Method for promoting spore production and propagation of arbuscular mycorrhizal fungi

Abstract

The invention belongs to the technical field of fungus propagation, and relates to a method for promoting spore production and propagation of arbuscular mycorrhizal fungi, which comprises the following steps: inoculating the arbuscular mycorrhizal fungi into a matrix, sowing host seeds in the matrix, carrying out normal maintenance, carrying out stress treatment after the host seeds are sown for 5-7 weeks, and collecting a sample after the culture period is ended, the stress treatment is drought stress treatment and / or nutrition stress treatment. According to the method for promoting spore production and propagation of the arbuscular mycorrhizal fungi, spore production can be effectively, efficiently and rapidly carried out, industrial limitation is broken through, and theoretical and experimental foundations are laid for solving the problems of long propagation period, high cost and the like of the AM fungi and rapid propagation of the AM fungi; the technical barrier of traditional fungicide production is broken through, the environmental response mechanism of a mycorrhizal symbiotic system is revealed from the level of ecology, and a theoretical basis is provided for assisting application of arbuscular mycorrhizal fungi in agriculture and achieving green development of agricultural production.

Description

Technical Field

[0001] This invention belongs to the field of fungal propagation technology and relates to a method for promoting the sporulation and propagation of arbuscular mycorrhizal fungi. Background Technology

[0002] Arbuscular mycorrhiza (AM) fungi are a key component of the plant rhizosphere microbiota, forming symbiotic relationships with 80% of terrestrial plants. They are a widespread symbiotic relationship between terrestrial plants and AM fungi. AM fungi are primarily composed of long, filamentous hyphae that extend into the plant root system, forming an arbuscular structure; these are called endophytic arbuscular mycorrhizae (AM fungi). Before the chlamydospores germinate, plant roots secrete specific compounds such as strigolactones and flavonoids, which induce AM fungal spore germination. After germination, the spores undergo a pre-infection growth stage, followed by hyphal branching, eventually forming appressoriums. These appressoriums can directly invade root hairs or cortical cells, proliferating and spreading within the root cell tissue. These spores are usually located in the soil or substrate and are released into the environment when forming a symbiotic relationship with the fungus. These spores play a crucial role in the dispersal and survival of the fungus in the soil. After infecting plants, AM fungi form typical arbuscule, intraradical hyphae, and vesicle structures within the plant's root cortex. Extraradical structures include spores and extraradical hyphae in the soil. AM fungi can obtain important nutrients such as carbon sources and lipids from plants, while plants can increase their absorption of nutrients from the soil through fungal hyphae, thereby promoting plant growth and development and improving the plant's resistance to stress. These symbiotic structures and symbiotic patterns can provide more ideal nutritional conditions for both parties, achieving the goal of mutual benefit and symbiosis.

[0003] In the process of sustainable development in modern agriculture, arbuscular mycorrhizal (AM) fungi can significantly enhance plant stress resistance and improve soil ecology, making them a core resource for the research and development of biological agents. Research on endophytic arbuscular mycorrhizal fungi is not only significant for a deeper understanding of symbiotic mechanisms, but also plays a crucial role in their application in agricultural development and ecological restoration.

[0004] Research on arbuscular mycorrhizal fungi (AM) has been ongoing for decades, with various symbiotic culture techniques established and applied to the propagation of AM fungi for different research objectives, such as soil pot culture and AM fungi-plant root in vitro culture. The traditional soil pot culture method uses a 1:1 zeolite-river sand substrate to plant sorghum, cultivating it for 180-300 days. The above-ground parts of the plant are then cut off, and the roots are chopped, mixed evenly with the substrate, and air-dried before being used as an inoculum. However, this method is time-consuming, costly, and the substrate is easily contaminated during cultivation. The AM fungi-plant root in vitro culture method involves a dual in vitro culture method using Ri-T-DNA transformation of carrot roots. However, the offspring lose their infectivity, require specialized equipment, are costly, and have significant limitations in practical applications.

[0005] In recent years, commercial AM fungal inoculants have developed rapidly and there is a large market demand. However, the quantity of AM fungal inoculants available on the market is small and the price is high. After purchase, they still need to be propagated, and the quality of the inoculants after propagation is inconsistent. The large-scale application of AM fungi is still limited.

[0006] Existing mycorrhizal propagation technologies are limited by problems such as high host dependence, long propagation cycle, and weak environmental adaptability. The technology system is immature and lacks investment, and large-scale industrial production and widespread commercial application of mycorrhizal agents have not yet been achieved. Summary of the Invention

[0007] The main objective of this invention is to overcome the deficiencies in the prior art. This invention adopts a stable soil pot cultivation method and, by focusing on the substrate, stress conditions, and host, provides a method to promote the sporulation and propagation of arbuscular mycorrhizal fungi.

[0008] To achieve the above objectives, the specific technical solution is as follows: This invention provides a method for promoting the sporulation and propagation of arbuscular mycorrhizal fungi, which involves inoculating arbuscular mycorrhizal fungi into a substrate, sowing host seeds in the substrate, and carrying out normal cultivation. Six weeks after sowing the host seeds, a stress treatment is applied, and samples are harvested after the cultivation period ends. The stress treatment is drought stress treatment and / or nutrient stress treatment.

[0009] One of the most important functions of arbuscular mycorrhizal fungi is to help plants absorb nutrients and water through hyphal expansion. When there is no environmental stress, plants can meet their own growth and reproduction needs or most of their needs by absorbing nutrients and water through their roots. In this case, arbuscular mycorrhizal fungi promote the growth of the host plant, which on the one hand further intensifies the competition of the underground parts for limited resources, and on the other hand accelerates the distribution of energy and matter to the above-ground parts. When stress factors are appropriately added, arbuscular mycorrhizal fungi will work harder to maintain the normal growth of the plant and complete their life cycle more quickly, thereby increasing their arbuscular abundance on the plant and the number of spores in the substrate, better helping the plant adapt to the stress environment, and also better combining with the host.

[0010] Furthermore, the drought stress treatment involves reducing watering for 3-8 days, preferably not watering for 5-7 days.

[0011] This invention employs drought stress treatment to expose plants that coexist with arbuscular mycorrhizal fungi to drought, water scarcity, resource scarcity, and harsh conditions. This promotes the infection of plant roots by arbuscular mycorrhizal fungi and allows for better symbiosis between plants and mycorrhizal fungi.

[0012] Furthermore, the nutritional stress treatment involves reducing the addition of nutrients for 3-8 days, preferably 3-7 days.

[0013] This invention employs nutrient stress treatment to expose plants that coexist with arbuscular mycorrhizal fungi to drought, water scarcity, resource scarcity, and harsh conditions. This promotes the infection of plant roots by arbuscular mycorrhizal fungi and allows for better symbiosis between plants and mycorrhizal fungi.

