Method for synergistically regulating soybean cadmium stress defense by microorganisms-signal molecules

Abstract

The present application relates to the technical field of soil pollution remediation, and in particular to a method for synergistically regulating soybean cadmium stress defense by microorganisms-signal molecules. The method comprises the following steps: preparing a composite inoculant with Cd stress resistance, wherein the composite inoculant comprises rhizobium and AM fungi; planting soybeans in Cd contaminated soil, and applying the composite inoculant when the first leaf of the soybean is fully unfolded; and applying a hydrogen sulfide donor solution to the soybean plant during the seedling stage to the podding stage. The present application utilizes the synergistic effect of synthetic flora and H2S signal molecules to construct an integrated technology route of "soil Cd control-plant Cd stress defense", which can not only promote the growth of soybeans, but also reduce the content of available Cd in the soil.

Description

Technical Field

[0001] This invention relates to the field of soil pollution remediation technology, specifically to a method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules. Background Technology

[0002] In some major soybean-producing areas, farmland is contaminated with cadmium (Cd) to varying degrees. Soybeans easily accumulate Cd in the aboveground parts and grains, which not only reduces yield but also threatens food safety and human health. Current control measures mainly include the application of chemical passivators, single-function microbial agents, and plant growth regulators. Chemical passivators require large dosages, easily alter soil physicochemical properties, and lack long-term stability; single-function microbial agents have limited colonization efficiency in complex field environments and are significantly affected by Cd toxicity and nutrient stress; applying plant growth regulators alone makes it difficult to simultaneously achieve the dual goals of reducing Cd availability and improving crop resistance.

[0003] Therefore, it is necessary to develop a cross-species symbiotic microbial synthesis community-H2S signaling molecule synergistic technology that is structurally sound and easy to operate, so as to enhance the Cd stress defense capacity of soybeans while reducing the bioavailability of Cd in the soil and the accumulation of Cd in soybeans, and realize the safe and efficient utilization of Cd-contaminated farmland. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies that rely solely on single-function microbial agents or plant regulators and cannot simultaneously achieve "controlling soil Cd migration and enhancing soybean Cd stress defense", the present invention aims to provide a method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules. This method is used to reduce soil Cd bioavailability and Cd accumulation in soybeans, and to enhance soybean's antioxidant defense capacity and production performance.

[0005] The technical solution adopted by this invention to solve the technical problem is:

[0006] A method for synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules includes the following steps: preparing a compound microbial inoculum with Cd stress resistance, the compound microbial inoculum comprising rhizobium and AM fungi; planting soybeans in Cd-contaminated soil, applying the compound microbial inoculum when the first true leaf of the soybean is fully expanded; and applying hydrogen sulfide donor solution to the soybean plants during the seedling stage to the grain-filling stage.

[0007] In the above-mentioned method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, the compound microbial inoculant is a mixture of rhizobium suspension and AM fungal spore suspension, and the volume ratio of the rhizobium suspension to the AM fungal spore suspension is 1:(1~3).

[0008] In the above-mentioned method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, the viable count of the rhizobium suspension is 1 × 10⁻⁶. 8 ~1×10 9 CFU / mL;

[0009] In the above-mentioned method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, the AM fungal spore suspension contains 40-80 spores / mL.

[0010] In the above-mentioned method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, the rhizobium is *Sinorhizobium meliloti* CCNWSX0020.

[0011] In the above-mentioned method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, the AM fungus is Rhizophagus irregularis.

[0012] In the above-mentioned method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, the compound microbial inoculant is applied by dripping it onto the rhizosphere of the soybean.

[0013] In the above-mentioned method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, the dosage of the compound microbial inoculant applied is 10-30 ml / plant.

[0014] In the above-mentioned method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, the soybeans are planted in greenhouses or in open fields, and the compound microbial inoculant is applied evenly to the soybean roots in Cd-contaminated soil through trenching.

[0015] In the above-mentioned method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, the Cd-contaminated soil is lightly to moderately Cd-contaminated soil with a total Cd content of 1.0~10.0 mg / kg.

[0016] In the above-mentioned method of synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, during soybean planting, the soil moisture content of the Cd-contaminated soil is adjusted to 60% to 80% of field capacity.

[0017] In the above-mentioned method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, the hydrogen sulfide donor solution is an 80-150 μmol / L solution prepared by dissolving NaHS in deionized water.

