Wheat root system synthetic flora and application thereof in blocking and controlling absorption of heavy metals by wheat

Abstract

The application belongs to the technical field of microorganisms, and specifically discloses a wheat root system synthetic microbial flora and application thereof in blocking and controlling absorption of heavy metals by wheat. The application provides a wheat root system synthetic microbial flora, which comprises Microbacterium arborescens M and Bacillus proteolyticus B. The strains of the wheat root system synthetic microbial flora are derived from wheat roots, have high homology with wheat, can be efficiently colonized in wheat, and can effectively block and control accumulation of heavy metals by wheat. The results of pot experiments and field experiments show that the wheat root system synthetic microbial flora can significantly reduce accumulation of cadmium and arsenic in the aboveground part of wheat and reduce the cadmium and arsenic content in wheat grains. The application is suitable for blocking and controlling absorption and accumulation of heavy metals by crops, and has important application value in repairing cadmium-arsenic compound contaminated farmland and ensuring wheat food safety.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a wheat root synthetic microbial community and its application in controlling the absorption of heavy metals in wheat. Background Technology

[0002] Wheat, as one of the most important cereal crops in northern my country, provides approximately 20% of the energy and protein for humans. Currently, farmland soils in my country suffer from varying degrees of heavy metal pollution, with cadmium (Cd) showing the highest exceedance rate at 18.03%, followed by arsenic (arsenic-like substances) at 2.95%. Heavy metal pollutants in soil can be absorbed by crop roots and transported to the aboveground parts, ultimately accumulating in edible parts such as grains, posing a potential threat to human health. Cadmium and arsenic exhibit significant differences in their occurrence forms and migration and transformation patterns in soil: cadmium exists primarily as cations, while arsenic mainly exists as arsenate anions. This characteristic significantly increases the difficulty of remediating soils contaminated with both cadmium and arsenic. While using remediation measures such as chemical passivation to reduce the availability of cadmium in the soil, it often leads to an increase in the availability of arsenic, thereby exacerbating the health risk of arsenic entering the human body through the food chain. Therefore, effectively controlling the cadmium and arsenic content in wheat grains within the national food safety standards for wheat fields contaminated with both cadmium and arsenic has become a major challenge in the field of farmland pollution control.

[0003] In recent years, research on microbial remediation of environments contaminated with single cadmium or arsenic has made some progress. Current soil remediation technologies mostly employ single strains to remediate single heavy metal pollution; however, their remediation effectiveness is unstable due to the significant differences in the environmental behavior of cadmium and arsenic in soil, failing to meet the requirements for safe utilization of soils contaminated with combined cadmium and arsenic pollution. Furthermore, existing research largely focuses on isolating and screening functional microorganisms from soil, which cannot efficiently colonize plants, thus hindering the precise regulation of plant absorption and translocation of heavy metals. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention isolates and constructs a microbial community containing Microbacterium M and Bacillus B from the root system of wheat during its critical growth period. This community enables the control of wheat's absorption and translocation of heavy metals in soil contaminated with cadmium and arsenic, which is of great significance for improving food safety.

[0005] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions: In a first aspect, the present invention provides a wheat root system synthetic microbial community, which includes microbes with preservation number CCTCC NO: M20252014 ( Microbacterium sp.) M and Bacillus with accession number CCTCC NO: M20252015 (sp.) M and Bacillus ( Bacillus sp.)B.

[0006] Preferably, the microbacterium M is a dendritic microbacterium ( Microbacterium arborescens M; the Bacillus B is Bacillus proteolyticus ( Bacillus proteolyticus B.

[0007] Preferably, the ratio of viable bacteria of the dendritic microbacterium M to the viable bacteria of the proteolytic bacillus B is 8-12:10.

[0008] This invention utilizes a targeted screening strategy during key growth stages to obtain a synthetic microbial community in wheat roots, composed of *Microbacterium dendriticum* M and *Bacillus proteolyticus* B. This synthetic community can efficiently fix cadmium and arsenic, and the combined use of the two strains can significantly reduce the accumulation of cadmium and arsenic in the aboveground parts of wheat. Furthermore, the strains used in this agent are isolated from wheat roots, exhibiting high homology with wheat and strong colonization ability. Compared with conventional physicochemical passivating agents, the synthetic microbial community in wheat roots provided by this invention has significant advantages such as low cost, ease of application, and eco-friendliness, and has important application value in remediating heavy metal-contaminated farmland and ensuring wheat food safety.