[0014] Furthermore, the normal maintenance involves watering once a day, and watering with Hoagland nutrient solution every three days starting from the 7th day after sowing; the Hoagland nutrient solution consists of calcium nitrate tetrahydrate 945 mg / L, potassium nitrate 506 mg / L, ammonium nitrate 80 mg / L, potassium dihydrogen phosphate 136 mg / L, magnesium sulfate heptahydrate 493 mg / L, pH=6.0; iron salt solution: ferrous sulfate heptahydrate 2.78 g, disodium ethylenediaminetetraacetate (EDTA-Na) 3.73 g 2.5 ml, pH=5.5; trace element solution: potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L.

[0015] Furthermore, the nutrient stress treatment uses Hoagland nutrient solution diluted 3-7 times.

[0016] Furthermore, the aforementioned method of promoting arbuscular mycorrhizal fungi to produce spores and proliferate was carried out through pot cultivation.

[0017] Furthermore, the arbuscular mycorrhizal fungus is *M. moses* (…). Funneliformis mosseae ).

[0018] This invention utilizes *Tetranychus mosieurii*, which significantly enhances plant stress resistance and improves soil ecology. It helps plants adapt to stressful environments, binds better to the host, and influences the growth and metabolism of the host plant. It helps plants absorb mineral elements such as N and P, thereby improving plant productivity and stress resistance. It also has ecological significance in promoting the formation of water-stable aggregates in the soil and improving the soil environment. Furthermore, it can produce almost all plant growth regulators, such as auxins, gibberellins, cytokinins, abscisic acid, and ethylene. These substances directly promote the rooting, germination, and growth of the host plant.

[0019] Furthermore, the inoculation amount of the arbuscular mycorrhizal fungi in the substrate is 245-300 spores / L of substrate. For example, 0.6L of substrate per pot is mixed with 3g of arbuscular mycorrhizal fungi inoculum (density: 54.55 spores / g), approximately 165 spores.

[0020] Furthermore, the matrix is ​​a variety of zeolite, river sand, vermiculite, perlite, and peat moss.

[0021] The use of the above-mentioned substrate in this invention can significantly increase the degree of mycorrhizal infection and sporulation.

[0022] Preferably, the matrix is ​​one of the following: zeolite + river sand, river sand + vermiculite + peat moss, zeolite + vermiculite + perlite, or zeolite + peat moss + vermiculite.

[0023] Further, in the zeolite + river sand matrix, the mass ratio of zeolite to river sand is (1-2):(1-2), preferably 1:1; in the river sand + vermiculite + peat moss matrix, the mass ratio of river sand, vermiculite, and peat moss is (1-2):(3-5):(1-2), preferably 1:4:1; in the zeolite + vermiculite + perlite matrix, the mass ratio of zeolite, vermiculite, and perlite is (1-3):(1-2)(1-2), preferably 2:1:1; in the zeolite + peat moss + vermiculite matrix, the mass ratio of zeolite, peat moss, and vermiculite is (1-2):(1-3)(1-2), preferably 1:2:1.

[0024] Furthermore, the host seed is one or more seeds selected from oats, pumpkin, clover, and sorghum.

[0025] Preferably, the host seed is oat, oat and clover, pumpkin, or sorghum and clover.

[0026] Furthermore, the culture period is 10-13 weeks, preferably 12 weeks.

[0027] In one specific embodiment of the present invention, the method for promoting the sporulation and propagation of arbuscular mycorrhizal fungi is as follows: (1) Mix river sand, vermiculite and peat moss in a mass ratio of (1-2):(3-5):(1-2), sterilize the mixed substrate and place it in a flowerpot; preferably, the mass ratio of river sand, vermiculite and peat moss is 1:4:1. (2) Inoculate the arbuscular mycorrhizal fungal agent into the substrate of the flowerpot; the inoculation amount of the arbuscular mycorrhizal fungus in the substrate is 245-300 spores / L substrate; (3) Mix sterilized oat seeds and clover seeds and sow them into the substrate. Water once a day after sowing. Starting from the 7th day after sowing, water with Hoagland nutrient solution every three days. After 6 weeks of sowing, subject the plants to drought stress treatment of 5 or 7 days without watering.

[0028] (4) Samples were collected after the 12-week cultivation period following sowing.

[0029] This invention does not apply Hoagland nutrient solution when subjecting the drought stress treatment of 5 or 7 days without watering.

[0030] In one specific embodiment of the present invention, the method for promoting the sporulation and propagation of arbuscular mycorrhizal fungi is as follows: (1) Mix zeolite and river sand in a mass ratio of (1-2):(1-2), sterilize the mixed substrate, and place it in a flowerpot; preferably, the mass ratio of zeolite and river sand is 1:1. (2) Inoculate the arbuscular mycorrhizal fungal agent into the substrate of the flowerpot; the inoculation amount of the arbuscular mycorrhizal fungus in the substrate is 245-300 spores / L substrate; (3) Mix sterilized sorghum seeds and clover seeds and sow them into the substrate. Water once a day after sowing. Starting from the 7th day after sowing, water with Hoagland nutrient solution every three days. Six weeks after sowing, dilute Hoagland nutrient solution 5 times and perform nutrient stress treatment for 3 or 7 days. (4) Samples were collected after the 12-week cultivation period following sowing.

[0031] In one specific embodiment of the present invention, the method for promoting the sporulation and propagation of arbuscular mycorrhizal fungi is as follows: (1) Mix zeolite, peat moss and vermiculite in a mass ratio of (1-2):(1-3)(1-2), sterilize the mixed substrate and place it in a flowerpot; preferably, the mass ratio of zeolite, peat moss and vermiculite is 1:2:1. (2) Inoculate the arbuscular mycorrhizal fungal agent into the substrate of the flowerpot; the inoculation amount of the arbuscular mycorrhizal fungus in the substrate is 245-300 spores / L substrate; (3) After sterilizing the pumpkin seeds, germinate them and sow them into the substrate. Water once a day after sowing. Starting from the 7th day after sowing, water with Hoagland nutrient solution every three days. Six weeks after sowing, dilute Hoagland nutrient solution 5 times and perform nutrient stress treatment for 3 or 7 days. (4) Samples were collected after the 12-week cultivation period following sowing.

[0032] Furthermore, in the above specific embodiments, a pot culture method is adopted, with 0.6L of substrate per pot and 3g of arbuscular mycorrhizal fungal agent inoculated (density: 54.55 cells / g); the culture period is 80 days.

[0033] Furthermore, the 3-day nutrient stress treatment refers to watering only for 2 days, watering once with a 5-fold diluted Hoagland nutrient solution on the 3rd day, and then carrying out normal care; the 7-day nutrient stress treatment refers to watering only for 6 days, watering once with a 5-fold diluted Hoagland nutrient solution on the 7th day, and then carrying out normal care.

