Preparation method and application of insect-resistant orderly-released rice seedling raising substrate

Abstract

The invention relates to the field of rice seedling raising cultivation, and discloses a preparation method of a pest-resistant orderly-released rice seedling raising substrate, which comprises the following steps: (a) preparing a composite microcapsule which comprises a functional microorganism core and an intelligent response type shell coating the functional microorganism core; wherein a compound microbial flora and a quorum sensing signal precursor are loaded in the functionalized microbial core; the intelligent response type shell contains a plant immunity inducer, and the material of the shell is set to be degraded under the action of rice root exudates. By designing a composite microcapsule structure, programmed and targeted release of active substances is realized, so that a plant immunity inducer acts in advance in the early stage of seedling raising, and a composite microbial flora is accurately released to a rhizosphere area only when a rice root system is in contact with the microcapsule, so that the effect of raising seedlings is achieved. And the utilization efficiency and the action accuracy of each functional component are obviously improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of rice seedling cultivation, in particular to a preparation method and application of a rice seedling substrate capable of orderly releasing against insect pests. BACKGROUND

[0002] Rice seedling cultivation is a basic and key link in the intensive production of rice, and the seedling substrate is an important material basis for providing physical support, water and nutrients for seedling growth.

[0003] In the prior art, the functional components of the seedling substrate are often directly physically mixed. This method causes the release of active substances to be disordered, and the release timing thereof is difficult to accurately match the specific needs of seedlings at different growth stages, resulting in low utilization rate of effective ingredients. In terms of disease control, traditional methods often rely on external intervention. For example, chemical agents are used to sterilize the substrate, or antagonistic microorganisms are added to the substrate. These are passive defense means and cannot effectively induce the seedlings to establish systemic resistance, so the adaptability of the seedlings to the external environment stress is often limited. For directly added biological agents, there are also technical limitations. Microorganisms are directly exposed to the complex substrate environment, and face the competition of indigenous microorganisms and the pressure of environmental factors, and their survival rate and rhizosphere colonization efficiency are usually not high, and the stability of biological efficiency is difficult to guarantee. In addition, even if part of the beneficial microorganisms can colonize, their functional expression also lacks effective regulation mechanism, and the secretion level of antagonistic substances or growth-promoting factors is often basic, rather than responsive adjustment according to needs, which limits the full play of their biological potential. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a preparation method and application of a rice seedling substrate capable of orderly releasing against insect pests, aiming to solve the problems of single function, disordered release of active substances and unstable biological efficiency of the seedling substrate in the prior art.

[0005] To achieve the above object, the present application is implemented by the following technical scheme: The present application provides, in a first aspect, a preparation method of a rice seedling substrate capable of orderly releasing against insect pests, comprising the following steps: (a) preparing a composite microcapsule, wherein the composite microcapsule comprises a functionalized microorganism core and an intelligent response type shell covering the outside of the core; the functionalized microorganism core is loaded with a composite microbial community and a quorum sensing signal precursor; the intelligent response type shell contains a plant immune inducer, and the material of the shell is arranged to disintegrate under the action of rice root exudates; (b) mixing the composite microcapsule prepared in step (a) with a basic physical substrate to obtain the combined rice seedling substrate.

[0006] In one specific embodiment, the preparation of the functionalized microbial core is characterized in comprising: (1) preparing a chitosan-modified biochar carrier; (2) loading the complex microbial flora and the quorum-sensing signal precursor onto the chitosan-modified biochar carrier by adsorption.

[0007] In one specific embodiment, the complex microbial flora comprises at least two of Trichoderma harzianum, Bacillus amyloliquefaciens and arbuscular mycorrhizal fungi.

[0008] In one specific embodiment, the preparation of the intelligent responsive shell is characterized in comprising: (1) dissolving alginate in water and adding the plant immune inducer thereto to form an embedding liquid; (2) adding a crosslinking agent containing chemical bonds that can be degraded by specific enzymes or organic acids in rice root exudates to the embedding liquid; (3) dispersing the functionalized microbial core in the embedding liquid of step (2) and forming the intelligent responsive shell by solidification treatment.