[0018] In the above-mentioned method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, the hydrogen sulfide donor solution is applied by rhizosphere irrigation and / or foliar spraying, and the application time interval is twice a week during the planting cycle.

[0019] In the above-mentioned method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, the amount of hydrogen sulfide donor solution applied each time is: 5-15 ml / plant for rhizosphere irrigation and 5-15 ml / plant for foliar spraying.

[0020] By employing the above technical solution, the present invention has at least the following advantages:

[0021] 1) This invention constructs a cross-kingdom synthetic microbial community through arbuscular mycorrhizal fungi and rhizobia, and combines it with the application of H2S signaling molecules to achieve integrated synergistic regulation of "reducing soil cadmium bioavailability and enhancing soybean cadmium stress defense," thus balancing inhibition and tolerance enhancement. This overcomes the limitation of applying individual microbial agents or regulators, which can only act on a single aspect. This invention can both reduce the content of available Cd in the soil and promote plant growth.

[0022] 2) The cross-species synthetic microbial community of this invention exhibits higher colonization capacity and stability in cadmium-contaminated rhizosphere, significantly increasing mycorrhizal infection rate and root nodule number, improving rhizosphere physiological and biochemical properties, and reshaping the dominant community of beneficial microorganisms, thereby maintaining a relatively long-lasting and stable cadmium reduction effect under complex field conditions. Exogenous H2S signaling significantly activates the soybean antioxidant enzyme system, reducing oxidative stress. Combined with the rhizosphere improvement effect of the synthetic microbial community, it can simultaneously reduce the Cd content in the aboveground parts of soybean and increase biomass and yield. The microbial agent used in this invention, along with NaHS, can be applied through conventional agronomic methods such as soil mixing, furrow application, and foliar spraying. The operation is simple and environmentally friendly, making it suitable for safe utilization and large-scale promotion in Cd-contaminated soybean producing areas through "remediation while production."

[0023] 3) This invention utilizes the synergistic effect of synthetic microbial communities and H2S signaling molecules to construct an integrated technical route of "soil Cd control - plant Cd stress defense": the cross-border synthetic microbial community enhances the complexation and fixation of Cd in the rhizosphere by increasing AM infection; H2S reduces the translocation and toxicity of Cd in the plant by inducing root nodules and mycorrhizal symbiosis and regulating root exudates and antioxidant enzyme activity. The two work synergistically to significantly reduce DTPA-Cd and increase biomass at the same time. This overcomes the defects of poor field stability and limited action links when applying microbial agents or regulators alone. It has the advantages of simple operation, green and environmentally friendly, and suitable for promotion in the main soybean producing areas contaminated with cadmium.

[0024] 4) Soybeans grown using the method of the present invention have at least 30% more aboveground and root biomass.

[0025] 5) Through the method of the present invention, a large number of mycelia, tufts and infection structures can be observed inside the soybean root system, the number of root nodules of soybean is increased by 45% and the mycorrhizal infection rate is increased by 124%.

[0026] 6) The method of this invention can significantly enhance root metabolic activity and promote the initiation of metabolic processes related to nutrient absorption, antioxidant defense and heavy metal response, providing a metabolic basis for improving soybean Cd tolerance.

[0027] 7) The method of the present invention can significantly upregulate differential metabolites, indicating that the synergistic treatment mainly manifests as an activation effect. The metabolites are mainly enriched in defense-related metabolic pathways such as flavonoid biosynthesis, antioxidant metabolism, and phenolic acids.

[0028] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0029] Figure 1 The following diagrams (a) and (b) show the growth of rhizobium colonies and AM fungal spores under different Cd stress indices, respectively, as provided in this embodiment of the invention.

[0030] Figure 2 The following diagrams illustrate soil Cd availability (a) and soybean tissue Cd content under different treatments, as provided in this embodiment of the invention.

[0031] Figure 3 These are real-life photos of the aboveground and root growth of soybeans provided in an embodiment of the present invention.

[0032] Figure 4 A bar chart (b) showing the number of soybean root nodules (a) and mycorrhizal infection rate provided in an embodiment of the present invention.

[0033] Figure 5 The above diagrams show AM fungal infection and root nodules in soybean roots provided in this embodiment of the invention. 'ad' represents the AM fungal infection status of soybean roots under different treatments, and 'ef' represents the root nodule growth status of soybeans under different treatments.