[0034] Compared with the prior art, the present invention has the following significant advantages: The method for promoting arbuscular mycorrhizal fungi (AM) sporulation and propagation provided by this invention can effectively, efficiently, and rapidly produce sporulations, breaking industry limitations and laying a theoretical and experimental foundation for solving the problems of long propagation cycles and high costs associated with AM fungi. It overcomes the technical barriers of traditional fungal agent production and further reveals the environmental response mechanism of mycorrhizal symbiotic systems from an ecological perspective, providing a theoretical basis for promoting the application of arbuscular mycorrhizal fungi in agriculture and achieving green development in agricultural production. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 This is a schematic diagram summarizing the embodiments and comparative examples of the present invention. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0038] Unless otherwise specified in the embodiments of the present invention, the techniques or conditions described in the literature in this field or the product instructions shall be followed; if the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through legitimate channels.

[0039] Flowerpot: Square, with a bottom diameter of 7.5cm, a top diameter of 10.2cm, and a height of 14cm; Arbuscular mycorrhizal fungi: Moses tuberculosis ( Funneliformis mosseae The strain originated from the Institute of Plant Nutrition and Resource Environment, Beijing Academy of Agricultural and Forestry Sciences, and is a strain from the "Arbuscular Mycorrhizal Fungi Germplasm Bank" (BGC). Hoagland nutrient solution: Calcium nitrate tetrahydrate 945 mg / L, potassium nitrate 506 mg / L, ammonium nitrate 80 mg / L, potassium dihydrogen phosphate 136 mg / L, magnesium sulfate heptahydrate 493 mg / L, pH=6.0; Iron salt solution: ferrous sulfate heptahydrate 2.78 g, disodium ethylenediaminetetraacetate (EDTA-Na) 3.73 g 2.5 ml, pH=5.5; Trace element solution: potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L.

[0040] The above substances are used in the embodiments or comparative examples of this invention.

