A strain of Bacillus fungi Y2 with the functions of reducing cadmium content in crops and promoting growth and its application.

Abstract

This invention provides a strain of Bacillus mycoides Y2 with functions of reducing cadmium content in crops and promoting growth, and its applications, relating to the fields of soil heavy metal pollution bioremediation and agricultural microbial application technology. Bacillus mycoides Y2 was deposited on July 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC35231. This strain can be applied to cadmium-contaminated soil via root application, reducing cadmium content in the edible parts of crops such as rice, rapeseed, and kale, and promoting crop growth and the formation of an iron film on the root surface of rice. Verification with contaminated soils in Hunan, Henan, and Tianjin has shown that it can stably colonize and adapt to different soils and crops. This strain and its accompanying formulation are green, efficient, highly stable, and widely applicable, providing key technical support for the safe utilization of cadmium-contaminated arable land.

Description

Technical Field

[0001] This invention relates to the field of soil heavy metal pollution bioremediation and agricultural microbial application technology, and in particular to a Bacillus fungi strain Y2 that has the functions of reducing cadmium content in crops and promoting growth, and its application. Background Technology

[0002] Industrial production and excessive agricultural inputs have led to the long-term accumulation of heavy metals in soil, threatening the ecological environment and the quality and safety of agricultural products, and hindering the green and high-quality development of agriculture. my country has clearly defined phased goals for soil pollution prevention and control, and urgently needs technological support for the safe utilization of polluted arable land.

[0003] Heavy metal pollution remediation is divided into physical, chemical and biological methods. Among them, microbial remediation has become a research hotspot due to its green, efficient and eco-friendly nature. Bacillus strains can reduce the content of available heavy metals in soil and promote crop growth. However, in practical applications, functional microbial agents generally suffer from low remediation efficiency, poor stability and insufficient environmental adaptability.

[0004] It is necessary to pay close attention to the rhizosphere microenvironment, screen functional microorganisms that interact closely with crop roots, develop functional strains with strong colonization ability, wide adaptability to environmental conditions and farmland types, and excellent cadmium control effect, address the shortcomings of existing microbial remediation technologies, improve the application effect of functional microbial agents and microbial fertilizers, and ensure the safe use of polluted arable land. Summary of the Invention

[0005] Based on the problems in the prior art, the present invention provides a Bacillus fungi strain Y2 with functions of reducing cadmium content in crops and promoting growth, and its applications.

[0006] The Bacillus mycoides Y2 strain described in this invention has been verified through pot experiments to reduce cadmium content in crops and promote growth. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC35231, deposit date of July 14, 2025, at No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0007] One of the objectives of this invention is to provide a strain of Bacillus mycoides Y2 that has the functions of reducing cadmium content in crops and promoting growth. The characteristic of this invention is that the Bacillus mycoides Y2 was deposited on July 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with the accession number CGMCC35231, and the deposit site is the Institute of Microbiology, Chinese Academy of Sciences.

[0008] The second objective of this invention is to provide an application of Bacillus mycoides Y2, as described above, in reducing the cadmium content of crops in cadmium-contaminated soil. The method of use includes:

[0009] During the seedling or heading stage of crops, a bacterial suspension of Bacillus mycoides Y2 was applied to cadmium-contaminated soil via root application.

[0010] The crops include dryland crops or paddy field crops. The dryland crops include rapeseed or Chinese cabbage, and the paddy field crops include rice.

[0011] Preferably, the crop is rapeseed;

[0012] The method of using the Bacillus mycoides Y2 includes:

[0013] The bacterial suspension of Bacillus mycoides Y2 was applied during the rapeseed seedling stage at a rate of 1.25 × 10⁻⁶. 10 5 × 10 plants 10 One per plant.

[0014] Preferably, the crop is rice;

[0015] The method of using the Bacillus mycoides Y2 includes:

[0016] The bacterial suspension of Bacillus mycoides Y2 was applied during the rice heading stage at a rate of 2.5 × 10⁻⁶. 10 1 × 10 plants 11 One per plant.

[0017] Preferably, the crop is a fast-growing vegetable;

[0018] The method of using the Bacillus mycoides Y2 includes:

[0019] The bacterial suspension of Bacillus mycoides Y2 was applied during the seedling stage of the fast-growing vegetables at a dosage of 1.25 × 10⁻⁶. 11 Units / square meter ~ 5×10 11 Units per square meter.

