A high-efficiency inoculation method for rice seedling-raising mycorrhizal fungi based on space-time coupling

Abstract

The application discloses a high-efficiency inoculation method of rice dry seedling mycorrhizal fungi based on space-time coupling, and relates to the field of rice dry seedling mycorrhizal fungi inoculation method. The application aims at solving the technical problems of the existing rice, which is difficult to utilize arbuscular mycorrhizal fungi and has poor root strength and poor structure stability. The application constructs a functional layer of fungicide with root tropism blocking function in the seedling tray, and cooperates with the implementation of critical water stress induction and waterlogging adaptive domestication process before transplanting, so that the rice root system completes high-abundance and deep-level mycorrhizal infection before the complete connection of the aeration organization. The composite network structure formed by the mycelium-root-substrate significantly enhances the root strength and structure stability of the seedling block, reduces the seedling loss rate in the mechanical transplanting process, realizes the synergistic integration of the microorganism growth promotion technology and the agricultural machinery and agronomy system, and the mycorrhizal infection rate in the root cortex of the rice seedling is still above 30% after the rice seedling is transplanted to the field waterlogging environment for 30 days. The application can be used in the field of agriculture.

Description

Technical Field

[0001] This invention relates to the fields of agricultural biotechnology and rice seedling cultivation engineering technology, specifically to a highly efficient inoculation method for mycorrhizal fungi in dry rice seedling cultivation. Background Technology

[0002] Rice is one of the most important food crops in my country and even the world. For a long time, in pursuit of high and stable yields, the rice production system has heavily relied on the input of chemical fertilizers, which has led to ecological problems such as soil degradation, reduced nutrient utilization efficiency, and agricultural non-point source pollution. Developing green alternative technologies centered on beneficial microorganisms is an important way to achieve a green transformation in rice production.

[0003] Arbuscular mycorrhizal fungi (AMF) are a class of beneficial fungi that can form a mutualistic symbiotic relationship with plant roots, playing an important role in promoting the absorption of mineral nutrients such as phosphorus, improving crop stress adaptability, and enhancing the rhizosphere microecology. However, AMFs are obligate aerobic microorganisms, and their spore germination and mycelial growth are highly dependent on soil oxygen supply. Because rice typically grows under prolonged flooding or periodic irrigation conditions in paddy fields, a common misconception exists that "AMFs cannot stably survive and function in rice systems," leading to slow progress in related research and application. Existing attempts to directly apply fungal agents to paddy fields often fail due to oxygen deficiency limiting spore germination or the inability of mycelia to establish effective connections with roots in a timely manner. Furthermore, with the continuous improvement of rice production mechanization, machine transplanting places higher demands on the root system strength and structural stability of seedlings. Under current seedling raising technology, seedling blocks are prone to scattering during seedling raising and mechanical handling, resulting in a higher rate of seedling loss. However, there is currently a lack of mature technical solutions that utilize microbial engineering to systematically improve the engineering performance of seedling blocks. Summary of the Invention

[0004] This invention aims to address the existing technical problems of difficulty in utilizing arbuscular mycorrhizal fungi in rice cultivation and the low root strength and poor structural stability of dry-seedling cultivation. It provides a highly efficient inoculation method for arbuscular mycorrhizal fungi in dry-seedling rice cultivation based on spatiotemporal coupling. This method utilizes the ecological window of good substrate aeration during the dry-seedling stage. By constructing a specific spatial structure and coordinating water regulation during the dry-seedling stage, a stable mycorrhizal symbiotic system is established before rice transplanting. This enables efficient and stable colonization of arbuscular mycorrhizal fungi in the rice root system, thereby overcoming the adverse effects of flooded environments on arbuscular mycorrhizal fungi colonization and simultaneously improving the engineering adaptability of seedlings, achieving stable colonization in dry-seedling cultivation.

[0005] The efficient inoculation method for mycorrhizal fungi in dry rice seedling raising based on spatiotemporal coupling of the present invention is carried out according to the following steps:

[0006] I. Pretreatment of inoculum source: Arbuscular mycorrhizal fungal host root segments obtained through propagation on host plants are selected and mixed with the carrier matrix to form a compound inoculum agent;

[0007] II. Construction of a root tropism-blocking functional layer: In a dry rice seedling tray, from bottom to top, a bottom soil layer 1, a compound microbial agent layer 2, a seed layer 3, and a covering soil layer 4 are laid in sequence; rice seeds are planted in the seed layer 3 with the rice seeds 1.0~2.0 cm away from the upper surface of the compound microbial agent layer 2; the compound microbial agent layer 2 is a microbial agent functional layer with root tropism-blocking function. The compound microbial agent layer 2 is set 1.0~2.0 cm directly below the rice seeds, so that the primary roots of rice must pass through the compound microbial agent layer 2 during their downward growth, thereby forming an inevitable infection zone on the root growth path;

