A new seedling raising substrate using plant root stubs as AM fungus inoculum, and a preparation method and application thereof

Abstract

This invention discloses a novel seedling substrate using plant root stubble as an AM fungal inoculant, its preparation method, and its application. The seedling substrate comprises plant root stubble; the plant root stubble is plant root system infected by arbuscular mycorrhizal fungi (AM fungi); the plant root stubble is derived from alfalfa and plantain. This invention is the first to directly use AM fungal-infected plant root stubble as an AM fungal inoculant in a seedling substrate. This seedling substrate is suitable for seedling planting projects in the field of ecological restoration. It can mainly increase the mycorrhizal fungal infection rate of plants, regulate plant root growth, promote nutrient absorption, enhance plant drought resistance, and enhance plant resistance to pathogens. It plays a particularly important role in seedling planting applications in ecological restoration projects with difficult site conditions.

Description

Technical Field

[0001] This invention belongs to the field of seedling substrate technology, and in particular relates to a novel seedling substrate using plant root stubble as AM fungal inoculant, its preparation method and application. Background Technology

[0002] Seedling substrate is the foundation of the seedling cultivation industry. Ecological restoration research has gradually become a hot topic, driving the rapid development of the seedling cultivation industry. Seedling substrate is the "chip" of seedling growth; its quality directly determines seedling survival rate, seedling resistance, and subsequent production efficiency. Traditional seedling substrates use peat as the core organic component, combined with inorganic minerals such as perlite and vermiculite, supplemented with natural materials such as garden soil, leaf mold, and compost. The main components of the seedling substrate primarily supplement nutrients and enhance fertility, rarely playing a role in strengthening plant resistance.

[0003] Arbuscular mycorrhizal fungi (AM fungi) are a type of soil fungi that can form a mutualistic symbiotic relationship with plant roots. They are core functional microorganisms in the rhizosphere micro-ecosystem of seedlings and play a prominent role in container seedling cultivation and ecological restoration. AM fungi mainly infect seedling roots, helping plants absorb nutrients, promoting root development, and enhancing plant resistance to stress and disease. They are key microbial resources for improving the quality of container seedlings, increasing transplant survival rates, and achieving green and efficient seedling cultivation, and can enhance the function of modern seedling substrates. More than 80% of terrestrial plants can form a symbiotic relationship with AM fungi. Infected plant roots are natural propagules of AM fungi. Collecting and drying the roots and then using them as AM fungal inoculants is a common method for AM fungal propagation, but few studies have directly applied it to production practice. Alfalfa and plantain are common perennial herbaceous plants in nature, characterized by high yield and rapid growth. Both are mycorrhizal infestation plants, with wild alfalfa and plantain root mycorrhizal infection frequencies reaching up to 98%, and infection intensity reaching approximately 80%. Collecting their root stubble is one way to recycle resources. Applying root stubble directly to seedling substrates is beneficial because infected root segments have the potential for AM fungal reinfection, allowing them to form a new symbiotic relationship with seedling roots and enhance seedling resistance. Furthermore, root stubble, as a seedling substrate additive, serves as a microbial carbon source, promoting microbial activity and enhancing nutrient biochemical processes within the substrate. Finally, adding root stubble can also increase the aeration of the seedling substrate to some extent. In conclusion, adding plant root stubble to seedling substrates can strengthen the substrate's function, thereby improving the survival rate of seedlings in ecological restoration projects.

[0004] Existing alternative seedling substrates are insufficient to meet the diverse needs of seedling cultivation. Traditional seedling substrates generally suffer from poor physical properties, such as insufficient aeration and porosity, imbalanced water and fertilizer retention, and functional deficiencies, particularly in their lack of ability to enhance seedling resilience. This is incompatible with the seedling needs in challenging site scenarios in ecological restoration. Seedlings cultivated in actual container substrates often exhibit poor root development and weak resilience, easily resulting in weak seedlings, which in turn increases management costs and subsequent replanting costs. With the national intensive seedling cultivation capacity required to exceed 300 billion seedlings by 2027, the demand for lightweight, functional, and low-cost substrates is even more urgent. However, existing technologies have not yet formed a mature solution that balances resource recycling, ecological protection, and seedling efficiency.