[0009] Secondly, the present invention provides an application of the above-mentioned wheat root synthetic microbial community in controlling the accumulation of heavy metals in wheat in heavy metal contaminated soil.

[0010] Preferably, the heavy metal includes at least one of cadmium or arsenic.

[0011] Preferably, the heavy metals include cadmium and arsenic.

[0012] Preferably, the wheat includes the aboveground parts of the wheat plant.

[0013] More preferably, the aboveground part of the wheat includes wheat grains.

[0014] Within the scope of the present invention, the wheat root synthetic microbial community provided by the present invention is used to control the accumulation of heavy metals in wheat in heavy metal contaminated soil. The cadmium and arsenic content in mature wheat grains can be reduced to within the limits of cadmium and arsenic content in the national food safety standards, meeting the requirements for safe consumption.

[0015] Thirdly, the present invention provides a method for controlling the absorption of heavy metals by wheat, specifically including: applying the above-mentioned wheat root synthetic microbial community by root irrigation during the middle and late stages of wheat growth.

[0016] Preferably, the mid-to-late stage of wheat growth includes at least one of the wheat booting stage, heading stage, or early grain-filling stage.

[0017] For example, the total number of effective viable bacteria applied is 4 × 10⁻⁶. 7 CFU / g-1×10 9 CFU / g soil.

[0018] This invention involves collecting root samples from wheat during key growth stages, isolating rhizobium with tolerance potential to cadmium and arsenic, and evaluating the effects of single bacteria and their synthetic flora on reducing accumulated cadmium and arsenic in wheat using a combination of seedling soil culture experiments. A synthetic flora resistant to cadmium and arsenic, composed of *Microbacterium dendriticum* M and *Bacillus proteolyticus* B, was screened and named the wheat root synthetic flora. This bacterial agent is isolated from wheat roots, making it easier to colonize the roots, resulting in a more direct effect and better environmental stability. Furthermore, the screening method targeting key growth stages significantly improves the efficiency and accuracy of screening, avoiding the blind spots of traditional culture methods. When cultured alone, *Bacillus proteolyticus* B showed a cadmium fixation rate of 62.42% and an arsenic fixation rate of 6.15%, while *Microbacterium dendriticum* M showed a cadmium fixation rate of only 0.87% and an arsenic fixation rate of 4.41%. However, after the two strains were mixed and cultured, the cadmium fixation rate significantly increased to 78.37%, demonstrating a clear synergistic effect. Applying this synthesized microbial community to wheat pots in cadmium- and arsenic-contaminated soil, the results showed that the composite microbial community could efficiently colonize the wheat roots, reducing cadmium and arsenic content in the aboveground parts of wheat by 33.06% and 25.05%, respectively, compared to the control group. These results indicate that the mixed culture of the two strains is significantly more effective than that of a single strain in controlling the absorption and accumulation of cadmium and arsenic in the aboveground parts of wheat, exhibiting a clear synergistic advantage.

