Application of method for preparing functional biological flora by coupling target biological flora and host original flora in rice cultivation

Abstract

The invention belongs to the field of agricultural microbial technology and agricultural product safety, and particularly relates to application of a method for preparing a functional biological flora in a targeted biological flora and host original flora coupling mode in rice cultivation. According to the method, bacillus subtilis, bacillus mucilaginosus, brevibacillus laterosporus and host original flora are subjected to coupled symbiotic culture to form specific functional flora, and the specific functional flora is compounded with nutrient carriers such as humic acid potassium fulvate and amino acid chelated trace elements to prepare the targeted biological bacterium water-soluble fertilizer. The microbial agent effectively reduces the bioavailability of heavy metals such as cadmium, lead and arsenic in soil and migration of the heavy metals to rice; meanwhile, the functional flora secretes hydrolase, oxidoreductase and the like, degrades pesticide residues such as organophosphorus and pyrethroid and decomposes the pesticide residues into non-toxic or low-toxicity substances, the method is specially used for solving the problem that heavy metal and pesticide residues in the rice in the filling stage of the rice exceed the standard, and the quality and edible safety of the rice are guaranteed from the source.

Description

Technical Field

[0001] This invention relates to the fields of agricultural microbiology and agricultural product technology, specifically to the application of a method for preparing functional microbial communities by coupling targeted microorganisms with the original host microbial community in rice cultivation. Background Technology

[0002] Rice is my country's most important staple food crop, and its product quality and safety are directly related to the national economy and people's livelihood. However, rice production generally faces two major safety hazards: 1. Heavy metal pollution: Industrial waste discharge and long-term use of pesticides and fertilizers containing heavy metals lead to the accumulation of heavy metals such as cadmium, lead, and arsenic in paddy field soil. During the grain-filling stage, rice plants readily transport these heavy metals from the roots to the grains, resulting in excessive levels of heavy metals in the rice and posing a serious threat to human health.

[0003] 2. Pesticide residues: To control pests and diseases, chemical pesticides such as organophosphates and pyrethroids are often applied during rice growth. If these pesticides are not completely degraded, they will remain in the rice, and long-term ingestion may cause chronic poisoning, damage to the nervous system, and even cancer risks.

[0004] In existing technologies, physicochemical remediation methods are costly and prone to causing secondary pollution, while ordinary microbial agents often have limited functions or poor adaptability to the rice rhizosphere environment, making it difficult to effectively control the translocation of heavy metals to grains and completely degrade pesticide residues during the critical grain-filling stage of rice. Therefore, there is an urgent need to develop a specialized microbial agent and its application method that can target the rice root-soil interface and systematically solve the problem of heavy metal and pesticide residues in rice through multi-mechanism synergy. Summary of the Invention

[0005] The purpose of this invention is to apply a functional microbial community, formulated by coupling a targeted microbial organism with the host's original microbial community, to rice cultivation. This functional microbial community comprises Bacillus subtilis, Bacillus mucilaginosus, and Bacillus laterosporus brevis, cultured symbiotically, along with the host's original microbial community. This microbial community is then compounded with a nutrient carrier containing humic acid, potassium humate, amino acid chelates, and trace elements under acidic conditions to produce a targeted microbial water-soluble fertilizer, which is primarily applied during the mid-to-late stages of rice growth.

[0006] The technical solution of the present invention to achieve the above objectives is as follows: The present invention provides a method for preparing functional microbial communities by coupling targeted microorganisms with the original host microbial community, which is applied in rice cultivation. The functional microbial communities obtained by coupling culture are prepared into microbial agents or water-soluble fertilizers for rice cultivation, so as to specifically solve the problem of excessive heavy metal and pesticide residues in rice.

