and JZ5-R primers with a SYBR Green ProTaq HS premixed qPCR kit. The reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; 60℃ annealing and extension for 30 s, for 40 cycles. The amplification specificity was verified by the melting curve, and the sample Ct value was substituted into the aforementioned copy number-Ct value standard curve to calculate the gene copy number (copies / g dry soil) of strain JZ-5 in the soil, thereby assessing its colonization dynamics and distribution characteristics in the soil.

[0088] The results showed that the bacterial load rapidly increased to 1.2 × 10⁻⁶ mmol / L 1 day after inoculation. 7 Copies / g dry soil, reaching peak bacterial count of 1.4 × 10⁻⁶ after 3 days. 7 The bacterial count remained at 1.1 × 10⁻⁶ copies / g dry soil after 7 days. 7 Copies / g dry soil demonstrate that JZ-5 can effectively colonize in soil and maintain high activity.

[0089] Example 5: Preparation of Bacillus strain JZ-5 inoculum

[0090] Bacillus strain JZ-5 was activated on LB solid medium and then inoculated onto LB solid test tube slant for later use. The bacteria were then transferred from the test tube to a 1000 mL shake flask containing 200 mL of LB liquid medium and cultured at 30°C with shaking until the logarithmic phase to obtain the shake flask strain. The primary seed tank is 50 L, with a feed volume of 40 L. The culture medium formula is: 8 g / L peptone, 5 g / L yeast extract, 8 g / L glucose, 1 g / L K₂HPO₄, 10 g / L NaCl, 2 g / L CaCO₃, 0.2 g / L MgSO₄, 0.1% soybean oil (v / v), and the remainder is water, pH 7.2-7.5. After feeding, the medium is autoclaved and cooled to 30℃. The cultured shake-flask inoculum is then inoculated into the 50 L primary seed tank at a 10% (v / v) inoculation rate. The stirring speed is 220 rpm, and the sterile air flow rate is 1:0.6-1.2. The medium is cultured until the logarithmic growth phase is reached. The seed culture that has reached the logarithmic growth phase is then inoculated into the secondary seed tank at a 10% (v / v) inoculation rate. The secondary seed tank is 500 L, with a feed volume of 40 L. L. The culture medium formula and culture conditions are the same as those of the primary seed tank. The seed liquid that has reached the logarithmic phase is inoculated into the production tank at an inoculation rate of 10% (v / v). The production tank has a capacity of 5 tons and a feed rate of 4.5 tons. The culture medium composition is the same as that of the seed tank. After feeding, the medium is autoclaved and cooled to 30°C. Sterile air is circulated to maintain a sterile state. The temperature is maintained at 30°C after inoculation. The aeration rate is 1:0.6–1.2, the stirring speed is 220 rpm, and the culture time is 96–108 h. The cell count at the end of fermentation is ≥1 billion cells / mL. After the fermentation broth is discharged from the tank, it can be directly dispensed as a liquid inoculum formulation. Alternatively, it can be dispensed after adsorption with peat moss to form a solid inoculum formulation.

Claims

1. A strain of Bacillus ( Bacillus sp. JZ-5, characterized in that, The Bacillus strain JZ-5 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025651.

2. A microbial preparation, characterized in that, It contains the Bacillus strain JZ-5 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The microbial preparation also contains a biologically acceptable carrier.

4. The microbial preparation according to claim 3, characterized in that, The biologically acceptable carriers include: culture media, enrichment agents, wetting agents, penetrants, dispersants, emulsifiers, stabilizers, adhesives, fillers, surfactants, controlled-release agents, synergists, binders, spreaders, anti-drift agents, safeners, or spray additives.

5. The use of the Bacillus strain JZ-5 of claim 1 or the microbial preparation of any one of claims 2 to 4 in the degradation of pyrethroid compounds.

6. The application according to claim 5, characterized in that, The pyrethroid compounds are selected from any one or more of cypermethrin, deltamethrin, fenvalerate, bifenthrin, permethrin, deltamethrin, flufenoxuron, and flufenoxuron.

7. The application of the Bacillus strain JZ-5 of claim 1 or the microbial preparation of any one of claims 2 to 4 in agricultural production.

8. The application according to claim 7, characterized in that, The application described is for the remediation of pyrethroid compound pollution in agricultural production.

9. The application according to claim 7, characterized in that, The application described is for promoting crop growth.

10. The application according to claim 8 or 9, characterized in that, The application described is for use in high-salt environments.

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