[0034] Figure 6 The different treatments provided in the embodiments of the present invention reduce the oxidative damage of MDA (a) and H2O2 (b) in soybean leaves;

[0035] Figure 7 Volcano plot of differential metabolites in soybean roots compared to the control, provided by embodiments of the present invention for the synergistic application of synthetic microbial community and signaling molecules;

[0036] Figure 8 This is a graph showing the enrichment of functional pathways of differential metabolites in soybean roots compared to the control, based on the synergistic application of synthetic microbial communities and signaling molecules provided in this embodiment of the invention. Detailed Implementation

[0037] To further illustrate the technical means and effects of the present invention in achieving the intended purpose, the following detailed description of the specific implementation methods, structures, features and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0038] This embodiment discloses a method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, comprising the following steps: preparing a compound microbial inoculum containing rhizobia and AM fungi; planting soybeans in Cd-contaminated soil, and applying the compound microbial inoculum when the first true leaf of the soybean is fully expanded; and applying hydrogen sulfide donor liquid to the soybean plants during the seedling stage to the grain-filling stage.

[0039] In this embodiment, arbuscular mycorrhizal (AM) fungi and rhizobia are important symbiotic microorganisms in the soybean rhizosphere, playing roles in promoting nutrient absorption, improving soil structure, and participating in Cd fixation. Hydrogen sulfide (H2S), as a novel gaseous signaling molecule, can not only regulate the symbiotic relationship between soybean cross-species symbiotic microorganisms such as rhizobia and AM fungi, but also alleviate Cd stress responses in soybeans by regulating the antioxidant system and maintaining intracellular homeostasis. This invention provides a method for synergistically regulating soybean Cd stress defense using a cross-species symbiotic microorganism-plant signaling molecule hydrogen sulfide, which can be used to reduce soil Cd bioavailability and Cd accumulation in soybeans, enhance soybean antioxidant defense capabilities, and improve production performance.

[0040] This invention promotes plant growth while reducing heavy metal absorption and oxidative stress in plants. The rhizobium and AM fungal hyphae of this invention reduce Cd toxicity and alleviate nutrient stress by fixing Cd and promoting symbiosis with soybeans. The signaling molecule H2S promotes a three-way symbiosis of soybean-rhizobium-AM fungi by increasing nodule and hyphal infection, thereby promoting soybean growth under Cd stress. In specific implementation, adding the compound microbial agent alone increased the number of rhizobium by 34% and the AM fungal infection rate by 83%. Simultaneously adding the compound microbial agent and NaHS synergistically increased the number of rhizobium by 45% and the AM fungal infection rate by 124%, synergistically reduced the soil DTPA-Cd content by 10% and reduced soybean heavy metal oxidative damage by approximately 30%, indicating that the compound microbial agent-H2S synergistically promotes soybean Cd stress defense and growth.

[0041] In this specific implementation, NaHS is used as the H2S donor for three reasons: ① H2S can activate the antioxidant enzyme system in plants, enhance oxidative stress defense, and inhibit the transport of heavy metals in plants; ② H2S can regulate the availability of heavy metals in the soil by promoting plant root metabolism (such as secreting organic acids), thereby alleviating heavy metal stress in plants; ③ H2S can promote leguminous crop-microbe symbiosis, induce changes in root secondary metabolites, and affect the heavy metal inhibition process and plant growth driven by rhizosphere microorganisms.

[0042] In this embodiment, the inoculant is applied when the soybean root system is extensive, which facilitates microbial colonization. During rapid plant growth, the plant needs to absorb a large amount of nutrients, which is also the time when it absorbs the most heavy metals, resulting in the most severe heavy metal stress. Applying NaHS at this time reduces soybean stress damage.