[0007] This invention utilizes targeted screening of diverse bacterial strains with both heavy metal passivation and pesticide degradation functions, coupled with symbiotic culture of the rice rhizosphere native host microbiota. This enhances the compatibility and colonization ability of the microbiota with the rice rhizosphere microecology. The functional microbiota is combined with an optimized nutrient carrier, and the pH is adjusted to a suitable range for the rice rhizosphere. This allows the strains to passivate heavy metals and degrade pesticide residues through metabolism, while also providing support for microbial growth and rice development through the nutrient carrier. This achieves a synergistic effect of "pollution control + nutrient supply," thus solving the problem of rice pollution.

[0008] Optionally, the functional microbial community is prepared by a method including the following steps: a. Construct a targeted biological strain resource bank and screen for functional single strains; b. Purify and compound the single strain; c. Couple and symbiotically culture the compound microbial community with the host original microbial community extracted from the rhizosphere soil of healthy rice. d. Mix the coupled cultured microbial community with the nutrient carrier and adjust the pH to 4.5-5.5 to prepare a microbial agent or water-soluble fertilizer.

[0009] The standardized process of "screening, purification and compounding, coupled culture and formulation preparation" ensures the functional stability and activity of the functional microbial community; the introduction of the original host microbial community enhances the colonization competitiveness of the compound microbial community in the rice rhizosphere and avoids the rejection effect between the exogenous microbial community and the soil microecology; the precise adjustment of pH value further adapts to the acidic environment of the rice rhizosphere and enhances the metabolic activity of the microbial community.

[0010] Optionally, the functional single strain mentioned in step a includes Bacillus subtilis, Bacillus mucilaginosus, and Bacillus brevis.

[0011] The three strains have synergistic and complementary functions: Bacillus subtilis can secrete organic acids and enzymes to passivate heavy metal ions and degrade organophosphorus pesticides, while inhibiting the growth of pathogens; Bacillus mucilaginosus can activate soil nutrients and reduce the bioavailability of heavy metals through adsorption and complexation, thereby enhancing the stress resistance of rice; Bacillus lateralis can efficiently degrade pyrethroid pesticides and secrete growth-promoting substances to promote rice growth. The combination of the three strains ensures the synergistic achievement of heavy metal passivation and degradation of the two major pesticide residues.

[0012] Optionally, the concentration requirement for each strain in the functional microbial community is: Bacillus subtilis ≥ 5 × 10⁻⁶. 8 CFU / ml, Bacillus subtilis ≥3×10 8 CFU / ml, Bacillus retrosporum ≥2×10 8 CFU / ml, host original flora ≥1×10 8 CFU / ml.

[0013] By clearly defining the minimum concentration standards for each strain, we can ensure that the microbial community has sufficient heavy metal passivation capacity and pesticide degradation efficiency, and avoid the decline in application effect due to insufficient strain concentration. The concentration control of the original host microbial community ensures its balanced symbiosis with the compound microbial community, and improves the overall colonization stability and metabolic activity of the compound microbial community.

[0014] Optionally, the nutrient carriers described in step d include potassium humate, amino acid chelated trace elements, potassium dihydrogen phosphate, sucrose or molasses, and pH adjusters.

[0015] The components of the nutrient carrier are functionally adapted: humic acid and potassium fulvate improve soil structure, promote microbial colonization and reproduction, and enhance soil water and fertilizer retention capacity; amino acid chelated trace elements are easily absorbed by rice, supplementing the trace elements needed for growth and improving rice's stress resistance; potassium dihydrogen phosphate provides phosphorus and potassium nutrition, promotes rice tillering and grain filling, and increases yield; sucrose or molasses provides a fast-acting carbon source for microbial growth and maintains microbial activity; pH ​​adjusters precisely regulate the system's acidity and alkalinity to suit the rice rhizosphere environment.

[0016] Optionally, the nutrient carrier in each ton of finished water-soluble fertilizer comprises: 40-60 kg of potassium humate, 5-15 kg of amino acid chelated trace elements, 15-25 kg of potassium dihydrogen phosphate, 10-20 kg of sucrose or molasses, and an appropriate amount of pH adjuster to maintain the pH of the system at 4.5-5.5.