[0020] The third objective of this invention is to provide an application of Bacillus mycoides Y2 as described above in promoting crop growth in cadmium-contaminated soil.

[0021] Preferably, the crop is rapeseed;

[0022] The method of using the Bacillus mycoides Y2 includes:

[0023] The bacterial suspension of Bacillus mycoides Y2 was applied during the rapeseed seedling stage at a rate of 1.25 × 10⁻⁶. 10 5 × 10 plants 10 One per plant.

[0024] The fourth objective of this invention is to provide a microbial preparation for cadmium pollution control and crop growth promotion, the preparation comprising Bacillus mycoides Y2 as described above and agriculturally acceptable excipients; the excipients include a carrier and a protectant, the carrier comprising sterile water or vermiculite, and the protectant comprising glycerol.

[0025] Preferably, the viable count of Bacillus mycoides Y2 in the formulation is 5.0 × 10⁻⁶. 8 cfu / mL ~1×10 11 cfu / mL.

[0026] The fifth objective of this invention is to provide an application of Bacillus mycoides Y2 as described above in promoting the formation of iron film on the root surface of rice in cadmium-contaminated soil.

[0027] The beneficial effects of this invention are:

[0028] This invention provides a Bacillus fungi Y2 that has the functions of reducing cadmium content in crops and promoting growth.

[0029] Experiments using contaminated soils from Hunan, Henan, and Tianjin have demonstrated that this strain can stably colonize the rhizosphere of different types of crops and effectively reduce cadmium accumulation in the edible parts of dryland crops (e.g., rapeseed) and paddy crops (e.g., rice) under various soil conditions. It also promotes the formation of an iron film on the root surface of rice in cadmium-contaminated soil and enhances crop growth under cadmium pollution stress. Furthermore, it is adaptable to soils in different regions such as Hunan, Henan, and Tianjin, exhibiting strong versatility.

[0030] The Bacillus mycoides Y2 and its supporting application technology of this invention, when applied to the remediation of cadmium-contaminated farmland, have the advantages of being green, efficient, stable, and widely applicable. Attached Figure Description

[0031] Figure 1 For the iron oxidation rate experiment of the strain;

[0032] Figure 2 The results are for the phosphorus-solubilizing function test of Bacillus mycosis fungi Y2.

[0033] Figure 3 Changes in rapeseed growth in the soil of Xiangtan, Hunan;

[0034] Figure 4 This study examines the changes in rapeseed growth in the soil of Weihui.

[0035] The Bacillus mycoides Y2 strain described in this invention has been verified through pot experiments to reduce cadmium content in crops and promote growth. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC35231, deposit date of July 14, 2025, at No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Detailed Implementation

[0036] The present application will now be described in further detail with reference to embodiments. In the following description, certain specific details are included to provide a comprehensive understanding of the various disclosed embodiments.

[0037] Example 1

[0038] Strains screening and mutagenesis

[0039] S1 The strain described in this invention was derived from the rhizosphere soil of cadmium-rich paddy fields in Xiangtan, Hunan Province. 1g of fresh soil was placed in a clean conical flask containing glass beads and 100ml of sterile water, and then shaken at 120rpm for 1 hour. The soil solution concentration at this point was 10%. -2 The purpose of adding glass beads at a concentration of g / L is to ensure that the soil is thoroughly ground, allowing the microorganisms within it to be better released into the solution.

[0040] S2 Soil Solution Dilution: Take 6 sterile test tubes and add 9 ml of sterile water to each, labeling them 1, 2, 3, and 4 respectively. Take 1 ml of soil solution from the conical flask and add it to test tube #1. Mix well. At this point, the concentration in test tube #1 is 10. -3 g / L, then take 1 ml of the liquid from test tube 1 and add it to test tube 2, and so on, until the soil solution concentration is 10. -3 g / L, 10 -4 g / L, 10 -5 g / L and 10 -6 g / L.