[0043] The timing of applying the aforementioned compound microbial inoculant and hydrogen sulfide donor solution is matched with the growth of soybeans. Specifically, the period when the first true leaf of soybean has fully unfolded is a critical window for rapid root development and the establishment of a rhizosphere symbiotic system. At this time, lateral roots and root hairs are formed in large numbers, rhizosphere exudate release is enhanced, and the efficiency of rhizobium infection and AM fungal colonization is high. This is conducive to the establishment of a stable dominant population of exogenous synthetic microorganisms in the rhizosphere and the early formation of a nutrient absorption and heavy metal control system. At the same time, the rhizosphere microbial community is not yet fully stable at this stage, and exogenous functional bacteria can more easily complete colonization, thereby improving the subsequent cadmium reduction and growth promotion effects. The period from seedling stage to grain filling stage is an important period for rapid accumulation of soybean biomass, active nitrogen fixation in root nodules, and Cd absorption and translocation to grains. Continuous application of H2S donor NaHS can promote root nodule formation and mycorrhizal infection, enhance nutrient absorption capacity, and reduce Cd toxicity and its risk of enrichment in grains by activating the antioxidant defense system and regulating the Cd translocation process, thereby continuously playing a role in promoting growth and reducing cadmium throughout the critical growth stages.

[0044] In addition, the timing of applying the inoculant and NaHS partially overlaps (i.e., the seedling stage of soybeans), which can promote plant growth while reducing plant heavy metal absorption and oxidative stress.

[0045] Furthermore, the methods for obtaining and culturing cadmium-tolerant rhizobia and AM fungi in this embodiment are as follows:

[0046] The symbiotic bacterial strain *Sinorhizobium meliloti* CCNWSX0020 and the symbiotic fungal strain AM used in this embodiment are *Rhizophagus irregularis*. *Sinorhizobium meliloti* CCNWSX0020 is deposited at the China Agricultural Microbiological Culture Collection Center (ACCC19736); the AM fungus *Rhizophagus irregularis* was provided by the Guangdong Provincial Microbiological Culture Collection Center. The rhizobium is a highly efficient nitrogen-fixing strain, *Sinorhizobium meliloti*, isolated from soybean root nodules in Cd-contaminated soil.

[0047] This embodiment also identified the cadmium tolerance of the two strains mentioned above. The specific method is as follows: Rhizobium was inoculated into TY liquid medium (5.0 g / L tryptone, 3.0 g / L yeast extract, 0.7 g / L CaCl2·2H2O), and cultured at 28℃ and 150 rpm in a shaker until the exponential growth phase. The OD of the bacterial suspension was then adjusted. 600 Approximately 0.1. Equal volumes of bacterial suspension were added to 0.1 mol / L NaCl solutions containing different concentrations of CdCl2 (0, 10, 50, 100, 150 mg / L), and incubated at 25 °C for 24 h. After incubation, the bacterial cells were washed twice with 0.1 mol / L NaCl, and 20 μL of the bacterial suspension was added dropwise to TY agar plates (TY solid medium: tryptone 5.0 g / L, yeast extract 3.0 g / L, CaCl2·2H2O 0.7 g / L, agar powder 15.0 g), and incubated at 32 °C for 48 h. Colony growth was observed. TY microbial culture medium was prepared according to the above formula and autoclaved at 121 °C for 30 min. The solid culture medium for the growth of AM fungal spores was dissolved in 10 mM sodium citrate, and the AM fungal spores were cultured at different concentrations of CdCl2 (0, 50, 150 mg / L) at 32 ℃ for 48 h, and the spore growth was observed. The spore suspension dissolved in sodium citrate was filtered through sterile cotton or gauze to remove any mycelia that might be mixed in, and the AM fungal spore suspension was obtained and stored in a 4C refrigerator for later use.

[0048] The screening results are as follows Figure 1 As shown, the growth of *Sinorhizobium meliloti* CCNWSX0020 and *Rhizophagus irregularis* under different concentrations of Cd stress in alfalfa is illustrated. Figure 1 As shown in (a), *Rhizobium sinense* in alfalfa was able to form clearly visible colonies when the Cd concentration was gradually increased from 0 mg / L to 150 mg / L. While the colony growth rate decreased with increasing Cd concentration, it still maintained good colony morphology and survival ability at 150 mg / L Cd treatment, indicating that this strain has strong Cd tolerance. Figure 1 As shown in (b), under Cd treatments of 0, 50, and 150 mg / L, *Rhizocystis heterophylla* spores were able to germinate normally and form hyphal networks, indicating that this AM fungus has high Cd tolerance. These results demonstrate that the rhizobia and AM fungi selected in this invention can survive and grow under high Cd stress conditions, and can serve as important functional strains for constructing cross-kingdom synthetic microbial communities in Cd-contaminated farmland, providing a guarantee for subsequent rhizosphere colonization and synergistic cadmium reduction.