[0019] This invention further applies the aforementioned root-derived microbial agent via root irrigation during the wheat's booting, heading, and early grain-filling stages. Pot experiments showed that the mixed formulation of the two strains was significantly more effective than a single strain in controlling the absorption and accumulation of cadmium and arsenic in wheat grains. Field trials demonstrated that this composite microbial community reduced the cadmium and arsenic content in wheat grains to within the limits specified in the National Food Safety Standard for Maximum Levels of Contaminants in Food (GB 2762-2022) – Cereals and Cereals. The wheat root-derived synthetic microbial community provided by this invention can effectively control the accumulation of heavy metals in wheat, making it suitable for safe utilization in heavy metal-contaminated farmland, and particularly valuable for the safe production of wheat in farmland with combined cadmium and arsenic contamination. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The results show the determination of the cadmium and arsenic fixation ability of strains B and M and their synthetic flora in Example 1 of the present invention. Figure 2This is the phylogenetic tree of strain B in Example 1 of the present invention; Figure 3 The colony morphology and scanning electron microscope image of strain B in Example 1 of this invention; Figure 4 This is a heatmap of the whole genome ANI analysis of strain B in Example 1 of the present invention; Figure 5 This is the phylogenetic tree of strain M in Example 1 of the present invention; Figure 6 The colony morphology and scanning electron microscope image of strain M in Example 1 of this invention; Figure 7 This is a heatmap of the whole genome ANI analysis of strain M in Example 1 of the present invention; Figure 8 The results of measuring the arsenic and cadmium content and translocation coefficient of wheat aboveground parts in different treatment groups under soil pot conditions in Example 1 of the present invention; Figure 9 The results of measuring the gene copy number of Bacillus and Microbacterium in wheat roots under hydroponic potting conditions in treatment groups 3 and 4 of Example 1 of the present invention; Figure 10 The results of cadmium and arsenic content determination in wheat grains of different treatment groups under soil pot cultivation conditions throughout the entire growth period in Example 2 of the present invention; Figure 11 The results of the determination of cadmium and arsenic content in wheat grains of different groups in the field test of Example 3 of the present invention are shown. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0023] Unless otherwise specified, the raw materials and reagents used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0024] Example 1 This embodiment provides a method for screening functional microbial communities with cadmium and arsenic passivation capabilities from wheat roots, and for isolating, identifying, and preserving the rhizobacteria with cadmium and arsenic tolerance potential screened by this method. The specific details are as follows: I. Screening of wheat root strains with tolerance potential to cadmium and arsenic 1. Sample Collection In April 2023, root samples were collected from wheat fields contaminated with cadmium and arsenic in Anxin County, Hebei Province, during the critical growth period of wheat. This invention preferentially uses wheat roots during the heading stage as the material for strain isolation.

[0025] 2. Culture of cadmium and arsenic resistant bacteria In a clean bench, soil adhering to the roots was washed off with sterile water, followed by surface disinfection with sodium hypochlorite solution to remove root surface microorganisms. The roots were then thoroughly homogenized in a sterile mortar using a sterile grinding rod. An appropriate amount of the sample was added to a sterile 10mM magnesium chloride solution and incubated for 1 hour in a shaker incubator at 180 rpm and 28°C to obtain a root suspension. Subsequently, the root suspension was serially diluted 10-10. -2 Take 0.1 mL of 10 -2 Gradient bacterial suspensions were spread onto TSA solid medium containing 0.1 mM Cd (CdCl2·2.5H2O) and 1 mM As (Na2HAsO4·7H2O). The medium was incubated upside down in a 28°C incubator for 48 h. The TSA medium formulation was as follows: per 1 L, it consisted of 5 g soybean peptone, 15 g tryptone, and 15 g agar powder, with distilled water added to a final volume of 1 L. The medium was then autoclaved at 121°C for 20 min.

[0026] 3. Separation and purification Select different colonies and streak them to ensure that you get a single colony. Use TSA medium plates for streaking. After the candidate bacteria grow, place them in a 4°C freezer and store one copy in a glycerol cryovial at -80°C.

[0027] 4. Screening of cadmium- and arsenic-fixed bacteria The single colonies obtained in step 3 were transferred to TSB medium and cultured for 12 hours. After culture, they were centrifuged at 4°C and 2940g for 10 minutes using a high-speed refrigerated centrifuge. The supernatant was discarded, and 2 mL of 10 mM magnesium chloride solution was added to wash the bacterial precipitate to remove residual culture medium. This process was repeated twice. The washed precipitate (i.e., bacterial cells) was resuspended in 10 mM magnesium chloride solution, and its OD value was measured. 600 Adjust OD 600 The concentration was set to 1, and then the culture medium containing 50 μM CdCl2·2.5H2O and 1 mM Na2HAsO4·7H2O was inoculated. The residual cadmium and arsenic ion content in the solution of a single strain was measured. Finally, a total of 22 strains with the ability to fix Cd and As were isolated. Among them, strains B and M showed the best results.