[0017] The dosage range of each ton of finished water-soluble fertilizer should be clearly defined to ensure that the nutrient supply matches the needs of microbial activity and avoid the application effect being affected by excessive or insufficient components. The dosage range should take into account the degree of soil pollution in different planting areas and the differences in rice varieties to improve product adaptability and application flexibility.

[0018] Optionally, the pH adjuster is citric acid.

[0019] Citric acid is a natural organic acid with high safety and will not cause secondary pollution. Its regulating effect is mild and long-lasting, and it can stably maintain the pH of the system within the suitable range of 4.5-5.5 in the rice rhizosphere. At the same time, it can help enhance the passivation effect of heavy metals and work synergistically with functional biological flora to improve the treatment efficiency.

[0020] Optionally, the application can reduce the residues of heavy metals cadmium, lead, and arsenic in rice by more than 30%, and the degradation rate of organophosphorus and pyrethroid pesticide residues exceeds 80%.

[0021] Quantify the application effect indicators, clarify the technical advantages of the invention, ensure that the application value is quantifiable and verifiable, and meet the actual needs of green and safe rice production.

[0022] Optionally, embodiments of the present invention also provide a targeted biological microbial water-soluble fertilizer for rice cultivation, which is a compound of functional biological microbial groups prepared by the method described in any of the above optional schemes and a nutrient carrier.

[0023] This targeted bio-fertilizer integrates the synergistic treatment effect of functional microbial communities with the auxiliary function of nutrient carriers. It is in liquid form, making it easy to apply through fertigation, drip irrigation, or foliar spraying. It is flexible and convenient to use and can be directly applied to the cultivation and management of rice at different growth stages, specifically addressing rice contamination issues.

[0024] Optionally, embodiments of the present invention also provide a cultivation method for reducing heavy metal and pesticide residues in rice, wherein the above-mentioned targeted biological bacteria water-soluble fertilizer is applied during the critical growth period of rice, especially from the late tillering stage to the early grain filling stage.

[0025] This product is applied during the critical growth period of rice (late tillering to early grain filling, when rice roots have strong absorption capacity and rice nutrients are actively accumulated) to ensure timely colonization of the microbial community and its metabolic function. Nutrients are supplied simultaneously, maximizing the passivation of heavy metals and the degradation of pesticides. It is simple to operate and easy for farmers to master and promote.

[0026] The beneficial effects of this invention are: 1. Multi-mechanism synergistic targeting: The bacterial community has a clear division of labor and synergistic effects. Bacillus subtilis competes for heavy metal absorption sites by producing siderophores; Bacillus mucilaginosus chelates and passivates heavy metals through extracellular polysaccharides; and Bacillus brevis laterospora immobilizes heavy metal ions by encapsulating them with extracellular polymers. These four mechanisms work together to block the migration of heavy metals into rice. At the same time, the phosphatases and esterases secreted by the bacterial community can efficiently degrade specific pesticides.

[0027] 2. Strong environmental adaptability: Through coupled culture with the original microbial community of the rice rhizosphere host, the colonization competitiveness and long-term activity of the functional microbial community in the paddy field ecosystem are significantly enhanced. Adjusting the product pH to 4.5-5.5 is not only conducive to the preservation of the microbial community, but also matches the microenvironment in which it exerts its optimal degradation activity.

[0028] 3. Precise control with remarkable results: Applying the product during the rice grain-filling stage, a critical window for controlling heavy metal and pesticide residues, can precisely intervene in the migration and accumulation of pollutants into the grains, thus ensuring the quality of rice from the source.

[0029] 4. Green and efficient: Based entirely on biotechnology, it does not introduce secondary pollution and greatly reduces dependence on chemical passivating agents and pesticides, making it an effective way to achieve green and safe rice production. Detailed Implementation

[0030] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to specific embodiments. Example

[0031] Preparation of Rice-Specific Targeted Bio-infectious Water-Soluble Fertilizer 1. Activation and propagation of strains: The original strains of Bacillus subtilis, Bacillus mucilaginosus, and Bacillus brevis lateralis are activated separately and fermented at high density in their respective suitable culture media until the required concentration is reached.