[0049] Specifically, the compound microbial inoculum is a mixture of rhizobium suspension and AM fungal spore suspension at a volume ratio of 1:(1~3);

[0050] In this embodiment, the viable count of the rhizobium suspension is 1 × 10⁻⁶. 8 ~1×10 9 CFU / mL; the spore count of AM fungal spore suspension is 40-80 spores / mL.

[0051] In this embodiment, the preparation method of the compound microbial inoculant is as follows: *Sinorhizobium meliloti* CCNWSX0020 and *Rhizophagus irregularis* (AM fungus) are cultured separately to obtain rhizobium inoculum and AM fungal spore inoculum; the rhizobium strain is inoculated into sterilized TY liquid medium and cultured in a constant temperature shaking incubator at 28 ℃ and 150 rpm for approximately 24 h, OD... 600 The value reached 0.1; the bacterial cells were collected by centrifugation (8000 g, 4 ℃, 10 min), washed with sterile deionized water, and then an equal volume of deionized water was added to prepare a bacterial suspension with a viable count of 1×10⁻⁶. 8 ~1×10 9 CFU / mL. Rhizobium inoculum solution and AM fungal spore suspension were mixed at a volume ratio of 1:(1~3) to prepare a synthetic inoculum. Rhizobium inoculation was performed at a concentration of 1×10⁻⁶ CFU / mL. 8 –1×10 9 CFU / mL, AM fungal spore content is 40–80 spores / mL.

[0052] In this embodiment, the hydrogen sulfide donor solution is an 80-150 μmol / L solution prepared by dissolving NaHS in deionized water. Specifically, NaHS is weighed and dissolved in deionized water to prepare a 100 mmol / L stock solution, which is stored at 4 °C protected from light. When using, the stock solution is diluted with deionized water to a 100 μmol / L hydrogen sulfide donor solution.

[0053] To verify the effectiveness of the method of synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, this embodiment provides a pot experiment. Specific experiments include:

[0054] 1) Soil preparation and treatment setup: Take topsoil (0–20 cm) from the lightly to moderately contaminated topsoil layer, air-dry it, pass it through a 2 mm sieve, and determine its basic physicochemical properties and total Cd content (approximately 1.0–5.0 mg / kg). Pack 2.5 kg of dry soil into each plastic pot, and apply basal fertilizer at the standard soybean fertilization rate in one application. After filling the pots, spray water to moisten the soil, ensuring the soil moisture content reaches 60%–80% of field capacity.

[0055] 2) The parallel experiment was set up with 4 treatments: control without inoculation and NaHS (CK), synthetic microbial community alone (MR), NaHS alone, and synthetic microbial community + NaHS synergistic treatment (MR+S). Each treatment had 4 replicates, which were randomly arranged.

[0056] 3) Select soybean variety Zhonghuang 13. Disinfect the seeds with 75% alcohol for 30 seconds, then with 2% NaClO solution for 2 minutes, rinse thoroughly with sterile water, and place on damp filter paper to germinate at 28℃. Sow 8 germinated seeds per pot, and thin out to 2 seedlings of uniform growth after emergence. When sowing or transplanting soybean seedlings in Cd-contaminated soil, retain 2–3 seedlings per hole. When the first true leaf of the soybean is fully expanded, drip or spray sterilized water onto the root zone to facilitate the colonization of cross-boundary synthetic microorganisms in the rhizosphere.

[0057] 4) Application of compound microbial inoculant and hydrogen sulfide donor solution: Day 0 is defined as when the first true leaf is fully expanded, at which point the soybean root system is relatively robust. 20 mL of the compound microbial inoculant is inoculated into the soybean rhizosphere. Equal volumes of sterile carrier are added to the CK and NaHS treatments. During the seedling to grain-filling stage, soybeans absorb a large number of elements from the soil, significantly increasing the risk of Cd accumulation. To reduce Cd accumulation in soybean tissues, when applying the hydrogen sulfide donor solution, in the NaHS and MR+S treatments, 10 mL of 100 μmol / L NaHS solution is slowly applied along the rhizosphere per plant, simultaneously with a foliar spray of the same concentration until the leaves are moist but not dripping. To ensure full utilization of NaHS by the soybeans, it is applied twice a week for 5 consecutive weeks. Equal volumes of deionized water are applied to the CK and MR treatments in the same manner. Throughout the growing season, water is replenished by weighing to maintain soil moisture content at 60%–80% of field capacity.