[0028] 5. Determination of the fixation capacity of strains B and M and their synthetic flora for cadmium and arsenic. Single colonies of strains B and M were picked and inoculated into 100 mL of TSB liquid medium, and cultured in a shaker at 28 °C until the viable count reached 4 × 10⁻⁶. 9CFU / mL was used as the seed culture. Strains B, M, and B+M (this group was simultaneously inoculated with both strains B and M, with a viable count of 1:1) were inoculated into 100 mL of fresh TSB liquid medium at a 1% (v / v) inoculation rate. 5 μL of 1M CdCl2·2.5H2O and 100 μL of 1M Na2HAsO4·7H2O were added to the medium to adjust the Cd concentration in the system. 2+ Final concentration to 50 μM, HAsO4 - The final concentration was 1 mM, and it was ensured that the total number of live bacteria inoculated in each group was equal.

[0029] The inoculated culture medium was placed in a shaker at 28°C and cultured for 72 hours. Samples were taken to determine the concentration of residual cadmium and arsenic in the solution. The cadmium and arsenic concentrations were determined using inductively coupled plasma mass spectrometry (ICP-MS). The results of the cadmium and arsenic fixation abilities of strains B, M, and B+M are shown below. Figure 1 As shown.

[0030] Depend on Figure 1 It can be seen that when cultured alone, strain B had a cadmium fixation rate of 62.42% and an arsenic fixation rate of 6.15%; strain M had a cadmium fixation rate of only 0.87% and an arsenic fixation rate of 4.41%. However, after the two strains were mixed and cultured, the cadmium fixation rate increased to 78.37%, showing a significant synergistic effect.

[0031] 6. Classification and identification (1) Identification of strain B Identification was performed using 16S rDNA, specifically by amplifying and sequencing the 16S rDNA of strain B using the universal prokaryotic 16S rDNA primers 27F and 1492R. The primer sequence information for 27F and 1492R is shown in Table 1, and the nucleotide sequence of its 16S rDNA is shown in SEQ ID NO. 1.

[0032] Table 1

[0033] Comparing its sequence with the NCBI GenBank nucleotide database revealed that the 16S rDNA of this strain is similar to that of Bacillus subtilis. Bacillus proteolyticusi The homology was 100.00%. A phylogenetic tree was constructed using MEGA 6.0 software, and its phylogenetic tree is as follows: Figure 2 As shown. By Figure 2 It was found that strain B isolated in this invention has a similarity to the reference sequence NR152692.1. Bacillus wiedmannii PP 757992.1 Bacillus albus MH 281748.1 Bacillus cereus NR 157735.1 Bacillus proteolyticus They gather into one branch, and are related to closely related species such as Bacillus mycoides , Bacillus thuringiensis The strains showed clear branching and no cross-clustering, indicating that their phylogenetic positions were relatively stable.

[0034] Strain B was cultured in TSB medium, and its colony microstructure was observed using scanning electron microscopy. The colony morphology and scanning electron micrographs are shown below. Figure 3 As shown. By Figure 3 As can be seen, on TSB medium, the colonies are off-white, round, and have irregular edges. Under scanning electron microscopy, strain B cells are short rods, uniform in size, 1.6-2.5 μm in length and 0.8-1.0 μm in width. The cell surface is smooth and flat, with weak filamentous connections between some cells. They are mostly arranged singly or in short chains. Oval spores are clearly visible inside the cells, and the cells are tightly packed. The morphological characteristics are consistent with the typical morphology of Bacillus.

[0035] Based on 16S rDNA sequence homology analysis, phylogenetic tree clustering results, and morphological identification, strain B was determined to belong to the genus Bacillus. Bacillus Therefore, strain B is named Bacillus B and deposited at the China Center for Type Culture Collection (CCTCC) of Wuhan University on September 12, 2025, with accession number CCTCC NO:M 20252015. The Latin name of this strain is... Bacillus sp.

[0036] After preservation, the following further studies were conducted: Due to the high conservation of the 16S rRNA sequence, relying solely on 16S rRNA is insufficient for accurate species-level differentiation. Therefore, genome-wide average nucleotide identity (ANI) analysis was used for species-level identification of Bacillus B. The heatmap of genome-wide ANI analysis for strain B is shown below. Figure 4 As shown. By Figure 4 It can be seen that Bacillus B and Bacillus proteolyticus The reference strain has an ANI value of 100%, indicating that the two belong to the same species. Therefore, Bacillus B is a type of Bacillus proteolyticus (Bacillus). Bacillus proteolyticus Therefore, strain B was subsequently named Bacillus proteinophilus B.