[0008] III. Induction of critical water stress: During the 1-leaf-1-heart stage to the 2-leaf-1-heart stage of rice seedlings, the relative moisture content of the seedling substrate was controlled at 60%~70% for seedling raising; by inhibiting the rapid longitudinal escape growth of the root system and prolonging the residence time of the root hair zone in the functional layer of the fungal agent, high-frequency contact between the rice root system and mycorrhizal fungi was induced and cortical infection was completed.

[0009] IV. Adaptation to flooding: Gradually irrigate the rice seedlings 3-5 days before transplanting to harden them off and complete the efficient inoculation of mycorrhizal fungi into the rice dry-seedling nursery. During this stage, the established mycorrhizal symbiotic structure is utilized to allow the mycorrhizal fungi to gradually adapt to the low-oxygen environment before the rice aeration tissues are fully connected.

[0010] Furthermore, the arbuscular mycorrhizal fungi mentioned in step one are Funneliformismosseae and / or Rhizophagus irregularis.

[0011] Furthermore, the carrier matrix mentioned in step one is a sterile phosphorus-poor matrix formed by mixing river sand and vermiculite in a volume ratio of 1:1.

[0012] Furthermore, the effective viable spore density in the functional layer of the microbial agent described in step two is not less than 20 spores / g substrate to ensure the source of infection.

[0013] Furthermore, the thickness of the composite microbial agent layer 2 in step two is 0.5~0.8 cm, and the application rate is 250~300 g / m², so as to ensure the formation of a continuous and uninterrupted mycelial interception network within the limited space of the seedling tray.

[0014] Furthermore, the seedling substrate mentioned in step two is prepared by mixing peat moss, vermiculite, and decomposed straw substrate in a volume ratio of 3:1:1.

[0015] This invention presents a highly efficient inoculation method for mycorrhizal fungi in dry-seedling rice cultivation based on spatiotemporal coupling. It fully utilizes the favorable aeration conditions during the dry-seedling stage to construct a functional layer of fungal agents along the rice root growth path. Through the synergistic effect of temporal regulation and spatial interception, it forces the primary roots and mycorrhizal fungi to complete deep infection within the critical physiological window. An effective viable spore density of no less than 20 spores / g substrate in the functional layer ensures a reliable source of infection. Combined with the appropriate layering thickness and application rate, it achieves complete physical interception without dead zones. Furthermore, the use of a breathable and phosphorus-poor carrier substrate allows for the formation of a continuous extra-root mycelial network within a limited space. This mycelial-root-substrate composite network structure formed within the functional layer enhances the root coiling force of machine-transplanted rice seedlings, preventing scattering during mechanical handling and transport, thereby reducing the rate of missed seedlings during machine transplanting. Even 30 days after transplanting to a flooded field, the mycorrhizal infection rate in the root cortex of rice seedlings remains above 30%.

[0016] This invention overcomes the misconception that mycorrhizal fungal inoculation in dry-seedling rice cultivation is difficult to apply to rice systems, achieving stable colonization of mycorrhizal fungi before flooding; significantly improving the continuity and persistence of mycorrhizal infection after rice transplanting; enhancing seedling rooting capacity and reducing seedling leakage rate during machine transplanting through a mycelium-root-substrate composite network structure; and achieving deep integration of microbial growth promotion technology with agricultural machinery and agronomic systems, making it applicable to the agricultural field. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the structure of the substrate inside the rice dry-seedling tray in Example 1; where 1 is the bottom soil layer, 2 is the compound microbial agent layer, 3 is the seed layer, 4 is the covering soil layer, and 5 is the seed.

[0018] Figure 2 This is a photograph of the root mycorrhizal infection of rice seedlings in Example 1 30 days after transplanting them into a flooded field. Detailed Implementation

[0019] The beneficial effects of the present invention will be verified using the following examples.

[0020] Example 1: The efficient inoculation method of mycorrhizal fungi for dry rice seedling raising based on spatiotemporal coupling in this example is carried out according to the following steps:

[0021] I. Inoculum source pretreatment: Funneliformismosseae inoculum obtained through propagation on host plants was selected. The inoculum contains spores, foliar hyphae, and effective propagules infecting root segments. The inoculum was mixed with a carrier matrix to form a compound inoculum, wherein the effective viable spore density was 35 spores / g matrix. The carrier matrix was a sterile, phosphorus-poor matrix prepared by mixing river sand and vermiculite in a volume ratio of 1:1.