[0041] Preparation of S3 solid modified ciliate culture medium: Add 0.3 g of ammonium chloride (NH4Cl), 2 g of manganese carbonate (MnCO3·H2O), 1 g of yeast extract, and 20 g of agar to 1000 ml of deionized water. Sterilize in an autoclave at 121℃ for 20 minutes. After cooling to 50℃, add 1 ml each of mixed vitamin solution and trace element solution, and 2 ml of ferric citrate solution.

[0042] Trace element solution formula (per liter): MgSO4·7H2O, 3g; NaCl, 1g; EDTA, 0.5g; MnSO4·H2O, 0.5g; FeSO4·7H2O, 0.1g; Co(NO3)2·6H2O, 0.1g; CaCl2, 0.1g; ZnSO4·7H2O, 0.1g; NiCl2·6H2O, 0.02g; CuSO4·5H2O, 0.01g; AlK(SO4)2, 0.01g; H3BO3, 0.01g; Na2MoO4·2H2O, 0.01g; Na2WO4·2H2O, 0.01g; Na2SeO3, 0.001g.

[0043] Mixed vitamin solution formulation (per liter): Folic acid, 2.000 mg; Vitamin B6, 10.000 mg; Vitamin B2, 5.000 mg; Biotin, 2.000 mg; Vitamin B1, 5.000 mg; Vitamin B3, 5.000 mg; Vitamin B5, 5.000 mg; Vitamin B12, 0.100 mg; Para-aminobenzoic acid, 5.000 mg; Alpha-lipoic acid, 5.000 mg; Potassium dihydrogen phosphate, 900 mg.

[0044] Ferric citrate solution (containing ferrous citrate): Dissolve 15g of trisodium citrate and 14.15g of ferrous sulfate heptahydrate in a small amount of water and add to a volumetric flask. Then, add water to bring the volume to 20ml. Filter sterilely through a 0.22μm filter membrane and aliquot into 2ml sterile centrifuge tubes. Store at -20℃ and thaw before use. This selective plate will cause colonies with iron-oxidizing capabilities to turn yellow or brown around their edges.

[0045] S4 Spreading, Separation, and Purification: Take 0.1 ml of soil solution from each test tube of different concentrations and spread it onto a modified ciliate solid medium. After sealing with film, incubate in a cell culture incubator at 25°C in the dark for 3 days. Multiple colonies with different morphologies and composite characteristics were observed on the plates. Different colonies were picked up with an inoculation loop and streaked onto new plates until a single, contaminant-free colony appeared. At this point, a strain Y0 was obtained.

[0046] S5 strain preservation: The selected single colony was inoculated into liquid LB medium and cultured in a shaker at 37°C and 120 rpm for 24 hours. The Y0 logarithmic fermentation broth and sterile glycerol were inoculated into sterile cryovials at a ratio of 3:7 to make glycerol tubes, which were then cryopreserved at -20°C.

[0047] S6 strain identification and mutagenesis: The Y0 fermentation broth was centrifuged in a 1.5ml sterile centrifuge tube, and the supernatant was discarded. 16S rDNA was measured for strain identification. The strain Y0 was identified as *Bacillus mycosis fungoides*. Bacillus mycoidesMutagenesis: The above strain Y0 was treated with 0.4% ethyl methanesulfonate (EMS) as a chemical mutagen to obtain Bacillus mycoides Y2. When streaked on a new modified ciliate culture dish, the color reaction on the plate was found to be more obvious, thus obtaining a Bacillus mycoides Y2 with better iron oxidation ability.

[0048]

[0049] Example 2

[0050] Detection of iron oxidation capacity and phosphorus solubility of strains

[0051] 1. Iron oxidation capacity test of the strain:

[0052] 100 μL of glycerol containing strain Y2 was inoculated into 50 mL of LB medium containing 520 mg / L ferrous iron at 0°C. The medium was then incubated on a shaker at 25°C and 120 rpm for 36 hours. The remaining ferrous iron content was determined by the o-phenanthroline spectrophotometric method. The ferrous iron was ferrous citrate, prepared according to the S3 ferric citrate solution in Example 1.