[0058] 5) During the soybean growth period, maintain routine water and fertilizer management. After harvesting the plants, test the available cadmium in the soil (DTPA-Cd), Cd content in different parts of the plant, number of root nodules, mycorrhizal infection rate, and changes in root metabolism to evaluate the synergistic regulatory effect. The results showed that after planting soybeans using the method of this embodiment, the soil DTPA-Cd content was significantly reduced, which reduced the Cd content in the soybean aboveground and root systems. Figure 2 The effect of MR+S treatment on resistive Cd is particularly significant.

[0059] Applying synthetic microorganisms and H2S in addition to conventional fertilization can reduce soil DTPA-Cd by 3%–10% and reduce the Cd content and bioaccumulation coefficient of soybean aboveground parts by 21%–38%.

[0060] After soybeans are planted using the method of this embodiment, the growth of the above-ground parts and root system of the soybeans is as follows: Figure 3As shown, inoculation with synthetic microorganisms and application of H2S promoted the growth of soybean aboveground parts and roots. The synergistic effect of synthetic microorganisms-H2S treatment was particularly significant, with aboveground and root biomass increasing by 30.7% and 30.1%, respectively.

[0061] This embodiment tested the number of soybean root nodules and mycorrhizal infection rate. Figure 4 The control (CK) treatment showed fewer mycelial and arbuscular structures in the roots, while the synthetic mycorrhizal-H2S synergistic treatment (MR+S) increased mycelial infection. Numerous mycelia, arbuscular structures, and infection structures were observed within the roots treated with MR+S, indicating a more fully established mycorrhizal symbiosis. Simultaneously, the MR+S treatment resulted in the highest number of root nodules, which were larger and more evenly distributed. Compared to CK, MR+S significantly increased the number of root nodules and the mycorrhizal infection rate. Figure 5 The number of root nodules and mycorrhizal infection rate increased by 45% and 124%, respectively; compared with the control (CK), the synthetic microbial community-H2S synergistic treatment (MR+S) significantly reduced oxidative damage to soybean leaves. Figure 6 MDA and H2O2 decreased by 26% and 29%, respectively. The results showed that H2S can promote the colonization and symbiotic process of rhizobia and AM fungi in the soybean rhizosphere, thereby strengthening the three-way interaction system of soybean-rhizobia-AM fungi, reducing soil DTPA-Cd content and Cd accumulation in soybean aboveground parts, enhancing soybean biomass and Cd stress defense capabilities, and providing a basis for enhancing nutrient absorption and heavy metal control capabilities under Cd stress.

[0062] In this embodiment, the MR+S treatment upregulated 5909 metabolites compared to the control. Figure 7 The downregulation of 347 metabolites indicates that the combined action of synthetic microorganisms and H2S can induce extensive metabolic reprogramming in the root system. The significant upregulation of differentially expressed metabolites suggests that the synergistic treatment primarily manifests as an activation effect. These results demonstrate that MR+S treatment can significantly enhance root metabolic activity, promote the initiation of metabolic processes related to nutrient absorption, antioxidant defense, and heavy metal response, and provide a metabolic basis for improving soybean Cd tolerance. Differentially expressed metabolites in soybean roots are mainly enriched in defense-related metabolic pathways such as flavonoid biosynthesis, antioxidant metabolism, and phenolic acids. Figure 8 Flavonoid metabolites not only serve as important signaling molecules for rhizobium recognition and colonization, but also participate in Cd complexation and antioxidant defense, which is beneficial for rhizosphere Cd complexation and intracellular antioxidant defense. Phenolic acid and organic acid metabolites can promote rhizosphere Cd fixation, alter Cd speciation, and regulate the structure of beneficial microbial communities. These metabolic findings confirm the mechanism of this invention, "synergistic regulation of soybean Cd stress defense by synthetic microbial communities and H2S."

[0063] In specific implementation, the synthetic microbial inoculant is applied by dripping it onto the rhizosphere of the soybean plant; the dosage of the synthetic microbial inoculant is 10-30 ml / plant. Specifically, in this embodiment, the dosage of the synthetic microbial inoculant is 20 ml / plant.