[0022] II. Construction of a Root-Taxis Interception Layer: In a standard 58 cm × 28 cm machine-transplanted rice tray, first lay a 2.0 cm thick seedling substrate as the subsoil layer 1, then lay a 0.5 cm thick layer of compound microbial agent 2, with an application rate of approximately 150 g / tray; next, lay a 1.5 cm thick seed layer 3, and plant pre-germinated rice seeds in the seed layer 3, with a sowing rate of 120 g / tray; the rice seeds should be 1.5 cm below the top surface of the compound microbial agent layer 2; finally, lay a 0.5 cm thick covering soil layer 4 on top of the seed layer 3 and level the surface; the compound microbial agent layer 2 is a functional layer with root-taxis interception function, and it is positioned 1.5 cm directly below the rice seeds. At a distance of cm, the primary roots of rice must pass through the composite microbial agent layer 2 during their downward growth, thus forming an inevitable infection zone along the root growth path; wherein the seedling substrate is prepared by mixing peat moss, vermiculite, and decomposed straw substrate in a volume ratio of 3:1:1.

[0023] III. Induction of critical water stress: During the first 15 days after rice emergence, i.e., from the 1-leaf-1-heart stage to the 2-leaf-1-heart stage, the relative moisture content of the seedling substrate is controlled at 65%, keeping the seedling substrate moist but not waterlogged. This is to inhibit the rapid longitudinal elongation of the root system and prolong the residence time of the root hair zone in the functional layer of the fungicide, thereby promoting full contact between the root system and arbuscular mycorrhizal fungi and cortical infection.

[0024] IV. Adaptation to Flooding: Four days before rice transplanting, the seedling trays are gradually flooded to harden the seedlings, allowing the rice roots, which have already formed a mycorrhizal symbiotic structure, to gradually adapt to the transition from an aerobic to a hypoxic environment. This lays the foundation for stable symbiosis under flooded conditions in the field after transplanting, and completes the efficient inoculation of mycorrhizal fungi into dry-seedling rice.

[0025] This embodiment takes the process of rice transplanting and seedling raising as the application object. Taking advantage of the ecological window period when the substrate has good aeration conditions during the dry seedling raising stage, by constructing a functional layer of microbial agents in the seedling tray and coordinating with water regulation, the rice roots can complete stable mycorrhizal infection before transplanting to the flooded environment of the field.

[0026] The rice seedlings obtained in this embodiment have tightly coiled root systems, making them less prone to scattering during machine transplanting and effectively reducing the seedling loss rate. The rice seedlings obtained in this embodiment do not scatter during mechanical handling and conveying, resulting in a seedling loss rate as low as 1.3% during machine transplanting.

[0027] The rice seedlings obtained in this embodiment, after being transplanted into a flooded field for 30 days, showed root mycorrhizal infection as shown in the following photograph. Figure 2 As shown, from Figure 2 It can be seen that the mycorrhizal infection rate in the root cortex remains at 32.4%, which is still relatively high.

[0028] Comparative Example 1: This comparative example differs from Example 1 in that the thickness of the composite microbial agent layer 2 in step two is 1.2 cm, and the application rate is 350 g / m². Other steps and parameters are the same as in Example 1.

[0029] The rice seedlings obtained in this comparative example showed reduced aeration due to the excessively thick inoculant layer, hindering root penetration and causing the roots to mainly concentrate above the inoculant layer. The root system exhibited poor compaction, resulting in decreased overall seedling integrity and localized breakage during mechanical handling. The machine-transplanted seedling leakage rate was 5.8%. Comparative Example 2: This comparative example differs from Example 1 in that the thickness of the composite inoculant layer 2 in step two was 0.3 cm, and the application rate was 100 g / m². Other steps and parameters were the same as in Example 1.

[0030] In this comparative example, due to insufficient thickness of the inoculant layer, a continuous barrier interface could not be formed, allowing some roots to bypass the inoculant layer and reduce the contact frequency. The root system showed moderate entanglement, resulting in poor stability of the seedling block structure and a 3.9% leakage rate during machine transplanting.

[0031] Comparative Example 3: This comparative example differs from Example 1 in that the compound microbial agent layer 2 is not set up during the seedling raising process. Instead, the same amount of compound microbial agent as in Example 1 is evenly mixed into the seed layer 3 so that the microbial agent is randomly distributed in the substrate. The remaining steps and parameters are the same as in Example 1.