[0032] 2. Extraction of original host microbiota: Samples were collected from the rhizosphere soil of uncontaminated healthy paddy fields, and the original host microbiota, rich in rice rhizosphere-specific microorganisms, was obtained through enrichment culture and centrifugation purification.

[0033] 3. Coupled symbiotic culture: The individual strains obtained in step 1 are mixed in proportion (blight: mucilage: lateral spores ≈ 5:3:2), and then mixed with the original host flora obtained in step 2 at a volume ratio of 10:1. The mixture is co-cultured at 31℃, pH 6.3 and aeration for 65 hours to form a stable composite functional flora.

[0034] 4. Formulation Preparation: The concentrated solution of the coupled-cultured compound functional microbial community is uniformly mixed with the following nutrient carrier, and the pH is adjusted to 5.0 with citric acid to prepare a water-soluble fertilizer: Potassium humate: 50 kg Amino acid chelated trace elements (Zn, Mn, B, Mo): 10 kg Potassium dihydrogen phosphate: 20 kg Molasses: 15 kg Compound microbial concentrate: Remaining amount, replenish to 1 ton to ensure that the concentration of each functional bacteria in the finished product meets the requirements.

[0035] Field application trials and efficacy verification In a paddy field in Hunan Province contaminated with heavy metals, an experimental group and a control group were set up. The experimental group was treated with the targeted biological bacteria water-soluble fertilizer prepared in this invention (diluted 500 times and applied to the roots) at the end of the tillering stage and the beginning of the heading and grain-filling stage, respectively. The control group was managed according to the local conventional treatment plan.

[0036] result Heavy metal residues: Post-harvest rice testing showed that the cadmium (Cd) content in the experimental group was 52.3% lower than that in the control group, the lead (Pb) content was 38.1% lower, and the arsenic (As) content was 45.6% lower.

[0037] Pesticide residues: For the applied chlorpyrifos (organophosphate) and lambda-cyhalothrin (pyrethroid), the residue degradation rates in the rice of the experimental group reached 85.7% and 91.2%, respectively.

[0038] Rice growth: The rice plants in the experimental group grew vigorously, and no obvious symptoms of heavy metal toxicity were found. The yield was the same as that of the control group. Example

[0039] Preparation of Rice-Specific Targeted Bio-infectious Water-Soluble Fertilizer 1. Activation and propagation of strains: The original strains of Bacillus subtilis, Bacillus mucilaginosus, and Bacillus brevis lateralis are activated separately and fermented at high density in their respective suitable culture media until the required concentration is reached.

[0040] 2. Extraction of original host microbiota: Samples were collected from the rhizosphere soil of uncontaminated healthy paddy fields, and the original host microbiota, rich in rice rhizosphere-specific microorganisms, was obtained through enrichment culture and centrifugation purification.

[0041] 3. Coupled symbiotic culture: The individual strains obtained in step 1 are mixed in proportion (blight: mucilage: lateral spores ≈ 5:3:2), and then mixed with the original host flora obtained in step 2 at a volume ratio of 10:1. They are co-cultured at 30℃, pH 6.0 and aeration for 60 hours to form a stable composite functional flora.

[0042] 4. Formulation Preparation: The concentrated solution of the coupled-cultured compound functional microbial community is uniformly mixed with the following nutrient carrier, and the pH is adjusted to 4.5 with citric acid to prepare a water-soluble fertilizer: Potassium humate: 40 kg Amino acid chelated trace elements (Zn, Mn, B, Mo): 5 kg Potassium dihydrogen phosphate: 15 kg Molasses: 10 kg Compound microbial concentrate: Remaining amount, replenish to 1 ton to ensure that the concentration of each functional bacteria in the finished product meets the requirements. Example

[0043] Preparation of Rice-Specific Targeted Bio-infectious Water-Soluble Fertilizer 1. Activation and propagation of strains: The original strains of Bacillus subtilis, Bacillus mucilaginosus, and Bacillus brevis lateralis are activated separately and fermented at high density in their respective suitable culture media until the required concentration is reached.