[0009] In one specific embodiment, the plant immune inducer is methyl jasmonate.

[0010] In one specific embodiment, the matrix of the intelligent responsive shell is alginate hydrogel, and the degradable chemical bonds contained in the crosslinked network thereof are ester bonds that can be cleaved by esterases secreted by rice roots.

[0011] In one specific embodiment, humic acid potassium is also added in the step of mixing the complex microcapsules with the basic physical matrix in step (b).

[0012] The second aspect of the present application provides a combined rice seedling raising substrate prepared by the method of any one of the embodiments of the first aspect for use in rice seedling raising.

[0013] The present application has the following action mechanism through the above technical solution: The core of the present application is to construct a complex microcapsule with a programmed, multi-stage action mechanism. The microcapsule provides time-sequential, different functional material support for the growth of rice seedlings.

[0014] In the early stage of seedling raising, the intelligent responsive shell structure is intact, and the plant immune inducer contained therein is slowly released into the substrate to act on rice seeds and seedlings to induce systemic resistance of the plant.

[0015] As the seedling grows, its root system extends and contacts the composite microcapsules. The specific substances (esterases) secreted by the root system act on the preset ester bonds in the shell material, causing the shell structure to disintegrate in the rhizosphere region. This process allows the contents of the functionalized microbial core, i.e., the composite microbial flora and the quorum sensing signal precursor, to be released at the site of the root system.

[0016] The released composite microbial flora colonizes and proliferates in the rhizosphere region. When the density of the flora reaches a predetermined threshold, Bacillus amyloliquefaciens metabolizes the quorum sensing signal precursor to produce and release a quorum sensing signal molecule. This signal molecule regulates the expression of relevant functional genes in the composite microbial flora on the one hand, and can be recognized by the plant root system to maintain or enhance the systemic resistance established by the plant immune inducer in the early stage.

[0017] Through the above structure and steps, the present application organically combines the processes of plant immune induction, microbial rhizosphere colonization, and functional regulation of microbial population in time sequence and space through one carrier.

[0018] The present application provides a preparation method and application of a rice seedling raising substrate for insect pest resistance and orderly release. It has the following beneficial effects: 1. The present application realizes programmed and targeted release of active substances by designing a composite microcapsule structure. Unlike the unordered release caused by simple mixing of active components in the prior art, the present application uses an intelligent response shell to allow the plant immune inducer to act in the early stage of seedling raising, and the composite microbial flora is accurately released in the rhizosphere region when the rice root system contacts the microcapsules, significantly improving the utilization efficiency and action accuracy of each functional component.

[0019] 2. The present application establishes a dual synergistic defense system combining "endogenous induction" and "exogenous barrier". Unlike the single external sterilization or inhibition mode of traditional technology, the present application first activates the systemic resistance of the seedling itself through the plant immune inducer, and then constructs a biological protection barrier composed of composite microbial flora in the root system, the two mechanisms complement and enhance each other, providing more comprehensive and persistent disease protection for the seedling.

[0020] 3. The present application significantly improves the survival rate and colonization efficiency of beneficial microorganisms in the substrate. By loading the microorganisms in the functionalized core and coating them in the intelligent response shell, the contact of the microorganisms with unfavorable environmental factors in the substrate before colonization is effectively isolated; at the same time, the mechanism of rhizosphere site release ensures that the microorganisms are directly delivered to the target area with rich nutrients, thereby solving the problem of unstable efficiency of microbial inoculants due to competition and environmental stress in the prior art.

[0021] 4. By introducing quorum sensing signal precursors, this invention endows the rhizosphere biobarrier with self-regulating and enhanced functional properties. When the population density of colonizing microorganisms reaches a specific threshold, the quorum sensing mechanism is triggered, regulating the synthesis and secretion of antimicrobial substances, thereby maximizing the protective function of the biobarrier. This intelligent regulation mechanism based on microbial population density is unattainable through simple physical mixing.