[0037] (2) Identification of strain M The 16S rDNA of strain M was amplified and sequenced using universal primers 27F and 1492R for prokaryotes. Its 16S rDNA sequence is shown in SEQ ID NO.2. Comparison of the sequence with the NCBI GenBank nucleotide database revealed that the 16S rDNA of this strain is similar to that of *Microbacterium dendriticum* NR 029265.1. Microbacterium arborescens The homology was 99.85%. A phylogenetic tree was constructed using MEGA 6.0 software, and its phylogenetic tree is as follows: Figure 5 As shown. By Figure 5 It was found that the strain M isolated in this invention is identical to the reference sequence NR 029265.1. Microbacterium arborescens They cluster together, with each node having a bootstrap value of 100.

[0038] Strain M was cultured in TSB solid medium, and its colony microstructure was observed using scanning electron microscopy. The colony morphology and scanning electron micrographs are shown below. Figure 6 As shown. By Figure 6 As can be seen, on TSB solid medium, the colonies of strain M are round, plump, raised, with neat edges, smooth surface, orange color, and dense texture. Under scanning electron microscopy, the bacteria of strain M are short rods, uniform in size, 0.5-1 μm in length and 0.3-0.5 μm in width, without spore formation, with smooth surface, mostly arranged singly or in short chains, and their morphological characteristics are consistent with the typical structure of the genus Microbacterium.

[0039] Based on 16S rDNA homology analysis, phylogenetic tree clustering results, and morphological characteristics, strain M can be identified as a microbacterium (Microbacterium tumefaciens). Microbacterium sp.) M. This strain is deposited at the China Center for Type Culture Collection (CCTCC) of Wuhan University on September 12, 2025, with accession number CCTCC NO:M 20252014. The Latin name of this strain is Microbacterium sp.

[0040] After preservation, the following further studies were conducted: Phylogenetic tree analysis revealed that this strain is related to... Microbacterium arborescens The strain is most closely related and clusters in the same branch. Due to the high conservation of the 16S rRNA sequence, relying solely on 16S rRNA is insufficient for accurate species-level differentiation; therefore, genome-wide average nucleotide identity (ANI) analysis was further employed for species-level identification of strain M. The heatmap of genome-wide ANI analysis for strain M is shown below. Figure 7 As shown. By Figure 7 It can be seen that strain M and Microbacterium arborescens The reference strain has an ANI value of 100%, indicating that the two belong to the same species. Therefore, strain M is confirmed to be *Microbacterium dendriticum* (…). Microbacterium arborescens Therefore, strain M was subsequently named Dendromycosis M.

[0041] Example 2 This embodiment provides a wheat root synthetic microbial powder, which includes Bacillus proteolyticus B powder and Microbacterium dendriticum M powder. The total viable count in this wheat root synthetic microbial powder is 1×10⁻⁶. 10 CFU / g, the ratio of viable bacteria of Bacillus proteolyticus B to Microbes dendriticis M is 1:1; Example 3 This embodiment provides a wheat root microbial community powder, which includes Bacillus proteolyticus B powder and Microbacterium dendriticum M powder. The total viable count in this wheat root microbial powder is 1×10⁻⁶. 11 The CFU / g ratio of viable Bacillus proteolyticus B to viable Dendromycosis M was 5:6.

[0042] Example 4 This embodiment provides a wheat root microbial powder, which includes Bacillus proteolyticus B powder and Microbacterium dendriticum M powder, and the total viable count in the wheat root microbial powder is 5 × 10⁻⁶. 10 The CFU / g ratio of viable Bacillus proteolyticus B to viable Dendromycosis M was 5:4.