[0005] Therefore, developing a new type of seedling substrate using plant root stubble as an AM fungal inoculant can alleviate the long-standing problem of traditional seedling cultivation relying on non-renewable resources such as peat and coconut coir to a certain extent, and realize the high-value recycling of natural plant resources. It can also improve seedling quality and planting efficiency by optimizing the physical structure and nutrient composition of the substrate. This has important practical significance and application value for promoting the transformation and upgrading of my country's seedling industry and ensuring ecological security. Summary of the Invention

[0006] In view of this, the present invention aims to provide a novel seedling substrate using plant root stubble as AM fungal inoculant, its preparation method and application, in order to solve at least one technical problem in the background art.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0008] A novel seedling substrate using plant root stubble as an AM fungal inoculant, wherein the seedling substrate comprises plant root stubble; the plant root stubble is plant root system infected by AM fungi; the plant root stubble is derived from alfalfa and plantain.

[0009] Furthermore, the amount of plant root stubble added to the seedling substrate is 10 g per planting hole, or 2 g per liter of culture medium.

[0010] The structure, morphology, and infection intensity of AM fungi in the plant roots vary significantly across different growth and development stages. In the early infection stage (seedling stage), the AM fungal infection intensity in the plant roots is low, approximately 10%–20%, with AM fungi primarily existing as root hyphae and spores. In the middle infection stage (vigorous growth stage), the AM fungal infection intensity in the plant roots increases rapidly, reaching approximately 20%–55%, with AM fungi again primarily existing as root hyphae and arbuscular structures. In the late infection stage (mature stage), the AM fungal infection intensity in the plant roots remains relatively stable, with some AM fungal structures beginning to degenerate and decompose; the infection intensity reaches approximately 55%–80%, with AM fungi again primarily existing as root hyphae, vesicles, and spores. The root stubble collected in this application was collected during the later stage of the symbiotic relationship between the plant and AM fungi (collection time was September to October). At this time, the AM fungal infection intensity of the plant roots was relatively high, about 55% to 80%. The root stubble mainly contained mature vesicles, mature spores, and mycelia within the roots. These AM fungi have strong morphological and structural reinfection capabilities and have the potential to be used as AM fungal inoculants.

[0011] The aforementioned new seedling substrate also includes peat, vermiculite, and perlite, with a volume ratio of 2~4:1:10~12;

[0012] Alternatively, a mixture of well-rotted coconut coir, perlite, and garden soil in a volume ratio of 1-3:1:0.5-1.5;

[0013] Or peat moss, vermiculite, and river sand, with a volume ratio of 1~3:1:0.5~1.5.

[0014] A novel seedling substrate preparation method using plant root stubble as an AM fungal inoculant includes the following steps:

[0015] S1: Collect plant roots infected with AM fungus and air dry them;

[0016] S2: Cut the air-dried plant roots into small pieces to obtain plant root stubble.

[0017] S3: Mix the plant root stubble obtained in step S2 with the seedling base substrate evenly to obtain the seedling substrate.

[0018] Furthermore, the plant roots mentioned in step S1 are collected in September or October under natural growth conditions. At this time, the infection intensity of AM fungi in the plant roots is at a high level. In the mixed root stubble of alfalfa and plantain, naturally growing AM fungi plants can be infected by multiple AM ​​fungi simultaneously. The mixed plant root stubble contains a mixed inoculum of multiple AM ​​fungi.

[0019] To collect plant roots infected with arbuscular mycorrhizal fungi, the soil should be shaken off, the roots kept intact, and allowed to air dry naturally without sterilization, so as to ensure that the AM fungi in the roots have the ability to reinfect the plant.

[0020] The mixed root stubble of alfalfa and plantain, naturally growing AM fungal plants can be infected by multiple AM ​​fungi simultaneously, and the mixed plant root stubble contains a mixed inoculum of multiple AM ​​fungi.

[0021] Furthermore, the plant roots should be cut into segments of 3-5 cm in length.

[0022] The above-mentioned seedling substrates are used to promote plant growth or enhance plant resistance.

[0023] Furthermore, the plant in question is either Haloxylon ammodendron or corn.

[0024] The above-mentioned seedling substrates are used in ecological restoration or crop cultivation.

[0025] Furthermore, the plant in question is either Haloxylon ammodendron or corn, both of which form a symbiotic relationship with AM fungi.

[0026] A method for planting seedlings involves adding the above-mentioned seedling substrate to the planting pit, mixing it evenly with the soil, and then transplanting bare-root seedlings.