[0022] 5. Through these multiple synergistic effects, the present invention helps cultivate stronger rice seedlings and reduces reliance on chemical pesticides. Rice seedlings cultivated using the present method exhibit higher survival rates and growth vitality due to their inherently strong stress resistance and healthy rhizosphere microecology. This not only improves rice seedling quality but also reduces the need for chemical fungicides in subsequent field management, resulting in favorable environmental benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the process of the present invention. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the present specification. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] Example: Example 1: Preparation of the Rice Seedling Raising Matrix with Orderly Release of Pest Resistance of the Present Invention 1.1. Preparation of composite microcapsules: (a) Preparation of chitosan-modified biochar functionalized carrier: Dried rice husks were pyrolyzed at 500°C for 2 hours under oxygen-limited conditions in a muffle furnace. After cooling, rice husk biochar was obtained. The biochar was then immersed in a 0.8 M hydrochloric acid solution and treated at 25°C for 18 hours. After treatment, the biochar was repeatedly rinsed with deionized water until the pH of the washings reached 7.0. The washed biochar was then oven-dried at 75°C. A 2.0% chitosan solution was prepared using a 1.5% acetic acid aqueous solution as the solvent. The dried biochar was added to the chitosan solution at a solid-to-liquid ratio of 1:10 (g / mL) and magnetically stirred at 30°C for 5 hours. Subsequently, 0.8 M sodium hydroxide solution was slowly added dropwise to adjust the pH of the mixture to 7.5, allowing chitosan to precipitate on the biochar. Finally, the product was washed with deionized water until neutral and oven-dried at 60°C to obtain a chitosan-modified biochar functionalized support.

[0026] (b) Preparation of functionalized microbial core: After liquid activation culture of Trichoderma harzianum and Bacillus amyloliquefaciens, they were mixed at a volume ratio of 1:1, and spore agent of C. moseleyi was added to adjust the final viable cell (spore) count of the mixed microbial suspension to 5x10 9 CFU / mL. N-acyl homoserine lactone (AHL) precursor was dissolved in the suspension to a final concentration of 30 µM. The functionalized carrier prepared in step (a) was mixed with the microbial suspension at a solid-liquid ratio (g / mL) of 1:4, and placed in a vacuum drying oven, maintaining a vacuum degree of -0.07 MPa for 30 minutes. The saturated carrier was pre-frozen at -80°C for 5 hours, followed by vacuum freeze-drying to obtain the functionalized microbial core.

[0027] (c) Construction of intelligent responsive shell and microencapsulation: A 2.0% (mass fraction) aqueous solution of sodium alginate was prepared. To the solution, methyl jasmonate (MeJA) and Tween-80 were added to a final volume fraction of 0.15% and 0.2%, respectively, and high-speed stirring was performed to form a uniform emulsion. Subsequently, polyethylene glycol diacrylate (PEGDA, Mn=700) was added to the emulsion to a final mass fraction of 0.3%. The functionalized microbial core particles prepared in step (b) were uniformly dispersed in the embedding solution. Using a peristaltic pump with a 1.0 mm diameter nozzle, the mixed solution was dropped into a 1.0 M calcium chloride solidification solution for 30 minutes. The formed microcapsules were removed, washed with sterile deionized water, and air-dried in a 25°C fume hood to obtain composite microcapsules with an average particle size of about 3.0 mm.

[0028] 1.2. Final preparation of combined seedling raising substrate: (a) Preparation of basic physical substrate: Coconut coir, rice husk charcoal, and vermiculite were mixed at a volume ratio of 5:3:2. The mixed substrate was subjected to steam sterilization at 110°C for 25 minutes, and cooled to room temperature for standby.

[0029] (b) Final mixing: The composite microcapsules prepared in step 1.1 and potassium humate powder were added to the sterilized basic substrate of step (a) at a ratio of 2.0% and 0.2% of the total dry weight of the substrate, respectively. A horizontal ribbon mixer was used for low-speed stirring for 15 minutes to ensure uniform distribution of all components, and the final product was packaged and sealed.