[0043] Example 1 This invention employs pot experiments to investigate the effects of treatments with *Bacillus proteolyticus* B, *Microbacterium dendriticum* M, and their combined microbial communities on the cadmium and arsenic content in the aboveground parts of wheat, and the translocation coefficient of cadmium and arsenic from roots to aboveground parts. Further investigation was conducted into the colonization of *Bacillus proteolyticus* B and *Microbacterium dendriticum* M. Specific details are as follows: 1. Effects of different treatment groups on cadmium and arsenic content in the aboveground parts of wheat and the translocation coefficient of cadmium and arsenic from roots to aboveground parts. Soil samples for the pot experiment were taken from a wheat field in Hebei Province contaminated with cadmium and arsenic (cadmium content 1.47 mg / kg, arsenic content 38.43 mg / kg, calcareous soil, alkaline pH). Soil samples were taken from a depth of 5-20 cm, visible impurities were removed, the soil was air-dried, and then sieved through a 5 mm sieve. 650 g soil samples were dispensed into each pot (9 cm in diameter, 14 cm in height). The experiment consisted of four treatment groups, with three replicates per treatment group. The settings for each group were as follows: Treatment group 1, root irrigation with a final concentration of 6 × 10⁻⁶ mg / kg soil. 7 CFU / g of *Microbacterium dendriticum* M; Treatment group 2, root irrigation with a final concentration of 6×10⁻⁶. 7 CFU / g of Bacillus proteolyticus B; Treatment group 3: Root irrigation with Microbacterium dendriticum M and Bacillus proteolyticus B, with a viable count ratio of 1:1 and a total viable count of 6 × 10⁻⁶. 7CFU / g soil; Treatment group 4, with an equal volume of sterile TSB liquid culture medium added, served as the control group without bacterial solution. The tested variety was Jimai 22, with 7 wheat plants planted in each pot. The wheat was inoculated by root irrigation at the 3-leaf stage, and samples were collected 7 days after inoculation.

[0044] Wheat samples were collected on day 7 after inoculation in potted experiments. The roots were carefully removed from the pots, ensuring the rhizomes were not broken. The plant surface was repeatedly rinsed with deionized water to remove all soil from the roots. Digestion was performed using a graphite furnace digestion method with HNO3 + H2O2. The cadmium and arsenic contents in different parts of Jimai 22 wheat were determined by inductively coupled plasma mass spectrometry (ICP-MS). The results of arsenic and cadmium content and translocation coefficients in the aboveground parts of wheat from different treatment groups under soil potted conditions are shown below. Figure 8 As shown.

[0045] Depend on Figure 8 It can be seen that, compared with treatment group 4 (control group), the cadmium and arsenic content in the aboveground wheat of treatment group 3, which was treated with root irrigation of Microbacterium dendriticum M and Bacillus proteolyticus B, decreased by 33.06% and 25.05%, respectively, and the translocation coefficients of cadmium and arsenic from the root system to the aboveground parts decreased by 16.41% and 33.80%, respectively, but had little impact on the rhizosphere soil microenvironment.

[0046] Compared with the groups that received root irrigation with Microbacterium dendriticum M (treatment group 1) or Bacillus proteolyticus B (treatment group 2), the cadmium content in the aboveground parts of wheat in treatment group 3 was significantly reduced (p < 0.05).

[0047] 2. Colonization status of Bacillus and Microbacterium genera. This invention investigates the colonization of *Bacillus proteoglycans* B and *Microbacterium dendriticum* M in wheat roots using hydroponic pot culture. The nutrient solution used in the hydroponic pot culture experiment was Hoagland's solution containing Cd and As stress, with Cd (CdCl2·2.5H2O) and As (Na2HAsO4·7H2O) stress concentrations of 5 μM and 10 μM, respectively. Each 6-well hydroponic container contained 1 L of nutrient solution. The experiment consisted of two treatment groups, with three replicates per treatment group. The treatment group 3 was as follows: *Microbacterium dendriticum* M and *Bacillus proteoglycans* B were directly added, with a viable count ratio of 1:1 and a total viable count concentration of 6 × 10⁻⁶. 7 CFU / mL nutrient solution; treatment group 4, with an equal volume of sterile TSB liquid culture medium added, served as the control group without bacterial solution. The tested variety was Jimai 22, with 6 wheat plants planted in each pot. The wheat was inoculated by root irrigation at the 3-leaf stage, and samples were collected 7 days after inoculation.