[0027] Compared with existing technologies, the novel seedling substrate using plant root stubble as AM fungal inoculant, its preparation method, and its application described in this invention have the following advantages:

[0028] 1. This invention is the first to use plant root stubble infected with AM fungi directly as AM fungal inoculant for seedling substrate, realizing the high-value recycling of natural plant resources and alleviating the dependence of traditional seedling cultivation on non-renewable resources such as peat and coconut coir.

[0029] 2. The plant root stubble in the seedling substrate of this invention can, on the one hand, serve as an AM fungal agent, forming a new symbiotic relationship with the seedling roots and enhancing the seedling resistance; on the other hand, it can serve as a microbial carbon source, promoting the activity of microorganisms in the substrate and enhancing the biochemical processes of nutrients in the substrate; at the same time, it can also increase the air permeability of the seedling substrate.

[0030] 3. Experiments have shown that after two months of planting *Haloxylon ammodendron* using the seedling substrate of this invention (alfalfa root stubble and plantain root stubble), the survival rate reached 95%, the plant height reached 44.2 cm, the crown width reached 46.7 cm, and the number of lateral branches reached 30. This is significantly better than the blank control group and the control group with only alfalfa stems and leaves and alfalfa root stubble added, and also higher than the control group with only alfalfa root stubble added and only plantain root stubble added. After five months of planting, the plant height, crown width, number of lateral branches, and maximum branch length of *Haloxylon ammodendron* increased by 13%, 23%, 60%, and 19% respectively compared with the blank control group. Under wild growing conditions, the more lateral branches and the longer the branches of *Haloxylon ammodendron*, the greater its ability to resist harsh conditions such as drought, wind and sand, and severe cold, and the stronger the plant resistance. In maize planting, the seedling substrate of this invention increased the fresh weight and dry weight of maize by 93% and 71% respectively, the mycorrhizal infection intensity of maize roots reached 37.1%, and the abundance of tufted branches reached 15.3%, while the blank control group had 0 in both aspects. The greater the dry weight of corn, the more dry matter the plant accumulates, the greater its ability to resist external stresses and pests, and the stronger its resistance. These results indicate that the seedling substrate of this invention can significantly promote plant growth and enhance plant resistance. Attached Figure Description

[0031] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0032] Figure 1 This is a side view of the field experimental site of the present invention (the blue line from left to right represents the distribution of planting pits for the experimental seedlings).

[0033] Figure 2 The growth diagram of Haloxylon ammodendron in this invention is shown (A is the blank control group, B is the control group 3, and C is the experimental group). Detailed Implementation

[0034] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0035] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0036] Example 1: A seedling substrate containing a mixture of alfalfa and plantain root stubble can promote the growth of Haloxylon ammodendron.

[0037] Alfalfa and plantain grown in the wild are collected in September and October. At this time, alfalfa and plantain are in the late stage of growth, and the infection of AM fungi in the roots reaches its peak. The infected root segments at this time have a high infection potential and can be reused as AM fungal inoculants. The specific operation is to dig out the alfalfa and plantain with a shovel, excavating the root system to a depth of about 0-30 cm. The plant is divided into two parts from the rootstock: one part is the above-ground stem and leaves, and the other part is the underground root system. The soil on the root stubble is shaken off manually. The above-ground parts and roots of alfalfa are retained separately, while only the roots of plantain are retained for later use. The collected above-ground stems, leaves, and roots were brought back to the laboratory and air-dried. The stems, leaves, and roots were then cut into pieces of about 3-5 cm using scissors and used to prepare seedling additives. Alfalfa stems and leaves and alfalfa root stubble were mixed at a mass ratio of 1:1 (i.e., 5 g of stems and leaves and 5 g of root stubble, totaling 10 g) for control group 3; and alfalfa root stubble and plantain root stubble were mixed at a mass ratio of 1:1 (i.e., 5 g of root stubble and 5 g of root stubble, totaling 10 g) for experimental group.

[0038] An in-situ field monitoring experiment was set up in the ecological restoration project area of ​​Urad Rear Banner, Xilingol League, Inner Mongolia, with geographical coordinates: 106.536117°E, 41.944197°N. The experiment included four control groups and one experimental group. Haloxylon ammodendron was planted on April 20th. The blank control group received standard planting of bare-root Haloxylon ammodendron seedlings without any added substances to the planting pits. Control group 1 received planting pits with only 10 g of alfalfa root stubble added; control group 2 received planting pits with only 10 g of plantain root stubble added; and control group 3 received planting pits with a mixture of stems, leaves, and root stubble (alfalfa stems and leaves: alfalfa root stubble mass ratio of 1:1). The experimental group received planting pits with 10 g of root stubble mixture (alfalfa root stubble and plantain root stubble mass ratio of 1:1). After the additives were thoroughly mixed with the soil in the planting pits, bare-root Haloxylon ammodendron seedlings were transplanted. Each treatment was replicated four times, with 15 planting pits selected for each replicate. After successful transplantation, the plants grew naturally for two months. The first measurement and data recording were conducted at the end of June. The main measurement indicators were the survival rate, plant height, branching status, and branching level of the Haloxylon ammodendron.