[0030] Comparative Example 1: Compared with Example 1, the difference is that only the basic physical substrate prepared in step 1.2(a) of Example 1 is used, without adding any composite microcapsules or potassium humate.

[0031] Comparative Example 2: The difference compared with Example 1 is that the step of preparing the composite microcapsules in step 1.1 is not performed, but all active components (including functionalized carriers, microorganisms, inducers, signal precursors) used for preparing the composite microcapsules in Example 1 are directly physically mixed with the basic physical matrix, and the rest are the same.

[0032] Comparative Example 3: The difference compared with Example 1 is that in step 1.1(c), methyl jasmonate (MeJA) is not added in the alginate embedding solution used for preparing the smart response shell, and the rest are the same.

[0033] Comparative Example 4: The difference compared with Example 1 is that in step 1.1(c), polyethylene glycol diacrylate (PEGDA) is not added in the alginate embedding solution used for preparing the smart response shell, and the rest are the same.

[0034] Comparative Example 5: The difference compared with Example 1 is that in step 1.1(b), N-acyl homoserine lactone (AHL) precursors are not added to the composite microorganism suspension when preparing the functionalized microorganism core, and the rest are the same.

[0035] Test Example 1: Seedling growth effect and disease resistance test 1. Test steps Six groups of seedling raising substrates prepared in Example 1 and Comparative Examples 1-5 were taken, and three replicates were set for each group. The substrates in each group were respectively loaded into standard seedling raising trays. Full and uniform rice seeds were selected, and after germination, they were uniformly sown. The seedling raising trays were placed in an artificial climate chamber for cultivation, and the controlled conditions were: temperature 28±2℃, relative humidity 75±5%, light / dark cycle 14 hours / 10 hours.

[0036] (a) Growth index determination: On the 25th day after sowing, 30 seedlings with uniform growth were randomly selected from each replicate of the seedling raising tray. The roots were washed with deionized water to remove the attached substrate, and the surface water was absorbed. A graduated ruler was used to measure the plant height and root length. Then the aboveground and underground parts of the seedlings were separated and placed in an oven at 105℃ for 30 minutes, and then dried at 75℃ to constant weight. The vigorous seedling index was calculated according to the formula "vigorous seedling index=(underground dry weight / aboveground dry weight)×whole plant dry weight".

[0037] (b) Disease resistance test: On the 20th day after sowing, the rice bakanae disease pathogen (Fusarium fujikuroi) was prepared into a concentration of 1×10 6A spore suspension of 1 x 105spores / mL was prepared. The seedlings in each group were sprayed with the spore suspension using a hand-held sprayer until the leaves were covered with fine droplets. After inoculation, the seedling trays were moved back to the original culture conditions for further cultivation for 7 days. On the 7th day after inoculation, the disease incidence of each treatment group was counted, and the disease index was evaluated according to the following grading standard: 0: No visible symptoms on the plants.

[0038] 1: Slight yellowing of the base or heart leaves of the plants, with slight overgrowth.

[0039] 2: Obvious overgrowth of the plants, with yellowing of the leaves.

[0040] 3: Severe overgrowth of the plants, with fine and weak yellowing, or base rot.

[0041] 4: Plant death.

[0042] The corresponding indexes were calculated according to the formula "disease incidence (%) = (number of diseased plants / total number of plants surveyed) x 100" and "disease index = [∑(number of plants at each level x corresponding disease level) / (total number of plants surveyed x 4)] x 100".

[0043] 2. Test data The data obtained from the above determination and calculation were recorded in Table 1.

[0044] Table 1 - Test results of seedling growth and disease resistance of Example 1 and Comparative Examples 1-5 As can be seen from the data in Table 1, compared with Comparative Example 1 containing no active ingredients and Comparative Example 2 in which the active ingredients were simply physically mixed, the substrate prepared in Example 1 of the present application had increased growth indexes such as plant height, root length and dry weight, higher seedling index, and significantly reduced disease incidence and disease index. This indicates that the composite microcapsule structure constructed in the present application can more effectively promote seedling growth and inhibit disease occurrence compared with no active ingredients or simple mixing of active ingredients, and the reason is that the structure provides protection for each active component and achieves ordered release.