[0041] Examples 1-32: Examples of Drought Stress Treatment Example 1 A method for promoting the sporulation and propagation of arbuscular mycorrhizal fungi is as follows: (1) Mix 0.1L of river sand, 0.4L of vermiculite and 0.1L of peat moss. After sterilizing the mixed substrate with γ-rays, place it in a flowerpot. (2) Inoculate approximately 165 spores from 3g of arbuscular mycorrhizal fungal inoculum (density: 54.55 spores / g) into the substrate of the flowerpot; (3) Mix 16 sterilized oat seeds and 25 clover seeds and sow them into the substrate. For the first 40 days, maintain normal care, that is, water once a day. From the 7th day after sowing, water with Hoagland nutrient solution every three days. On the 41st day after sowing, perform drought stress treatment for 5 days without watering, and do not water with Hoagland nutrient solution during this period. (4) At 12 weeks after sowing, samples were collected from both the above-ground and underground parts, and weighed separately. The infection rate of the fresh roots was determined. The substrate was allowed to air dry to constant weight and then placed in uncontaminated resealable bags for spore density determination. (RVac5) Example 2 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 1, the only difference is that in step (3), a drought stress treatment of 7 days without watering is performed 41 days after sowing, and Hoagland nutrient solution is not applied during this period. (RVac7) Example 3 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 1, except that in step (1), the substrate is a mixture of 0.3L zeolite and 0.3L river sand. (ZRac5) Example 4 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 3, the only difference is that in step (3), a drought stress treatment of 7 days without watering is performed 41 days after sowing. (ZRac7) Example 5 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 1, except that in step (1), the substrate is a mixture of 0.3L zeolite, 0.15L vermiculite, and 0.15L perlite. (ZVac5) Example 6 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 5, the only difference is that in step (3), a drought stress treatment of 7 days without watering is performed 41 days after sowing. (ZVac7) Example 7 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 1, except that in step (1), the substrate is a mixture of 0.15L zeolite, 0.3L peat moss, and 0.15L vermiculite. (ZPac5) Example 8 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 7, the only difference is that in step (3), a drought stress treatment of 7 days without watering is performed 41 days after sowing. (ZPac7) Example 9 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 1, except that in step (3), 16 sterilized oat seeds are sown into the substrate. (RVaa5) Example 10 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 9, the only difference is that in step (3), a drought stress treatment of 7 days without watering is performed 41 days after sowing. (RVaa7) Example 11 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 9, except that in step (1), the substrate is a mixture of 0.3L zeolite and 0.3L river sand. (ZRaa5) Example 12 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 11, the only difference is that in step (3), a drought stress treatment of 7 days without watering is performed 41 days after sowing. (ZRaa7) Example 13 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 9, except that in step (1), the substrate is a mixture of 0.3L zeolite, 0.15L vermiculite, and 0.15L perlite. (ZVaa5) Example 14 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 13, the only difference is that in step (3), a drought stress treatment of 7 days without watering is performed 41 days after sowing. (ZVaa7) Example 15 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 9, except that in step (1), the substrate is a mixture of 0.15L zeolite, 0.3L peat moss, and 0.15L vermiculite. (ZPaa5) Example 16 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 15, the only difference is that in step (3), a drought stress treatment of 7 days without watering is performed 41 days after sowing. (ZPaa7) Example 17 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 1, except that in step (3), one sterilized pumpkin seed is first germinated and then sown into the substrate after germination. (RVbb5) Example 18 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 17, the only difference is that in step (3), a drought stress treatment of 7 days without watering is performed 41 days after sowing. (RVbb7) Example 19 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 17, except that in step (1), the substrate is a mixture of 0.3L zeolite and 0.3L river sand. (ZRbb5) Example 20 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 19, the only difference is that in step (3), a drought stress treatment of 7 days without watering is performed 41 days after sowing. (ZRbb7) Example 21 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 17, except that in step (1), the substrate is a mixture of 0.3L zeolite, 0.15L vermiculite, and 0.15L perlite. (ZVbb5) Example 22 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 21, the only difference is that in step (3), a drought stress treatment of 7 days without watering is performed 41 days after sowing. (ZVbb7) Example 23 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 17, except that in step (1), the substrate is a mixture of 0.15L zeolite, 0.3L peat moss, and 0.15L vermiculite. (ZPbb5) Example 24 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 23, the only difference is that in step (3), a drought stress treatment of 7 days without watering is performed 41 days after sowing. (ZPbb7) Example 25 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 1, except that in step (3), 16 sterilized and pre-germinated sorghum seeds and 25 clover seeds are mixed and sown into the substrate. (RVdc5) Example 26 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 25, the only difference is that in step (3), a drought stress treatment of 7 days without watering is performed 41 days after sowing. (RVdc7) Example 27 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 25, except that in step (1), the substrate is a mixture of 0.3L zeolite and 0.3L river sand. (ZRdc5) Example 28 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 27, the only difference is that in step (3), a 7-day drought stress treatment without watering is performed 41 days after sowing. (ZRdc7) Example 29 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 25, except that in step (1), the substrate is a mixture of 0.3L zeolite, 0.15L vermiculite, and 0.15L perlite. (ZVdc5) Example 30 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 29, the only difference is that in step (3), a drought stress treatment of 7 days without watering is performed 41 days after sowing. (ZVdc7) Example 31 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 25, except that in step (1), the substrate is a mixture of 0.15L zeolite, 0.3L peat moss, and 0.15L vermiculite. (ZPdc5) Example 32 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 31, the only difference is that in step (3), a drought stress treatment of 7 days without watering is performed 41 days after sowing. (ZPdc7) Examples 33-64: Examples of Nutritional Stress Treatment Example 33 A method for promoting the sporulation and propagation of arbuscular mycorrhizal fungi is as follows: (1) Mix 0.15L of zeolite, 0.3L of peat moss and 0.15L of vermiculite. After sterilizing the mixed substrate with γ-rays, place it in a flowerpot. (2) Inoculate approximately 165 spores from 3g of arbuscular mycorrhizal fungal inoculum (density: 54.55 spores / g) into the substrate of the flowerpot; (3) Mix 16 sterilized oat seeds and 25 clover seeds and sow them into the substrate. For the first 40 days, maintain normal care, that is, water once a day. From the 7th day after sowing, water with Hoagland nutrient solution every three days. On the 41st day after sowing, dilute Hoagland nutrient solution 5 times and perform nutrient stress treatment for 3 days. That is, water only for 3 days and water with Hoagland nutrient solution diluted 5 times once on the 4th day. (4) At 12 weeks after sowing, samples were collected from both the above-ground and underground parts, and weighed separately. The infection rate of the fresh roots was determined. The substrate was allowed to air dry to constant weight and then placed in uncontaminated self-sealing bags for spore density determination. (ZPac3) Example 34 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 33, the only difference is that in step (3), a 7-day nutrient stress treatment is applied 41 days after sowing, that is, watering is only done for 7 days, and Hoagland nutrient solution diluted 5 times is applied once on the 8th day. (ZPac7-2) Example 35 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 33, except that in step (3), 16 sterilized and pre-germinated sorghum seeds and 25 clover seeds are mixed and sown into the substrate. (ZPdc3) Example 36 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 35, the only difference is that in step (3), a 7-day nutrient stress treatment is applied 41 days after sowing. (ZPdc7-2) Example 37 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 33, except that in step (3), 16 sterilized oat seeds are sown into the substrate. The seeds are mixed into the substrate. (ZPaa3) Example 38 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 37, the only difference is that in step (3), a 7-day nutrient stress treatment is applied 41 days after sowing. (ZPaa7-2) Example 39 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 33, the only difference is that in step (3), one sterilized pumpkin seed is first germinated, and after germination, it is sown into the substrate. (ZPbb3) Example 40 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 39, the only difference is that in step (3), a 7-day nutrient stress treatment is applied 41 days after sowing. (ZPbb7-2) Example 41 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 33, except that in step (1), the substrate is a mixture of 0.3L zeolite and 0.3L river sand. (ZRac3) Example 42 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 41, the only difference is that in step (3), a 7-day nutrient stress treatment is performed 41 days after sowing. (ZRac7-2) Example 43 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 33, except that in step (1), the substrate is a mixture of 0.3L zeolite, 0.15L vermiculite, and 0.15L perlite. (ZVac3) Example 44 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 43, the only difference is that in step (3), a 7-day nutrient stress treatment is applied 41 days after sowing. (ZVac7-2) Example 45 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 33, except that in step (1), the substrate is a mixture of 0.1L river sand, 0.4L vermiculite, and 0.1L peat moss. (RVac3) Example 46 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 45, the only difference is that in step (3), a 7-day nutrient stress treatment is applied 41 days after sowing. (RVac7-2) Example 47 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 45, except that in step (3), 16 sterilized oat seeds are sown into the substrate. (RVaa3) Example 48 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 47, the only difference is that in step (3), a 7-day nutrient stress treatment is applied 41 days after sowing. (RVaa7-2) Example 49 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 41, except that in step (3), 16 sterilized oat seeds are sown into the substrate. (ZRaa3) Example 50 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 49, the only difference is that in step (3), a 7-day nutrient stress treatment is performed 41 days after sowing. (ZRaa7-2) Example 51 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 43, except that in step (3), 16 sterilized oat seeds are sown into the substrate. (ZVaa3) Example 52 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 51, the only difference is that in step (3), a 7-day nutrient stress treatment is performed 41 days after sowing. (ZVaa7-2) Example 53 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 41, except that in step (3), one sterilized pumpkin seed is first germinated and then sown into the substrate. (ZRbb3) Example 54 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 53, the only difference is that in step (3), a 7-day nutrient stress treatment is performed 41 days after sowing. (ZRbb7-2) Example 55 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 45, except that in step (3), one sterilized pumpkin seed is first germinated and then sown into the substrate. (RVbb3) Example 56 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 55, the only difference is that in step (3), a 7-day nutrient stress treatment is applied 41 days after sowing. (RVbb7-2) Example 57 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 43, the only difference is that in step (3), one sterilized pumpkin seed is first germinated and then sown into the substrate. (ZVbb3) Example 58 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 57, the only difference is that in step (3), a 7-day nutrient stress treatment is performed 41 days after sowing. (ZVbb7-2) Example 59 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 41, except that in step (3), 16 sterilized and pre-germinated sorghum seeds and 25 clover seeds are mixed and sown into the substrate. (ZRdc3) Example 60 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 59, the only difference is that in step (3), a 7-day nutrient stress treatment is applied 41 days after sowing. (ZRdc7-2) Example 61 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 45, except that in step (3), 16 sterilized and pre-germinated sorghum seeds and 25 clover seeds are mixed and sown into the substrate. (RVdc3) Example 62 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 61, the only difference is that in step (3), a 7-day nutrient stress treatment is performed 41 days after sowing. (RVdc7-2) Example 63 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 43, except that in step (3), 16 sterilized and pre-germinated sorghum seeds and 25 clover seeds are mixed and sown into the substrate. (ZVdc3) Example 64 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 63, the only difference is that in step (3), a 7-day nutrient stress treatment is applied 41 days after sowing. (ZVac7-2) Comparative Examples 1-16: Comparative Examples of Normal Maintenance Comparative Example 1 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 1, the only difference is that in step (3), normal maintenance is performed without drought or nutrient stress treatment. (RVac0) Comparative Example 2 The method for spore production and propagation of arbuscular mycorrhizal fungi in this comparative example is basically the same as that in Example 3, the only difference is that in step (3), normal maintenance is performed without drought stress treatment. (ZRac0) Comparative Example 3 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 5, the only difference is that in step (3), normal maintenance is performed without drought stress treatment. (ZVac0) Comparative Example 4 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 7, the only difference is that in step (3), normal maintenance is performed without drought stress treatment. (ZPac0) Comparative Example 5 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 9, the only difference is that in step (3), normal maintenance is performed without drought stress treatment. (RVaa0) Comparative Example 6 The method for spore production and propagation of arbuscular mycorrhizal fungi in this comparative example is basically the same as that in Example 11, the only difference is that in step (3), normal maintenance is performed without drought stress treatment. (ZRaa0) Comparative Example 7 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 13, the only difference is that in step (3), normal maintenance is performed without drought stress treatment. (ZVaa0) Comparative Example 8 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 15, the only difference is that in step (3), normal maintenance is performed without drought stress treatment. (ZPaa0) Comparative Example 9 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 17, the only difference is that in step (3), normal maintenance is performed without drought stress treatment. (RVbb0) Comparative Example 10 The method for spore production and propagation of arbuscular mycorrhizal fungi in this comparative example is basically the same as that in Example 19, the only difference is that in step (3), normal maintenance is carried out without drought stress treatment. (ZRbb0) Comparative Example 11 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 21, the only difference is that in step (3), normal maintenance is performed without drought stress treatment. (ZVbb0) Comparative Example 12 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 23, the only difference is that in step (3), normal maintenance is performed without drought stress treatment. (ZPbb0) Comparative Example 13 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 25, the only difference is that in step (3), normal maintenance is performed without drought stress treatment. (RVdc0) Comparative Example 14 The method for spore production and propagation of arbuscular mycorrhizal fungi in this comparative example is basically the same as that in Example 27, the only difference is that in step (3), normal maintenance is performed without drought stress treatment. (ZRdc0) Comparative Example 15 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 29, the only difference is that in step (3), normal maintenance is performed without drought stress treatment. (ZVdc0) Comparative Example 16 The method for spore production and propagation of arbuscular mycorrhizal fungi in this embodiment is basically the same as that in Example 31, the only difference is that in step (3), normal maintenance is performed without drought stress treatment. (ZPdc0) In each embodiment and comparative example, the amount of water or nutrient solution applied after the first thorough watering is the amount of water lost the previous day.