[0053] Simultaneously, a blank control group, a CK group, a Bacillus subtilis group, and a Y0 group were set up;

[0054] The preservation number of Bacillus subtilis is CMCC(B)63501=ATCC 6633. Similarly, 100 μL of glycerol containing Bacillus subtilis and Bacillus mycosis fungoides was inoculated into 50 mL of LB medium with a ferrous iron concentration of 520 mg / L at 0°C. The medium was then incubated on a shaker at 25°C and 120 rpm for 36 hours. The remaining ferrous iron content was determined using the o-phenanthroline spectrophotometric method. Figure 1 It can be seen that the color of the ferrous culture medium for microbial iron oxidation changes from green to red, and the better the iron oxidation effect, the deeper the red color.

[0055] The test results are shown in Table 1:

[0056]

[0057] Table 1 shows that after strain Y0 was mutagenized by treatment with 0.4% ethyl methanesulfonate (EMS) and added to a medium containing ferrous iron, the ferrous iron content was significantly reduced compared to other strains after 36 hours of incubation, indicating that the iron oxidation capacity of the strain was enhanced after mutagenesis. Microbial-driven iron oxidation promotes rhizosphere iron cycling and regulates the formation of iron oxides in the soil. Iron oxides, as important immobilization media for heavy metals such as cadmium in the soil, can affect the bioavailability of cadmium in the rhizosphere. Microbial promotion of iron oxidation is also closely related to the formation of root membrane structures in crops, especially the iron membrane on the root surface of rice, which is an important barrier for intercepting cadmium migration and transport. Microbial promotion of iron oxidation and its attachment on the root surface promotes the formation of iron and manganese on the root surface, thereby improving the cadmium control capacity of the crop organs themselves.

[0058] 2. Phosphorus solubility test

[0059] Preparation of bacterial suspension: Prepare LB liquid medium and dispense 50 ml into 200 ml Erlenmeyer flasks. Transfer 100 μL of the preserved strain from the glycerol tube into each Erlenmeyer flask and incubate at 120 rpm for 24 hours to prepare the bacterial fermentation broth. Centrifuge at 4000 rpm for 10 min and discard the supernatant. Adjust the OD600 values ​​of Y2 and Y0 bacterial suspensions to 0.6 with sterile water, and adjust the OD600 value of Bacillus subtilis bacterial suspension to 0.62. At this point, the viable count of the three strains is approximately 5.0 × 10^8 cfu / ml.

[0060] LB medium formula: 1000mL distilled water, 10g sodium chloride, 5g yeast extract, 10g tryptone, sterilize at 121℃ for 30min.

[0061] Phosphate-solubilizing function test: Prepare Pikovskaya's solid culture medium: 1000mL distilled water, (NH4)2SO4, 0.5g; NaCl, 0.2g; MgSO4·7H2O, 0.1g; MnSO4, 0.02g; FeSO4·7H2O, 0.02g; KCl, 0.2g; yeast extract, 0.5g; glucose, 10g; Ca3(PO4)2, 5g; agar, pH 7.4. Sterilize at 121℃ for 30min, cool to about 50℃, pour into sterile petri dishes to prepare solid plates. After cooling, spread the Y2 bacterial solution on the plates and incubate in a cell culture room for 3 days. Observe whether a clear zone forms. If so, it proves that strain Y2 has phosphate-solubilizing function.

[0062] Depend on Figure 2 It is known that Bacillus mycoides Y2 has phosphate-solubilizing function.

[0063] This indicates that Y2 can improve the utilization rate of phosphorus by plants by dissolving phosphate in the soil, thereby promoting plant growth.

[0064] Example 3

[0065] Verification of cadmium reduction ability in rice seedlings

[0066] Early indica X24 rice seeds were disinfected with 20% H2O2, washed with clean water, and then spread on seedling trays. After being cultured in a seedling box at 30℃ for 1 day, the germinated seeds were transferred to a soil culture box containing moist vermiculite for further cultivation.

[0067] This embodiment is divided into the Y2 low concentration group, Y2 medium concentration group, Y2 high concentration group, water group, wild-type Bacillus mycoides Y0 group and Bacillus subtilis group.

[0068] In the Y2 low-concentration group, on the second day of growth of X24 rice seedlings, 1.5 × 10^9 strains of Bacillus mycoides Y2 with an OD value of about 0.6 were added to the seedling trays. During this period, 1 / 8 of Hoglund's nutrient solution with a cadmium concentration of 0.5 ppm was added and cultured for 14 days. After harvesting and drying to constant weight, the seedlings were digested with MOS-grade nitric acid and then diluted to 50 mL with distilled water. The cadmium content in the seedlings was detected by ICP-MS.