[0039] from Figure 2It is evident that the experimental group of *Haloxylon ammodendron* showed better growth and a greater number of branches. Table 1 shows that the mixture of alfalfa and plantain root stubble significantly improved the survival rate of *Haloxylon ammodendron*, and also increased plant height, crown width, and the number of lateral branches. Compared with the blank control group, the survival rate of the experimental group increased by 15%, and the plant height, crown width, and number of lateral branches increased by 13.3 cm, 20.4 cm, and 13, respectively, representing increases of 43%, 78%, and 76%. Compared with control group 1, the survival rate of the experimental group increased by 3%, and the plant height, crown width, and number of lateral branches increased by 1.1 cm, 5.2 cm, and 3, respectively, representing increases of 3%, 13%, and 11%. Compared with control group 2, the survival rate of the experimental group increased by 4%, and the plant height, crown width, and number of lateral branches increased by 1.5 cm, 3.7 cm, and 5, respectively, representing increases of 4%, 9%, and 20%. Compared with control group 3, the survival rate of the experimental group increased by 2%, and the plant height, crown width, and number of lateral branches increased by 1.7 cm, 3.4 cm, and 13.4 cm, respectively. The increases in crown width and lateral branches were 4%, 8%, and 7%, respectively; this indicates that the addition of alfalfa and plantain root stubble mixture to the experimental group did indeed promote the growth of Haloxylon ammodendron, and the increase in crown width and lateral branches reflects the enhanced ability to resist wind and sand.

[0040] Table 1. Survival rate and growth of bare-root Haloxylon ammodendron seedlings two months after transplantation.

[0041] Experimental treatment Survival rate (%) Plant height (cm) Crown width (cm) Number of lateral branches of the plant (individual) Blank control group 80 30.9 26.3 17 Control group 1 92 43.1 41.5 27 Control group 2 91 42.7 43.0 25 Control group 3 93 42.5 43.3 28 experimental group 95 44.2 46.7 30

[0042] Example 2: Adding a mixture of alfalfa and plantain root stubble to the seedling substrate can enhance the resistance of Haloxylon ammodendron.

[0043] The experimental design was the same as above. Five months after transplanting bare-root seedlings (end of September), the height, crown width, number of lateral branches, and maximum branch length of *Haloxylon ammodendron* were recorded and monitored. Table 2 shows that the mixture of alfalfa and plantain root stubble significantly increased plant height, crown width, number of lateral branches, and maximum branch length. Compared with the blank control group, the experimental group showed increases of 5.6 cm, 10.8 cm, 15 lateral branches, and 3.1 cm in plant height, crown width, number of lateral branches, and maximum branch length, respectively, representing increases of 13%, 23%, 60%, and 19%. Compared with control 1, the experimental group showed increases of 4.3 cm, 10.7 cm, 10 lateral branches, and 2.1 cm in plant height, crown width, number of lateral branches, and maximum branch length, respectively, representing increases of 10%, 22%, 33%, and 12%. Compared with control 2, the experimental group showed increases of 4.8 cm, 10.8 cm, 12 lateral branches, and 2.5 cm in plant height, crown width, number of lateral branches, and maximum branch length, respectively. The height, crown width, number of lateral branches, and maximum branch length of the experimental group of Haloxylon ammodendron increased by 3.4 cm, 7.6 cm, 13, and 1.4 cm, respectively, with increases of 8%, 15%, 39%, and 8%. The increase in crown width and number of lateral branches indirectly reflects the increase in plant resistance, indicating that the root-stubble mixture can indeed enhance seedling resistance and promote plant growth.

[0044] Table 2. Growth of transplanted bare-root Haloxylon ammodendron seedlings five months later.