[0042] Each embodiment and comparative example had four parallel treatments, and the data were averaged.

[0043] Spore density refers to the number of arbuscular mycorrhizal fungal spores contained in a unit mass of soil, plant root system, or other samples. It represents the abundance and distribution of arbuscular mycorrhizal fungi in a specific environment, and to some extent reflects the fungi's reproductive capacity and ecological activity. It is one of the important indicators for measuring mycorrhizal fungal resources in soil.

[0044] Spore density determination method: Randomly take 10g of the root-substrate mixture from the propagation substrate, place it in a 50ml centrifuge tube, add 30ml of distilled water, and stir thoroughly to form a soil suspension. Centrifuge at 3000r / min for 4min. After centrifugation, discard the supernatant, add 30ml of 60% sucrose solution to the centrifuge tube, stir thoroughly, and centrifuge again at 2000r / min for 2min to extract spores. Pour the supernatant from the centrifuge tube into a double-layer sieve (20-mesh upper sieve and 400-mesh lower sieve). Completely transfer the material from the lower sieve to a petri dish, and record the total number of spores under a dissecting microscope. Calculate the spore density using the formula (spore density = number of AM fungal spores / soil mass) for result processing and analysis.

[0045] Infection rate determination method: Fixed host plant roots were treated using a series of steps: boiling until transparent, decolorizing, acidifying, staining, and fading. After decolorization, sterile water was used for preservation. Thirty root segments were placed on glass slides and observed under an optical microscope for mycorrhizal morphology, invasion points, vesicles, etc. Infection points, hyphae, and vesicles observed within the root segments were recorded as infected. The abundance of arbuscular mycorrhizae was classified and recorded according to the mycorrhizal infection grading standard. The infection rate was calculated based on the number of infected roots, and the number of root segments below the infection rate was recorded for further result processing and analysis.

[0046] Based on the data analysis of Examples 1-32 (drought stress treatment) and Comparative Examples 1-16 (normal maintenance), the following conclusions can be drawn: 1.1 Effects on plant growth Regarding the substrate, substrate factors had a highly significant impact on the fresh weight of both aboveground and underground parts. In the oat and clover mixed-sowing (ac) host, the aboveground fresh weight of the substrate treatment of river sand + vermiculite + peat moss (1:4:1) (RV) was significantly increased compared with the substrate treatment of zeolite + river sand (1:1) (ZR), with the highest aboveground fresh weight under RVac5d drought stress, reaching an average of 26.00 g. Similarly, in the host ac, the underground fresh weight of the substrate treatment was significantly increased compared with ZR, with the highest underground fresh weight under RVaa7d drought stress, reaching 9.35 g. Regarding the host, the host also had a highly significant impact on the fresh weight of both aboveground and underground parts of the plant. In the substrate zeolite + peat moss + vermiculite (1:2:1) (ZP), compared with dc (sorghum and clover mixed sowing), the host ac significantly increased the impact on biomass. The fresh weight of the underground parts in all three ZPac treatments was significantly higher than that in the three ZVdc treatments. However, the biomass of the dc host showed no significant change under 5-day and 7-day drought stress. The effect of drought stress on plant growth was not significant. The effects of both substrate and host on the underground parts were extremely significant, and the interaction of substrate, host, and drought stress on the fresh weight of the underground parts was extremely significant. Under drought stress, the optimal combination for aboveground fresh weight was RVac for 5 days, and the optimal combinations for underground fresh weight were RVaa for 7 days, ZPac for 5 days, and ZPac for 7 days. The effects of substrate, host, and drought stress on plant growth are shown in Table 1. The effects of different substrates, hosts, and drought stress on plant growth are shown in Table 2. The effects of different substrates, hosts, and drought stress on arbuscular mycorrhizal sporulation are shown in Table 3. The effects of different substrates, hosts, and drought stress on arbuscular mycorrhizal fungal infection values ​​are shown in Table 4.

[0047] Table 1. Effects of substrate, host, and drought stress on plant growth.

[0048] Note: Data in the table are significant at the 0.05 level. ***P<0.001, **P<0.01, *P<0.05. NS or no labeling indicates no significant difference.

[0049] Table 2. Effects of different substrates, host, and drought stress on plant growth. Note: Data in the table are mean ± standard error (n=3). ZR represents a substrate mix of zeolite and river sand (1:1), RV represents a substrate mix of river sand, vermiculite, and peat moss (1:4:1), ZV represents a substrate mix of zeolite, vermiculite, and perlite (2:1:1), ZP represents a substrate mix of zeolite, peat moss, and vermiculite (1:2:1), aa represents monoculture oats, ac represents oat and clover mixture, dc represents sorghum and clover mixture, 5d represents five days, and 7d represents seven days. For example, ZRaa5d drought stress means monoculture oats on a zeolite + river sand (1:1) substrate for 5 days of drought stress.