[0069] The Y2 medium concentration group was prepared by adding 2.5 × 10^9 strains of Bacillus mycoides Y2 with an OD value of approximately 0.6 to the seedling trays on the second day of growth of X24 rice seedlings. During this period, 1 / 8 of Hoglund's nutrient solution with a cadmium concentration of 0.5 ppm was added and cultured for 14 days. After harvesting and drying to constant weight, the seedlings were digested with MOS-grade nitric acid and then diluted to 50 mL with distilled water. The cadmium content in the seedlings was detected by ICP-MS.

[0070] The Y2 high-concentration group was prepared by adding 5 × 10^9 strains of Bacillus mycoides Y2 with an OD value of approximately 0.6 to the seedling trays on the second day of growth of X24 rice seedlings. During this period, 1 / 8 of Hoglund's nutrient solution with a cadmium concentration of 0.5 ppm was added and cultured for 14 days. After harvesting and drying to constant weight, the seedlings were digested with MOS-grade nitric acid and then diluted to 50 mL with distilled water. The cadmium content in the seedlings was detected by ICP-MS.

[0071] In the water group, water was applied to the seedling culture trays instead of bacterial solution. During the experiment, water was applied to the seedling culture trays on the second day of growth of X24 rice seedlings. During this period, 1 / 8 of Hoglund nutrient solution with a cadmium concentration of 0.5 ppm was added. After culturing for 14 days, the seedlings were collected, dried to constant weight, digested with MOS grade nitric acid, and then diluted to 50 mL with distilled water. The cadmium content in the seedlings was detected by ICP-MS.

[0072] The Bacillus subtilis group was prepared by adding 1.5 × 10⁹ Bacillus subtilis cells to seedling trays on the second day of growth of X24 rice seedlings. During this period, 1 / 8 Hoglund nutrient solution with a cadmium concentration of 0.5 ppm was added, and the seedlings were cultured for 14 days. After harvesting and drying to constant weight, the seedlings were digested with MOS-grade nitric acid and then diluted to 50 mL with distilled water. The cadmium content in the seedlings was determined by ICP-MS. The Bacillus subtilis preservation number is CMCC(B)63501=ATCC 6633.

[0073] Wild-type Bacillus mycoides Y0 group was obtained by adding 1.5 × 10^9 wild-type Bacillus mycoides Y0 to seedling trays on the second day of growth of X24 rice seedlings. During this period, 1 / 8 of Hoglund's nutrient solution with a cadmium concentration of 0.5 ppm was added and cultured for 14 days. After harvesting and drying to constant weight, the seedlings were digested with MOS grade nitric acid and then diluted to 50 mL with distilled water. The cadmium content in the seedlings was detected by ICP-MS.

[0074] The test results are shown in Table 2:

[0075] Table 2 Results of cadmium reduction test in rice seedlings

[0076]

[0077] As shown in Table 2, Bacillus mycoides Y2 can effectively reduce the absorption and accumulation of cadmium in rice seedlings, thereby reducing the cadmium content in the seedlings.

[0078] Example 4

[0079] Verification of cadmium reduction ability of potted rice

[0080] X24 early indica rice was planted in soil with excessive cadmium in Xiangtan, Hunan Province. In this example, the rice was divided into three groups: Y2 low concentration group, Y2 medium concentration group, Y2 high concentration group, clear water group, wild-type Bacillus mycoides Y0 group, and Bacillus subtilis group.

[0081] In this embodiment, the low concentration group of Y2 involved applying 1×10^10 strains of strain Y2 to the potted soil during the rice heading stage. At the rice maturity stage, fresh rice root samples were collected to extract the iron film on the root surface, and the grains and panicle rachis were collected, dried, and then digested to detect the cadmium content.

[0082] In the Y2 medium concentration group, 2.5 × 10^10 strains of strain Y2 were applied to the potted soil during the rice heading stage. At the rice maturity stage, fresh rice root samples were collected, the root surface iron film was extracted, grains were collected, and the panicle rachis was dried, digested, and the cadmium content was detected.