[0048] Wheat samples were collected on day 7 after inoculation in hydroponic potted plants. The roots were carefully removed from the hydroponic container, ensuring the rhizomes were not broken. The plant surface was repeatedly rinsed with deionized water to remove the nutrient solution from the roots. The gene copy number of the strain in the roots of Jimai 22 was determined using qPCR. DNA was extracted and purified from wheat root samples of treatment groups 3 and 4. Bacterial sequence alignment was performed using the NCBI database to identify the bacteria with the highest homology, and specific primers were designed accordingly. The target regions of the bacteria were amplified using the specific primers. The primer information for Bacillus and Microbacterium is shown in Table 2. The results of determining the gene copy number of Bacillus and Microbacterium in wheat roots under hydroponic potted conditions in treatment groups 3 and 4 are shown below. Figure 9 As shown.

[0049] Table 2

[0050] Depend on Figure 9 It can be seen that in the hydroponic pot experiment, both Bacillus proteolyticus B and Microbacterium dendriticum M can colonize efficiently in the root system. Seven days after inoculation, compared with the control group, the root gene copy numbers of Bacillus and Microbacterium in treatment group 3 were significantly increased by 316.52% and 75.17%, respectively, showing good root colonization ability and synergistic survival characteristics.

[0051] Example 2 This invention employs a pot experiment to investigate the effects of treatments with Bacillus proteolyticus B, Microbacterium dendriticum M, and their combined bacterial groups on the cadmium and arsenic content of wheat grains. The specific details are as follows: Soil samples for the pot experiment were taken from a wheat field in Hebei Province contaminated with cadmium and arsenic (cadmium content 1.47 mg / kg, arsenic content 38.43 mg / kg, calcareous soil, alkaline pH). Soil samples were taken from a depth of 5-20 cm, visible impurities were removed, the soil was air-dried, and then sieved through a 5 mm sieve. 5 kg of soil samples were placed in each pot (17 cm in diameter, 20 cm in height). The experiment consisted of four treatment groups, with three replicates per treatment group. The settings for each group were as follows: Treatment group 1, root irrigation with a final concentration of 6 × 10⁻⁶ mg / kg soil. 7 CFU / g of *Microbacterium dendriticum* M; Treatment group 2, root irrigation with a final concentration of 6×10⁻⁶. 7 CFU / g of Bacillus proteolyticus B; Treatment group 3: Root irrigation with Microbacterium dendriticum M and Bacillus proteolyticus B, with a viable count ratio of 1:1 and a total viable count of 6 × 10⁻⁶. 7CFU / g soil; Treatment group 4, with an equal volume of sterile TSB liquid culture medium added, served as the control group without bacterial solution. The tested variety was Jimai 22, with 12 wheat plants planted in each pot. Root irrigation inoculation treatment began during the wheat heading stage, with a second inoculation performed 7 days later, for a total of 3 inoculations. Samples were collected at maturity.

[0052] Mature wheat grains were collected and washed sequentially with tap water, distilled water, and ultrapure water. The grains were then blanched at 85℃ for 30 minutes and dried at 60℃ to constant weight. After pulverizing using a stainless steel grinder, the cadmium and arsenic contents of the wheat grains were determined. Accuracy and precision were controlled using the national primary standard reference material (GBW100493, wheat flour), and blank samples were analyzed simultaneously to eliminate reagent interference. The results of cadmium and arsenic content determination in wheat grains of different groups are shown below. Figure 10 As shown.

[0053] Depend on Figure 10 It was found that, compared with treatment group 4 (control group), the cadmium and arsenic content of wheat grains in treatment group 3, which was treated with root irrigation of *Microbacterium dendriticum* M and *Bacillus proteolyticus* B, decreased by 29.90% and 72.90%, respectively. Compared with the groups treated with root irrigation of *Microbacterium dendriticum* M (treatment group 1) or *Bacillus proteolyticus* B (treatment group 2), the cadmium and arsenic content of wheat grains in treatment group 3 was significantly reduced (p < 0.05).