[0050] Table 3. Effects of different substrates, hosts, and drought stress on sporulation of arbuscular mycorrhizal fungi. Note: Data in the table are mean ± standard error (n=3). ZR represents a substrate mix of zeolite and river sand (1:1), RV represents a substrate mix of river sand, vermiculite, and peat moss (1:4:1), ZV represents a substrate mix of zeolite, vermiculite, and perlite (2:1:1), ZP represents a substrate mix of zeolite, peat moss, and vermiculite (1:2:1), aa represents monoculture oats, ac represents oat and clover mixture, dc represents sorghum and clover mixture, 5d represents five days, and 7d represents seven days. For example, ZRaa5d drought stress means monoculture oats on a zeolite + river sand (1:1) substrate for 5 days of drought stress.

[0051] Table 4. Effects of different substrates, hosts, and drought stress on arbuscular mycorrhizal fungal infection values.

[0052] Note: Data in the table are mean ± standard error (n=3). ZR represents a substrate mix of zeolite and river sand (1:1), RV represents a substrate mix of river sand, vermiculite, and peat moss (1:4:1), ZV represents a substrate mix of zeolite, vermiculite, and perlite (2:1:1), ZP represents a substrate mix of zeolite, peat moss, and vermiculite (1:2:1), aa represents monoculture oats, ac represents oat and clover mixture, dc represents sorghum and clover mixture, 5d represents five days, and 7d represents seven days. For example, ZRaa5d drought stress means monoculture oats on a zeolite + river sand (1:1) substrate for 5 days of drought stress.

[0053] 1.2 Effects on arbuscular mycorrhizal fungal infection The results showed significant differences in spore density and propagation multiples between the substrate and the host organism (ac). Compared with the ZR substrate, the RV substrate treatment significantly increased spore density, with the average spore density of RVac under normal cultivation reaching 47.67 spores / 10g (11.56-fold propagation). In the dc host, the infection rate under normal cultivation with the ZR substrate (92.22%) was significantly higher than that under normal cultivation with the ZV substrate (63.33%).

[0054] Regarding the host, the difference between host and spore density was extremely significant. In the ZP substrate, compared with dc, the ac host had a significantly higher impact on biomass, and the differences in spore density and propagation multiple between the substrate host and drought stress were extremely significant.

[0055] Regarding stress, drought stress significantly affected spore density and propagation multiples. Comparing normal care and drought stress treatments, drought stress generally influenced infection rate-related indicators. The infection rate of ZRaa under normal care was 86.67%, decreasing to 83.33% after 5 days of drought stress (ZRaa5d) and 81.11% after 7 days (ZRaa7d), though the decrease was not significant. The arbuscular abundance of infected root segments under RVdc care was 6.12%, decreasing to 6.87% after 5 days of drought stress (RVdc5d) and 7.44% after 7 days (RVdc7d), showing no significant pattern of change. This indicates that drought stress has a complex impact on various indicators of arbuscular mycorrhizal fungal infection. Among the three treatments, in the ZP substrate, drought stress was most effective for aa hosts after 5 days, reaching a maximum of 73.33 spores / 10g (propagation reaching 17.78 times). Regarding infection and arbuscular mycorrhizal formation, 7 days of drought stress showed better results, with the highest infection rate reaching 85.56% for ZRac at 7 days and the highest whole-root arbuscular abundance reaching 26.20% for ZRaa at 7 days. Data showed significant fluctuations among different treatments within the same substrate, with large variations in spore density and mycorrhizal infection rate in the ZR substrate. For example, under normal cultivation, ZRaa had a spore density of 47 spores / 10g (11.39-fold multiplication) and a mycorrhizal infection rate of 86.67%; while ZRdc under normal cultivation had a spore density of 37g (8.97-fold multiplication) and a mycorrhizal infection rate of 92.22%. The interaction of the three factors significantly affected spore density and multiplication rate. Regarding spore density, ZPac at 5 days of drought stress showed better sporulation under stress. The effects of substrate, host, and drought stress on sporulation of arbuscular mycorrhizal fungi are shown in Table 5.

[0056] Table 5. Effects of substrate, host, and drought stress on sporulation of arbuscular mycorrhizal fungi.

[0057] Note: Data in the table are significant at the 0.05 level. ***P<0.001, **P<0.01, *P<0.05. NS or no labeling indicates no significant difference.

[0058] The results show that the fresh weight of the underground parts is positively correlated with spore density. Appropriate drought stress significantly improves sporulation. The fresh weight of the underground parts shows a highly significant positive correlation with each other, indicating that better treatment of the underground parts' fresh weight has a positive correlation with spore density. The correlation between spore density and other indicators is not significant, indicating that the association between spore density and other factors is not obvious in this invention. The arbuscular abundance of infected root segments is significantly positively correlated with infection intensity and highly significantly positively correlated with the arbuscular abundance of the entire root system, indicating that as infection intensity increases, the arbuscular abundance of the entire root system increases significantly, verifying the conclusion mentioned earlier that the arbuscular abundance of the entire root system increases with increasing infection intensity. The arbuscular abundance of infected root segments is highly significantly positively correlated with the arbuscular abundance of the entire root system, indicating that an increase in the arbuscular abundance of infected root segments will significantly increase the arbuscular abundance of the entire root system. The correlation analysis of drought stress is shown in Table 6.

[0059] Table 6 Correlation analysis of drought stress

[0060] Note: Data in the table are significant at the 0.05 level. ***P<0.001, **P<0.01, *P<0.05. NS or no labeling indicates no significant difference.

[0061] In summary, the combination of RV and ac is most beneficial to host growth, sporulation, and infection rate. 5 days of drought stress is the optimal drought stress for the propagation of AM fungi; while 7 days of drought stress results in lower plant biomass, but increases in infection rate, sporulation, and arbuscular density.

[0062] 2. Analysis of data from Examples 33-64 (nutritional stress treatment) and Comparative Examples 1-16 (normal care) shows that: The results show that substrate factors have a highly significant impact on the fresh weight of both aboveground and underground parts of plants (p<0.001). RV substrate has a significant advantage in promoting plant growth, with higher fresh weights of both aboveground and underground parts compared to ZR substrate. For example, the average fresh weight of aboveground parts under 7 days of nutrient stress was 29.67 g for RGaa and 16.27 g for underground parts under 7 days of nutrient stress for RVac. Regarding the host, host factors have a significant impact on plant growth. Compared to aa and dc, ac host significantly increases the impact on biomass. Nutrient stress has no significant effect on aboveground growth, but a significant effect on underground biomass, indicating that underground parts of plants are more sensitive to nutrient stress. The interaction between substrate and host factors, as well as the interaction between substrate, host, and nutrient stress factors, all have a significant impact on the fresh weight of underground parts. The combination of RV and aa is most beneficial to the fresh weight of both aboveground and underground parts of plants. The effects of substrate, host, and nutrient stress on plant growth are shown in Table 7. The effects of different substrates, hosts, and nutrient stress on plant growth values ​​are shown in Table 8. The effects of different substrates, hosts, and nutrient stress on arbuscular mycorrhizal sporulation are shown in Table 9. The effects of different substrates, hosts, and nutrient stress on arbuscular mycorrhizal fungal infection values ​​are shown in Table 10.