[0045] Experimental treatment Plant height (cm) Crown width (cm) Number of lateral branches of the plant Maximum branch length (cm) Blank control group 42.8 47.9 25 16.2 Control group 1 44.1 48.0 30 17.2 Control group 2 43.6 47.9 28 16.8 Control group 3 45.0 51.1 33 17.9 experimental group 48.4 58.7 40 19.3

[0046] Example 3: Adding a mixture of alfalfa and plantain root stubble to the seedling substrate can promote corn growth.

[0047] In the indoor experiments, a mixture of zeolite and quartz sand was used as the culture medium. The zeolite particle size was 0.8–1.2 mm, and the quartz sand particle size was 1.0–2.0 mm. They were thoroughly mixed at a volume ratio of 1:1. Before the experiment, both the zeolite and quartz sand mixtures were dried at 105℃ for 2 hours to remove moisture and impurities. Seedling trays were used as the culture vessels. The blank control group did not have any stems, leaves, or root stubble added to its culture medium. Control group 1 was supplemented with 2 g of a mixture of air-dried alfalfa stems, leaves, and root stubble. The experimental group was supplemented with 2 g of a mixture of air-dried alfalfa root stubble and plantain root stubble. Corn was planted in the culture medium and cultured for 4 weeks under suitable moisture and light conditions. During the culture period, 10% Hogland nutrient solution (10 ml) was applied once a week to promote maceration. After 4 weeks of culture, the plant dry weight, fresh weight, and root weight were measured and the data were recorded. The results showed that, compared with the blank control, the growth of maize in the experimental group was promoted, with fresh weight and dry weight increasing by 93% and 71%, respectively (Table 3). This indicates that the seedling substrate with added root stubble mixture can promote plant growth. Compared with the blank control, the infection intensity and arbuscular abundance of mycorrhizal fungi in the experimental group of maize increased from no infection to 37.1% and 15.3%, respectively (Table 4). This indicates that the function of beneficial microorganisms was enhanced, and AM fungi help plants resist biotic and abiotic stresses, indirectly enhancing plant resistance and promoting plant growth.

[0048] Table 3 Fresh and dry weight of maize plants after 4 weeks of cultivation.

[0049] Experimental treatment Fresh weight of corn (g) Dry weight of corn (g) Blank control group 8.11 0.79 Control group 1 14.50 1.26 experimental group 15.68 1.35

[0050] Table 4. Intensity of mycorrhizal fungal infection and abundance of maize rhizomes after 4 weeks of maize culture.

[0051] Experimental treatment Mycorrhizal infection intensity (%) Clump abundance (%) Blank control group 0 0 Control group 1 30.6 14.8 experimental group 37.1 15.3

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A novel seedling substrate using plant root stubble as an AM fungal inoculant, characterized in that: The seedling substrate contains plant root stubble; the plant root stubble is plant root system infected with AM fungus; the plant root stubble is derived from alfalfa and plantain.

2. The novel seedling substrate using plant root stubble as AM fungal inoculant according to claim 1, characterized in that: The amount of plant root stubble added to the seedling substrate is 10 g per planting hole, or 2 g per liter of culture substrate.

3. The method for preparing a novel seedling substrate using plant root stubble as an AM fungal inoculant, as described in claim 1 or 2, is characterized in that: Includes the following steps: S1: Collect plant roots infected with AM fungus and air dry them; S2: Cut the dried plant roots into small pieces to obtain plant root stubble; S3: Mix the plant root stubble obtained in step S2 with the seedling base substrate evenly to obtain the seedling substrate.

4. The method for preparing a novel seedling substrate using plant root stubble as AM fungal inoculant according to claim 3, characterized in that: The plant roots described in step S1 are collected in September or October under natural growth conditions.

5. The method for preparing a novel seedling substrate using plant root stubble as AM fungal inoculant according to claim 3, characterized in that: Plant roots should be cut into segments 3-5cm in length.

6. The use of the seedling substrate according to any one of claims 1 to 3 in promoting plant growth or enhancing plant resistance.

7. The application according to claim 6, characterized in that: The plant in question is either Haloxylon ammodendron or corn.

8. The application of the seedling substrate according to any one of claims 1 to 3 in ecological restoration or crop cultivation.

9. The application according to claim 8, characterized in that: The plants mentioned are Haloxylon ammodendron or corn, both of which form a symbiotic relationship with AM fungi.

10. A method for planting seedlings, characterized in that, Add the seedling substrate as described in any one of claims 1 to 2 to the planting pit, mix it evenly with the soil, and then transplant the bare-root seedlings.

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