[0045] Comparing the results of Comparative Example 1 with Comparative Examples 3 and 4, it can be observed that Comparative Example 3 lacking a plant immune inducer had a higher disease index than Example 1, and Comparative Example 4 lacking a rhizosphere-responsive release mechanism had inferior growth indexes and disease resistance indexes to Example 1. This indicates that the combination of the plant immune inducer and the rhizosphere-responsive release mechanism in the intelligent response shell is an important part of achieving the desired effect. The former induces the systemic resistance of the plants in advance, and the latter ensures that the microbial flora is accurately released at the action site during the critical period of root colonization.

[0046] Comparing the results of Example 1 and Comparative Example 5, the disease index of Comparative Example 5, which lacks the quorum sensing signal precursor, is higher than that of Example 1. This indicates that after the complex microbial flora successfully colonizes, the quorum sensing mechanism mediated by the quorum sensing signal precursor can further enhance the disease inhibition function of the microbial community, effectively supplementing and strengthening the function of the entire cooperative defense system.

[0047] While embodiments of the present application have been shown and described, it is to be understood that the embodiments described are merely divergences of the principles and spirit of the present application and that numerous modifications, changes, substitutions, and alterations can be made thereto without departing from the spirit and scope of the present application as defined by the appended claims and their equivalents.

Claims

1. A method for preparing a rice seedling raising substrate with orderly release of pest resistance, characterized in that: The following steps are involved: (a) preparing a composite microcapsule comprising a functionalized microbial core and an intelligent responsive shell encapsulating the core; The functionalized microbial core is loaded with a complex microbial flora and a quorum sensing signal precursor; the intelligent responsive shell contains a plant immune inducer, and the shell material is configured to degrade under the action of rice root secretions; (b) mixing the composite microcapsules obtained in step (a) with a basic physical matrix to obtain the combined rice seedling raising matrix.

2. The method for preparing a rice seedling raising substrate with orderly release of insect pests according to claim 1, characterized in that: The functionalized microbial core comprises a carrier, and the carrier is chitosan-modified biochar.

3. The method for preparing a rice seedling raising substrate with orderly release of pest resistance according to claim 1, characterized in that: The complex microbial flora includes at least two of Trichoderma harzianum, Bacillus amyloliquefaciens and arbuscular mycorrhizal fungi.

4. The method for preparing a rice seedling raising substrate with orderly release of pest resistance according to claim 1, characterized in that: The matrix of the intelligent responsive shell is alginate hydrogel.

5. The method for preparing a rice seedling raising substrate with orderly release of pest resistance according to claim 1, characterized in that: The plant immunity inducer is methyl jasmonate.

6. The method for preparing a rice seedling raising substrate with orderly release of pest resistance according to claim 1, characterized in that: The alginate hydrogel contains chemical bonds that can be degraded by specific enzymes or organic acids in rice root exudates.

7. The method for preparing a rice seedling raising substrate with orderly release of pest resistance according to claim 2, characterized in that: The step of preparing the functionalized microbial core in step (a) comprises: loading the composite microbial flora and the quorum sensing signal precursor onto the chitosan-modified biochar carrier by adsorption.

8. The method for preparing a rice seedling raising substrate with orderly release of pest resistance according to claim 7, characterized in that: The step of preparing the composite microcapsule in step (a) further includes: using an alginate solution containing the plant immunity inducer to embed the functionalized microbial core to form the intelligent responsive shell.

9. The method for preparing a rice seedling raising substrate with orderly release of pest resistance according to claim 1, characterized in that: In the step (b) of mixing the composite microcapsules with the basic physical matrix, potassium humate is also added.

10. Use of a rice seedling raising substrate with orderly release of insect pest resistance prepared by the method of claim 1 in rice seedling raising.

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