[0064] In specific implementation, the Cd-contaminated soil refers to lightly to moderately Cd-contaminated soil with a total Cd content of 1.0 to 10.0 mg / kg.

[0065] In practice, during soybean planting, the soil moisture content of the Cd-contaminated soil is adjusted to 60%–80% of field capacity, which is beneficial to soybean growth and the activity of the compound microbial inoculant.

[0066] The hydrogen sulfide donor solution is applied by rhizosphere irrigation and / or foliar spraying, with the application interval being twice a week during the planting cycle; NaHS is dissolved in deionized water to prepare a 100 mM hydrogen sulfide donor solution; it is sprayed twice a week from the soybean seedling stage to the grain filling stage, with an application rate of 10 mL per plant, so that H2S forms a stable signal concentration in different tissues of soybean.

[0067] The amount of hydrogen sulfide donor solution applied each time is: 5-15 ml / plant for rhizosphere irrigation; and 5-15 ml / plant for foliar spraying. The optimal concentration of NaHS in the hydrogen sulfide donor solution is 100 μM. Spraying twice a week for 4-6 consecutive weeks can significantly increase the number of root nodules by 20%-50% and the mycorrhizal infection rate by 30%-120%, and increase the aboveground and root biomass of soybean by 10%-31%.

[0068] This invention also provides the application of the above method in the safe utilization of Cd-contaminated farmland, namely, applying synthetic microbial communities to Cd-contaminated soil before soybean planting or during the seedling stage, and periodically applying NaHS during soybean growth. Through the synergistic regulation of "cross-border synthetic microbial communities-H2S" on soybean-AM fungi-rhizobium symbiosis, rhizosphere microbial community and soybean metabolic system, the goal of reducing soybean Cd content and increasing yield can be achieved.

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for the synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules, characterized in that, Includes the following steps: A compound microbial inoculum with Cd stress resistance was prepared, wherein the compound microbial inoculum includes rhizobia and AM fungi; Soybeans were planted in Cd-contaminated soil, and the compound microbial inoculum was applied when the first true leaf of the soybean was fully expanded. Hydrogen sulfide donor solution is applied to the soybean plants from the seedling stage to the grain-filling stage.

2. The method for synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules according to claim 1, characterized in that, The compound microbial inoculant is a mixture of rhizobium suspension and AM fungal spore suspension, with a volume ratio of 1:(1~3) between the rhizobium suspension and the AM fungal spore suspension.

3. The method for synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules according to claim 2, characterized in that, The viable count of the rhizobium suspension was 1 × 10⁻⁶. 8 ~1×10 9 CFU / mL; The AM fungal spore suspension contains 40-80 spores / mL.

4. The method for synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules according to claim 1, characterized in that, The rhizobium is *Alfalfa Rhizobium sinense* (… Sinorhizobium meliloti CCNWSX0020); The AM fungus is *Rhizocystis heterophylla* (… Rhizophagus irregularis ).

5. The method for synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules according to claim 1, characterized in that, The compound microbial inoculant is applied by dripping it onto the rhizosphere of the soybean. The dosage of the compound bacterial inoculant is 10-30 ml / strain.

6. The method for synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules according to claim 1, characterized in that, The soybeans are planted in greenhouses or in open fields, and the compound microbial inoculant is applied evenly to the soybean roots in Cd-contaminated soil through trenching.

7. The method for synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules according to claim 1, characterized in that, The Cd-contaminated soil is lightly to moderately Cd-contaminated soil with a total Cd content of 1.0 to 10.0 mg / kg. During soybean cultivation, the soil moisture content of the Cd-contaminated soil was adjusted to 60% to 80% of field capacity.

8. The method for synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules according to claim 1, characterized in that, The hydrogen sulfide donor solution is an 80-150 μmol / L solution prepared by dissolving NaHS in deionized water.

9. The method for synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules according to claim 1, characterized in that, The hydrogen sulfide donor solution is applied by rhizosphere irrigation and / or foliar spraying, and the application interval is twice a week during the planting cycle.

10. The method for synergistic regulation of soybean cadmium stress defense by microorganisms and signaling molecules according to claim 9, characterized in that, The amount of hydrogen sulfide donor solution applied each time is: 5-15 ml / plant for rhizosphere irrigation; and 5-15 ml / plant for foliar spraying.

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