[0054] Example 3 In field trials, this invention verified the effects of applying Bacillus proteolyticus B and Microbacterium dendriticum M on cadmium and arsenic in wheat grains. The specific details are as follows: This invention conducts a field trial on cadmium-contaminated calcareous soil (average Cd content of 2.19 mg / kg soil) in Xingtang County, Shijiazhuang City, to verify the remediation effect. The specific details are as follows: After harvesting corn in October, the land was plowed and prepared, and wheat variety (Jimai 22) was sown. During the booting, heading, and early grain-filling stages, *Bacillus proteolyticus* B and *Microbacterium dendriticum* M were applied via root irrigation at a final concentration of 6 × 10⁻⁶ total viable bacteria in the soil. 7CFU / g (referred to as the bacterial treatment group), and a control group was set up, differing from the previous group only in that no bacteria were applied. Soil bulk density method was used to calculate the soil volume of the 0–20 cm topsoil layer in farmland, with a soil mass per unit area (one mu) of topsoil mass taken as 150 tons. Normal field management was implemented. Grain samples were collected at maturity, and the collected wheat grains were washed sequentially with tap water, distilled water, and ultrapure water. They were then blanched at 85℃ for 30 min, dried at 60℃ to constant weight, and pulverized using a stainless steel pulverizer. The cadmium and arsenic content in the wheat grains was determined. Accuracy and precision were controlled using the national first-class standard material (GBW100493, wheat flour), and blank samples were analyzed simultaneously to remove reagent interference. The results of cadmium and arsenic content determination in wheat grains of different groups are as follows: Figure 11 As shown.

[0055] Depend on Figure 11 It can be seen that in the group with wheat root synthetic microbiota composed of Bacillus proteolyticus B and Microbacterium dendriticum M, the cadmium content of mature wheat grains decreased from 0.15 mg / kg in the control group to 0.09 mg / kg, a decrease of 37.89%; the arsenic content of grains decreased from 0.046 mg / kg in the control group to 0.030 mg / kg, a decrease of 34.57%. The wheat grains obtained by applying the wheat root synthetic microbiota provided by this invention meet the national food safety standards for cadmium and arsenic content (0.1 mg / kg and 0.5 mg / kg), and meet the requirements for safe consumption.

[0056] In summary, the wheat root synthetic microbial community provided by this invention, composed of Bacillus proteolyticus B and Microbacterium dendriticum M, can not only fix cadmium ions in solution, but also simultaneously inhibit the absorption of cadmium and arsenic by wheat in the soil-wheat system, reduce the accumulation of cadmium and arsenic in the aboveground parts of wheat, and reduce the cadmium and arsenic content of wheat grains to within the national food safety standard limits. It has great application potential in the remediation of cadmium and arsenic compound pollution environment and the safe production of wheat in cadmium and arsenic compound pollution farmland.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A wheat root-synthesizing microbial community, characterized in that: It contains microbes with accession number CCTCC NO: M 20252014. Microbacterium sp.) M and Bacillus with accession number CCTCC NO: M 20252015 ( Bacillus sp . B.

2. The synthetic microbial community as described in claim 1, characterized in that: The microbacterium M is dendritic microbacterium M; the Bacillus B is protein-degrading Bacillus.

3. The wheat root synthetic microbial community as described in claim 2, characterized in that: The ratio of viable counts of the dendritic microbacterium M to the proteolytic bacillus B is 8-12:

10.

4. The application of the wheat root synthetic microbial community according to any one of claims 1-3 in controlling the accumulation of heavy metals in wheat in heavy metal contaminated soil.

5. The application as described in claim 4, characterized in that: The heavy metals include at least one of cadmium or arsenic.

6. The application as described in claim 4, characterized in that: The heavy metals include cadmium and arsenic.

7. The application as described in claim 4, characterized in that: The wheat includes the above-ground parts of the wheat plant.

8. The application as described in claim 7, characterized in that: The above-ground parts of wheat include wheat grains.

9. A method for controlling the absorption of heavy metals by wheat, characterized in that: During the middle and late stages of wheat growth, the wheat root synthetic microbial community described in any one of claims 1-3 is applied by root irrigation.

10. The method for controlling the absorption of heavy metals by wheat as described in claim 9, characterized in that: The mid-to-late stages of wheat growth include at least one of the following stages: the wheat booting stage, the heading stage, or the early grain-filling stage.

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