[0032] I. Pretreatment of bacterial source: This step is the same as in Example 1;

[0033] 2. In a standard 58 cm × 28 cm transplanting tray, calculate the amount of seedling substrate needed for a 2.0 cm thick bottom soil layer 1. Then, calculate the amount of compound microbial agent needed for a 0.5 cm thick layer 2, with an application rate of approximately 150 g / tray. Next, calculate the amount of seedling substrate needed for a 1.5 cm thick seed layer 3. Mix the calculated seedling substrate and compound microbial agent evenly and spread them in the transplanting tray. Then, plant rice seeds that have undergone pre-germination treatment, with a sowing rate of 120 g / tray. Finally, spread a 0.5 cm thick covering soil layer 4 on top of the seed layer 3 and smooth the surface. The seedling substrate is prepared by mixing peat moss, vermiculite, and decomposed straw substrate in a volume ratio of 3:1:1.

[0034] III. Induction of Critical Moisture Stress: This step is the same as in Example 1;

[0035] IV. Adaptation to flooding: This step is the same as in Example 1; complete the inoculation of mycorrhizal fungi for dry rice seedling raising.

[0036] The rice seedlings obtained in this comparative example showed a random distribution of roots within the substrate, lacking a directional crossing process. The contact between the roots and the inoculant was highly accidental, failing to form a stable, high-frequency contact interface. The root clusters were significantly less compacted than in the example, resulting in poor overall seedling integrity. During mechanical handling and transport, the seedlings were prone to edge loosening or partial detachment, leading to a 4.6% seedling leakage rate during machine transplanting.

[0037] The rice seedlings obtained in this comparative example, after being transplanted to a flooded field for 30 days, showed a mycorrhizal infection rate of only 23.8% in the root cortex, significantly lower than the 32.4% in Example 1. This indicates that simply applying the inoculant uniformly is insufficient to achieve efficient infection and to construct a mycelium-root-substrate composite network structure, thus failing to effectively improve the structural stability of the seedling block.

Claims

1. A highly efficient inoculation method for mycorrhizal fungi in dry rice seedling raising based on spatiotemporal coupling, characterized in that, The method is performed in the following steps: I. Pretreatment of inoculum source: Arbuscular mycorrhizal fungal host root segments obtained through propagation on host plants are selected and mixed with the carrier matrix to form a compound inoculum agent; II. Construction of root tropism interception functional layer: In the dry rice seedling tray, from bottom to top, lay the bottom soil layer (1), the compound microbial agent layer (2), the seed layer (3) and the covering soil layer (4); plant the rice seeds in the seed layer (3) with the rice seeds 1.0~2.0 cm away from the upper surface of the compound microbial agent layer (2); III. Induction of critical water stress: During the rice seedling stage from 1 leaf and 1 heart to 2 leaves and 1 heart, the relative moisture content of the seedling substrate is controlled at 60% to 70% for seedling raising; IV. Adaptability to flooding: Gradually irrigate the seedlings 3-5 days before rice transplanting to harden them off and complete the efficient inoculation of mycorrhizal fungi into the rice dry-seedling nursery.

2. The efficient inoculation method for mycorrhizal fungi in dry rice seedling raising based on spatiotemporal coupling according to claim 1, characterized in that, The arbuscular mycorrhizal fungi mentioned in step one are *Funneliformis mosseae* and / or *Rhizophagus irregularis*.

3. A highly efficient inoculation method for mycorrhizal fungi in rice dry-seedling raising based on spatiotemporal coupling, as described in claim 1 or 2, is characterized in that... The carrier matrix mentioned in step one is a sterile phosphorus-poor matrix made by mixing river sand and vermiculite in a volume ratio of 1:

1.

4. A highly efficient inoculation method for mycorrhizal fungi in dry rice seedling raising based on spatiotemporal coupling, as described in claim 1 or 2, characterized in that, In step two, the effective live spore density in the functional layer of the microbial agent is not less than 20 spores / g substrate.

5. A highly efficient inoculation method for mycorrhizal fungi in dry rice seedling raising based on spatiotemporal coupling, as described in claim 1 or 2, characterized in that, The thickness of the composite microbial agent layer 2 mentioned in step two is 0.5~0.8 cm, and the application rate is 250~300 g / m².

6. A highly efficient inoculation method for mycorrhizal fungi in rice dry-seedling raising based on spatiotemporal coupling, as described in claim 1 or 2, characterized in that, The seedling substrate mentioned in step two is prepared by mixing peat moss, vermiculite, and decomposed straw in a volume ratio of 3:1:1.

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