[0063] Under nutrient stress, the optimal combination of substrate RV and host aa is the one that maximizes host growth.

[0064] Table 7. Effects of substrate, host, and nutrient stress on plant growth.

[0065] Note: Data in the table are significant at the 0.05 level. ***P<0.001, **P<0.01, *P<0.05. NS or no labeling indicates no significant difference.

[0066] Table 8. Effects of different substrates, hosts, and nutrient stress on plant growth.

[0067] Note: Data in the table are mean ± standard error (n=3). ZR is a matrix mixture of zeolite and river sand (1:1), RV is a matrix mixture of river sand, vermiculite, and peat moss (1:4:1), ZV is a matrix mixture of zeolite, vermiculite, and perlite (2:1:1), ZP is a matrix mixture of zeolite, peat moss, and vermiculite (1:2:1), aa is monoculture of oats, ac is a mixture of oats and clover, and dc is a mixture of sorghum and clover.

[0068] Table 9. Effects of different substrates, hosts, and nutritional stress on sporulation of arbuscular mycorrhizal fungi. Note: Data in the table are mean ± standard error (n=3). ZR represents a substrate mix of zeolite and river sand (1:1), RV represents a substrate mix of river sand, vermiculite, and peat moss (1:4:1), ZV represents a substrate mix of zeolite, vermiculite, and perlite (2:1:1), ZP represents a substrate mix of zeolite, peat moss, and vermiculite (1:2:1), aa represents monoculture oats, ac represents oat and clover mixture, dc represents sorghum and clover mixture, 5d represents five days, and 7d represents seven days. For example, ZRaa5d drought stress means monoculture oats on a zeolite + river sand (1:1) substrate for 5 days of drought stress.

[0069] Table 10. Effects of different substrates, hosts, and nutrient stress on arbuscular mycorrhizal fungal infection values. Note: Data in the table are mean ± standard error (n=3). ZR represents a substrate mix of zeolite and river sand (1:1), RV represents a substrate mix of river sand, vermiculite, and peat moss (1:4:1), ZV represents a substrate mix of zeolite, vermiculite, and perlite (2:1:1), ZP represents a substrate mix of zeolite, peat moss, and vermiculite (1:2:1), aa represents monoculture oats, ac represents oat and clover mixture, dc represents sorghum and clover mixture, 5d represents five days, and 7d represents seven days. For example, ZRaa5d drought stress means monoculture oats on a zeolite + river sand (1:1) substrate for 5 days of drought stress.

[0070] Regarding the substrate, it had a highly significant impact on spore density and mycorrhizal infection frequency. In the ZR host dc substrate, under 7 days of nutrient stress, the average spore density of ZRac7d-2 reached 88.33 spores / 10g (a 21.42-fold increase in proliferation). Nutrient stress significantly affected both spore density and infection efficiency; comparing normal cultivation with nutrient stress treatment, nutrient stress altered some indicators in most cases. The mycorrhizal spore density of RVdc under normal cultivation was 28.00 spores / 10g, which increased significantly to 48.00 under nutrient stress for RVdc 3d-2 and 75.00 spores / 10g under nutrient stress for RVdc 7d-2. The highest spore density reached was 88.33 spores / 10g (175-fold multiplication) under drought stress for ZRac 7d-2. The substrate, host, and stress treatments significantly affected arbuscular mycorrhizal fungal infection. The host significantly influenced spore density, infection intensity, and arbuscular abundance throughout the root system. All pairwise interactions among these three factors significantly affected spore density and infection intensity. Under nutrient stress, the treatment most suitable for sporulation was ZRdc 7d-2 (21.42-fold multiplication), the treatment most suitable for infection intensity was ZVaa (normal cultivation) reaching 39.96%, and the treatment most suitable for arbuscular abundance was ZVac (normal cultivation) reaching 17.57%.

[0071] The effects of substrate, host, and nutrient stress on sporulation of arbuscular mycorrhizal fungi are shown in Table 11. The effects of substrate, host, and nutrient stress on infection by arbuscular mycorrhizal fungi are shown in Table 12.

[0072] Table 11. Effects of substrate, host, and nutrient stress on sporulation of arbuscular mycorrhizal fungi.

[0073] Note: Data in the table are significant at the 0.05 level. ***P<0.001, **P<0.01, *P<0.05. NS or no labeling indicates no significant difference.

[0074] Table 12 Effects of substrate, host, and nutrient stress on arbuscular mycorrhizal fungal infection

[0075] Note: Data in the table are significant at the 0.05 level. ***P<0.001, **P<0.01, *P<0.05. NS or no labeling indicates no significant difference.

[0076] The aboveground fresh weight and underground fresh weight of the plant were significantly positively correlated, indicating a strong consistency in the growth trends of aboveground and underground fresh weight. Infection intensity and the abundance of arbuscular masses in the entire root system were negatively correlated with plant biomass. As infection intensity increased, the abundance of arbuscular masses in the entire root system was likely to increase. The correlation coefficient between the abundance of arbuscular masses in infected root segments and the abundance of arbuscular masses in the entire root system was 0.490**, showing a significant positive correlation at the P<0.001 level, indicating a close association between the two. The abundance of arbuscular masses in the entire root system increased with increasing abundance of arbuscular masses in infected root segments and infection intensity. The correlation analysis of nutrient stress is shown in Table 13.

[0077] Table 13 Correlation analysis of nutritional stress

[0078] Note: Data in the table are statistically significant at the 0.05 level. ***P<0.001, **P<0.01, *P<0.05. No significant differences are indicated by NS or no labeling. 3. Data analysis of Examples 39, 40, 53, 54, 55, 56, 57, 58 and Comparative Examples 9-12 leads to the conclusion that: Different substrates significantly affected the aboveground and underground biomass of pumpkin. The average fresh weight of the aboveground parts of RV substrates was the highest at 53g. The values ​​for ZR and ZP substrates were similar and significantly lower than those of RV substrates, but higher than those of ZV substrates. This indicates that ZR and ZP substrates were less effective at promoting the growth of the aboveground parts of pumpkin than RV substrates, but slightly better than ZV substrates. The average fresh weight of the underground parts of ZV substrates was 7.11g, significantly higher than other substrates. This indicates that ZV substrates have a unique advantage in promoting the growth of pumpkin roots (underground parts). The average fresh weights of the underground parts of ZR, RV, and ZP substrates were 5.7g, 5.22g, and 5.97g, respectively, similar and significantly lower than those of ZV substrates. This suggests that these three substrates were less effective at promoting the growth of the underground parts of pumpkin than ZV substrates, although the differences among them were relatively small. The effects of different substrates on pumpkin growth are shown in Table 14.