[0044] 2. Extraction of original host microbiota: Samples were collected from the rhizosphere soil of uncontaminated healthy paddy fields, and the original host microbiota, rich in rice rhizosphere-specific microorganisms, was obtained through enrichment culture and centrifugation purification.

[0045] 3. Coupled symbiotic culture: The individual strains obtained in step 1 are mixed in proportion (blight: mucilage: lateral spores ≈ 5:3:2), and then mixed with the original host flora obtained in step 2 at a volume ratio of 10:1. They are co-cultured at 32℃, pH 6.5, and aeration for 72 hours to form a stable composite functional flora.

[0046] 4. Formulation Preparation: The concentrated solution of the coupled-cultured compound functional microbial community is uniformly mixed with the following nutrient carrier, and the pH is adjusted to 5.5 with citric acid to prepare a water-soluble fertilizer: Potassium humate: 60 kg Amino acid chelated trace elements (Zn, Mn, B, Mo): 15 kg Potassium dihydrogen phosphate: 25 kg Molasses: 20 kg Compound microbial concentrate: Remaining amount, replenish to 1 ton to ensure that the concentration of each functional bacteria in the finished product meets the requirements.

[0047] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. The application of a method for preparing functional microbial communities by coupling targeted microorganisms with the host's original microbial community in rice cultivation, characterized in that, The functional microbial communities obtained from coupled culture are prepared into microbial agents or water-soluble fertilizers for rice cultivation, in order to specifically solve the problem of excessive heavy metal and pesticide residues in rice.

2. The application as described in claim 1, characterized in that, The functional microbial community is prepared by a method including the following steps: a. Construct a targeted biological strain resource bank and screen for functional single strains; b. Purify and compound the single strain; c. Couple and symbiotically culture the compound microbial community with the host original microbial community extracted from the rhizosphere soil of healthy rice. d. Mix the coupled cultured microbial community with the nutrient carrier and adjust the pH to 4.5-5.5 to prepare a microbial agent or water-soluble fertilizer.

3. The application as described in claim 2, characterized in that, The functional single strains mentioned in step a include Bacillus subtilis, Bacillus mucilaginosus, and Bacillus brevis.

4. The application as described in claim 3, characterized in that, The concentration requirements for each strain in the functional microbial community are as follows: Bacillus subtilis ≥ 5 × 10⁻⁶ 8 CFU / ml, Bacillus subtilis ≥3×10 8 CFU / ml, Bacillus retrosporum ≥2×10 8 CFU / ml, host original flora ≥1×10 8 CFU / ml.

5. The application as described in claim 2, characterized in that, The nutrient carriers mentioned in step d include potassium humate, amino acid chelated trace elements, potassium dihydrogen phosphate, sucrose or molasses, and pH adjusters.

6. The application as described in claim 5, characterized in that, The nutrient carrier in each ton of finished water-soluble fertilizer comprises: 40-60 kg of potassium humate, 5-15 kg of amino acid chelated trace elements, 15-25 kg of potassium dihydrogen phosphate, 10-20 kg of sucrose or molasses, and an appropriate amount of pH adjuster to maintain the pH of the system at 4.5-5.

5.

7. The application as described in claim 6, characterized in that, The pH adjuster is citric acid.

8. A targeted biological microbial water-soluble fertilizer for rice cultivation, characterized in that, It is a compound of functional microbial flora prepared by any one of claims 2-7 and a nutrient carrier.

9. A cultivation method for reducing heavy metal and pesticide residues in rice, characterized in that, During the critical growth period of rice, especially from the late tillering stage to the early grain-filling stage, apply the targeted biological bacteria water-soluble fertilizer as described in claim 9.