[0083] In the high-concentration group of Y2, 5×10^10 strains of strain Y2 were applied to the soil of potted plants during the heading stage of rice. At the rice maturity stage, fresh rice root samples were collected to extract the iron film on the root surface, and the grains and panicles were collected, dried, and then digested to detect the cadmium content.

[0084] During the heading stage of rice, the water group applied water to potted plants. At the rice maturity stage, fresh rice root samples were collected to extract the iron film on the root surface. The grains were collected, dried, and then digested to detect cadmium content.

[0085] During the heading stage of rice, 2.5 × 10^10 Bacillus subtilis strains were applied to the soil in pots. At the rice maturity stage, fresh rice root samples were collected, the iron film on the root surface was extracted, and the grains and rachis were collected, dried, and then digested to detect cadmium content.

[0086] In the experiment, during the heading stage of rice, 2.5 × 10^10 strains of wild-type Bacillus mycoides Y0 were applied to the soil of potted plants. At the rice maturity stage, fresh rice root samples were collected to extract the iron film on the root surface, and the grains and rachis were collected, dried, and then digested to detect the cadmium content.

[0087] The test results are shown in Table 3.

[0088] Table 3. Verification results of cadmium reduction ability of potted rice

[0089]

[0090] In Example 4, the application of Bacillus fungi Y2 to the roots effectively promoted the formation of an iron film on the rice root surface, reaching up to nearly three times that of the blank control group. The iron film on the rice root surface can retain heavy metals such as cadmium, which is an important barrier for rice vegetative organs to intercept cadmium pollution. It is beneficial to control the absorption of cadmium by rice and the cadmium content in the grain. It can effectively reduce the translocation of cadmium from polluted soil to the above-ground parts of rice. Moreover, the application rate of Bacillus Y2 is positively correlated with its cadmium reduction effect. In the case of pollution where the cadmium content of rice exceeds the standard by more than 100%, increasing the application rate of Bacillus Y2 to a certain range can control the cadmium content of rice within the national food safety standard (0.2 mg / kg), and the cadmium content in the grain can be reduced by as much as 67.24%.

[0091] Example 5

[0092] Verification of the cadmium-reducing ability of potted rapeseed

[0093] This embodiment was divided into four groups: a low-concentration Y2 group, a medium-concentration Y2 group, a high-concentration Y2 group, a clean water group, a wild-type Bacillus mycoides Y0 group, and a Bacillus subtilis group. For each group, two different types of soil with varying degrees of cadmium contamination were selected from Xiangtan, Hunan and Xinxiang, Henan, to plant the "Green Beauty" rapeseed variety. The total cadmium content of the cadmium-contaminated soil in Xiangtan, Hunan was 0.6 mg / kg, while the cadmium-contaminated soil in Xinxiang, Henan was Weihui soil, with a total cadmium content of 2.3 mg / kg.

[0094] The Y2 low-concentration group was applied to the roots of rapeseed seedlings at a concentration of 1.25 × 10^10 Bacillus mycoides. Fresh rapeseed samples were collected after the rapeseed matured, and the weight of each plant was measured. Some samples were dried to constant weight and then digested to detect cadmium content.

[0095] In the Y2 medium concentration group, 2.5 × 10^10 Bacillus mycoides Y2 were applied to the soil of rapeseed pots during the seedling stage. After the rapeseed matured, fresh rapeseed samples were collected, and the weight of each plant was weighed. Some samples were dried to constant weight and then digested to detect cadmium content.

[0096] The Y2 high-concentration group was applied to the roots of rapeseed plants at a concentration of 5 × 10^10 Bacillus mycoides. During the seedling stage, the plants were planted in pots. Once the rapeseed matured, fresh samples were collected, and the weight of each plant was measured. Some samples were dried to constant weight and then digested to detect cadmium content.

[0097] In the water group, water was applied to the soil of rapeseed pots instead of bacterial solution. During the experiment, water was applied to the pots during the rapeseed seedling stage. Rapeseed biomass and cadmium content were measured at maturity.

[0098] Bacillus subtilis was applied to the roots of rapeseed seedlings at a rate of 1.25 × 10^10 cells per plant. Fresh rapeseed samples were collected upon maturity, and the weight of each plant was measured. Some samples were dried to constant weight and then digested to determine cadmium content.

[0099] Wild-type Bacillus mycoides Y0 group was applied to the soil of rapeseed pots at a dose of 1.25 × 10^10 cells via root application during the seedling stage. Fresh rapeseed samples were collected after maturity, and the weight of each plant was measured. Some samples were dried to constant weight and then digested to determine cadmium content.

[0100] The test results are shown in Tables 4 and 5.

[0101] Table 4. Verification results of cadmium reduction capacity of potted rapeseed in soil in Xiangtan, Hunan Province

[0102]

[0103] Table 5. Verification results of cadmium reduction capacity of rapeseed grown in pots in Weihui soil.

[0104]

[0105] Based on the data in Tables 4 and 5, and Figure 3 and Figure 4 It has been shown that root application of Bacillus mycoides Y2 can effectively reduce cadmium accumulation in rapeseed, a dryland crop, in soils with varying degrees of pollution, while simultaneously increasing rapeseed biomass. In soils severely contaminated with cadmium, the toxic damage caused by heavy metal pollution inhibits normal crop growth and affects the quality and safety of agricultural products. The application of Bacillus mycoides Y2 effectively inhibits the absorption and accumulation of cadmium in rapeseed, reduces the toxic damage of cadmium to rapeseed tissue cells, promotes the growth of rapeseed in contaminated farmland, and reduces the cadmium content in its edible parts, achieving a dual effect of "cadmium reduction" and "growth promotion."

[0106] Example 6

[0107] Verification of cadmium reduction capacity of field-grown vegetables in Dongli District

[0108] In this embodiment, fast-growing vegetables were planted in cadmium-contaminated fields in Dongli District, Tianjin. The groups were divided into low concentration Y2 group, medium concentration Y2 group, high concentration Y2 group, water group, wild-type Bacillus mycoides Y0 group and Bacillus subtilis group.

[0109] In the water group, water was applied to the cadmium-contaminated vegetable field soil instead of bacterial solution. During the experiment, water was applied to the soil during the seedling stage of the fast-growing vegetables. The fast-growing vegetables were collected at maturity, dried, and then digested to determine their cadmium content.

[0110] During the seedling stage of Chinese cabbage, 1.25 × 10^11 Bacillus subtilis were applied per square meter in the field where Chinese cabbage was planted. When the Chinese cabbage matured, it was collected, dried, and then digested to detect the cadmium content.

[0111] Wild-type Bacillus mycoides Y0 group was applied to the field where Chinese cabbage was planted during the seedling stage. Each square meter of the field was filled with 1.25 × 10^11 strains of strain Y0. The Chinese cabbage was collected at the maturity stage, dried, and then digested to detect the cadmium content.

[0112] In the Y2 low-concentration group, 1.25 × 10^11 strains of strain Y2 were applied per square meter in the field where fast-growing vegetables were planted during the seedling stage. Fast-growing vegetables were collected at the maturity stage, dried, and then digested to detect cadmium content.

[0113] In the Y2 medium concentration group, 2.5 × 10^11 strains of strain Y2 were applied per square meter in the field where fast-growing vegetables were planted during the seedling stage. Fast-growing vegetables were collected at the maturity stage, dried, and then digested to detect cadmium content.

[0114] In the high-concentration group of *Gnaphalium affine*, 5 × 10^11 strains of strain Y2 were applied per square meter in the field where *Gnaphalium affine* was planted during the seedling stage. After the *Gnaphalium affine* matured, the plants were collected, dried, and then digested to detect the cadmium content.

[0115] The test results are shown in Table 6.

[0116] Table 6. Verification results of cadmium reduction capacity of field-grown vegetables in Dongli District

[0117]

[0118] As shown in Table 6, field trials further verified that root application of Y2 can effectively reduce the cadmium content of crops, and the cadmium-reducing effect of Y2 increases with the increase of the amount of bacteria added.

[0119] The *Bacillus fungi* Y2 strain of this invention achieves an iron oxidation rate of 91.83% and possesses phosphorus-solubilizing capabilities, providing a functional basis for its cadmium-reducing and growth-promoting effects. It exhibits significant cadmium-reducing effects in various crops, including rice, rapeseed, and kale. The cadmium content in rice grains was reduced by up to 75.51%, in rapeseed by up to 47.95%, and in kale by up to 40.39%, with cadmium-reducing effects generally superior to *Bacillus subtilis* and the original strain Y0. This strain also exhibits significant growth-promoting effects, increasing rapeseed biomass in cadmium-contaminated soil by up to 142.84% and promoting the formation of iron films on rice roots by up to 290.44%, further enhancing the cadmium-reducing effect by strengthening the rhizosphere barrier. Verification in soils of different regions and cadmium-contaminated levels, including Xiangtan in Hunan, Weihui in Henan, and Dongli in Tianjin, shows that the Y2 strain can stably colonize and adapt to different planting scenarios, such as dryland and paddy fields, demonstrating strong environmental adaptability and versatility. In summary, Bacillus mycoides Y2 and its supporting application technologies and microbial preparations are green, efficient, stable, and easy to operate. They effectively solve the pain points of low efficiency and poor adaptability of existing microbial remediation technologies, providing reliable strain resources and technical support for the safe utilization of different types of cadmium-contaminated farmland. They have both environmental benefits and agricultural production value, and have broad application prospects.

[0120] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A strain of Bacillus fungi that can reduce cadmium content in crops and promote growth ( Bacillus mycoides Y2, characterized in that, The Bacillus mycoides Y2 was deposited on July 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC35231, at the Institute of Microbiology, Chinese Academy of Sciences.

2. An application of Bacillus mycoides Y2 as described in claim 1 in reducing cadmium content in crops in cadmium-contaminated soil, characterized in that, Usage instructions include: During the seedling or heading stage of crops, a bacterial suspension of Bacillus mycoides Y2 was applied to cadmium-contaminated soil via root application. The crop mentioned is rapeseed, Chinese cabbage, or rice.

3. The application as described in claim 2, characterized in that, The crop in question is rapeseed; The method of using the Bacillus mycoides Y2 includes: The bacterial suspension of Bacillus mycoides Y2 was applied during the rapeseed seedling stage at a rate of 1.25 × 10⁻⁶. 10 5 × 10 plants 10 One per plant.

4. The application as described in claim 2, characterized in that, The crop in question is rice; The method of using the Bacillus mycoides Y2 includes: When applied during the rice heading stage, the dosage of the Bacillus mycoides Y2 bacterial suspension is 2.5 × 10⁻⁶. 10 1 × 10 plants 11 Individuals / plant; When applied to rice seedlings, the dosage of the Bacillus mycoides Y2 bacterial suspension is 1.5 × 10⁻⁶. 9 5 × 10 plants 9 One per plant.

5. The application as described in claim 2, characterized in that, The crop in question is a fast-growing vegetable; The method of using the Bacillus mycoides Y2 includes: The bacterial suspension of Bacillus mycoides Y2 was applied during the seedling stage of the fast-growing vegetables at a dosage of 1.25 × 10⁻⁶. 11 Units / square meter ~ 5×10 11 Units per square meter.

6. An application of Bacillus mycoides Y2 as described in claim 1 in promoting crop growth in cadmium-contaminated soil, characterized in that, The crop mentioned is rapeseed, Chinese cabbage, or rice.

7. The application as described in claim 6, characterized in that, The crop in question is rapeseed; The method of using the Bacillus mycoides Y2 includes: The bacterial suspension of Bacillus mycoides Y2 was applied during the rapeseed seedling stage at a rate of 1.25 × 10⁻⁶. 10 5 × 10 plants 10 One per plant.

8. A microbial preparation for cadmium pollution control and crop growth promotion, characterized in that, The formulation comprises Bacillus mycoides Y2 as described in claim 1 and agriculturally acceptable excipients; the excipients include a carrier and a protectant, wherein the carrier is sterile water or vermiculite and the protectant is glycerol.

9. The microbial preparation according to claim 8, characterized in that, The viable count of Bacillus mycoides Y2 in the formulation is 5.0 × 10⁻⁶. 8 cfu / mL ~1×10 11 cfu / mL.

10. The application of Bacillus mycoides Y2 as described in claim 1 in promoting the formation of iron film on the root surface of rice in cadmium-contaminated soil.

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