[0079] Table 14 Effects of different substrates on pumpkin growth

[0080] Note: The data in the table are average ± standard error (n=3). ZR is a matrix combination of zeolite + river sand (1:1), RV is a matrix combination of river sand + vermiculite + peat moss (1:4:1), ZV is a matrix combination of zeolite + vermiculite + perlite (2:1:1), and ZP is a matrix combination of zeolite + peat moss + vermiculite (1:2:1).

[0081] ZP substrate had the highest spore density, at 110 spores / 10g (a 26.67-fold increase in propagation), significantly higher than ZV, ZR, and RV substrates. RV substrate had the fewest spores, significantly lower than the other substrates (p<0.05).

[0082] The results indicate that the ZP substrate is most favorable for the accumulation of arbuscular mycorrhizal fungal spores, possibly due to its suitable spore survival and reproduction characteristics in terms of nutrient composition and physical structure. The mycorrhizal infection frequencies of the ZR, RV, ZV, and ZP substrates were 76.67%, 85.55%, 80%, and 75.55%, respectively. This shows that under the conditions of this invention, the different substrates did not significantly affect the mycorrhizal infection frequency, and all could achieve a high infection frequency, significantly higher than other substrates. This indicates that the ZV substrate is most favorable for the formation and development of arbuscular mycorrhizal fungi in infected root segments, possibly related to the influence of this substrate on the root microenvironment, thereby affecting the interaction between fungi and roots. The effects of different substrates on sporulation in pumpkin are shown in Table 15, and the effects of different substrates on infection in pumpkin are shown in Table 16.

[0083] Table 15 Effects of different substrates on sporulation efficiency of pumpkin

[0084] Note: The data in the table are average ± standard error (n=3). ZR is a matrix combination of zeolite + river sand (1:1), RV is a matrix combination of river sand + vermiculite + peat moss (1:4:1), ZV is a matrix combination of zeolite + vermiculite + perlite (2:1:1), and ZP is a matrix combination of zeolite + peat moss + vermiculite (1:2:1).

[0085] Table 16 Effects of different substrates on pumpkin infection efficacy

[0086] Note: The data in the table are average ± standard error (n=3). ZR is a matrix combination of zeolite + river sand (1:1), RV is a matrix combination of river sand + vermiculite + peat moss (1:4:1), ZV is a matrix combination of zeolite + vermiculite + perlite (2:1:1), and ZP is a matrix combination of zeolite + peat moss + vermiculite (1:2:1).

[0087] The fresh weight of the underground parts was inversely proportional to the fresh weight of the aboveground parts. Spore density decreased with increasing aboveground fresh weight. Arbuscular abundance in infected root segments increased with increasing underground fresh weight. The arbuscular abundance of the entire root system was significantly positively correlated with the arbuscular abundance of infected root segments. In actual pumpkin cultivation, RV substrate is preferred for promoting vigorous growth of aboveground parts (such as stems and leaves); ZV substrate is a better choice if the focus is on root development. Correlation analysis of pumpkins grown in the same substrate is shown in Table 17.

[0088] Table 17 Correlation analysis of pumpkins in different substrates

[0089] Note: Data in the table are significant at the 0.05 level. ***P<0.001, **P<0.01, *P<0.05. NS or no labeling indicates no significant difference.

[0090] In summary, symbiotic relationships between arbuscular mycorrhizal (AM) fungi and host plants can promote the absorption of nutrients by the host plant roots, enhance the plant's resistance to abiotic stress and tolerance to soil-borne diseases, and regulate the synthesis and distribution of plant hormones. Different hosts and substrates have a highly significant impact on plant growth. Nutrient-poor sandy soil, vermiculite, and perlite were found to be unsuitable for mycorrhizal infection and plant growth, while substrate combinations with added peat moss under the same host and stress conditions showed increased infection rates, spore production, and mycelial density. After invading host plants, arbuscular mycorrhizal fungi require adequate nutrients to maintain initial growth; simultaneously, organic matter stimulates spore germination and mycelial infection. The substrates for propagating arbuscular mycorrhizal fungi using RV (river sand + vermiculite + peat moss 1:4:1) and ZP (zeolite + peat moss + vermiculite 1:2:1) supplemented with peat moss exhibit good sporulation and mycelial density. The good aeration and high water retention of the soil facilitate mycorrhizal extension and expansion, promoting the development of mycelia and spores of arbuscular mycorrhizal fungi. The overall sporulation rate of the ac hosts (oats and clover) is higher than that of other hosts. The combination of RV and ac in this invention is most beneficial to plant biomass growth, sporulation efficiency, and infection rate.

[0091] Drought is one of the most common abiotic stressors in nature. The infection rate of AM fungi initially increases and then decreases with increasing drought stress. The results of this invention indicate that 5 days of drought stress is the optimal drought stress for AM fungal propagation, with the highest sporulation. Under 7 days of drought stress, plant biomass is not high, but the degree of infection, sporulation, and arbuscular density increase. The combination of RV and ac is most beneficial to host growth, sporulation, and infection rate. Similarly, some studies suggest that the increase or decrease in fungal numbers under high or low temperatures is a result of increased abundance of thermotolerant or thermophilic bacterial populations, while a decrease in fungal numbers is due to high temperatures reducing the tolerance of their propagules to stress. Although the effects of drought stress on plant growth are not significantly different, this does not mean that drought stress has no impact on plants. In reality, plants initiate a series of complex physiological and biochemical response mechanisms when faced with drought stress in order to maintain their growth and survival. However, under the conditions of this invention, plants may adapt to the drought environment by adjusting their physiological state, such as changing stomatal conductance and regulating the synthesis of osmotic regulators, thereby making the differences in growth rate less significant.

[0092] Further analysis was conducted on the effects of the dual factors of substrate and host, as well as the interaction of the three factors of substrate, host, and drought stress, on the fresh weight of the underground parts. The dual factors of substrate and host had a highly significant impact on the underground parts, indicating that their interaction is crucial for the growth and development of plant roots. A suitable substrate and host combination can provide favorable growth conditions for the roots, promoting root expansion and nutrient absorption. The highly significant interaction of the three factors of substrate, host, and drought stress on the fresh weight of the underground parts suggests that plant growth is comprehensively regulated by multiple factors under complex environmental conditions. This multi-factor interaction may involve multiple levels of plant physiology, ecology, and genetics, providing a new perspective for a deeper understanding of the mechanisms by which plants adapt to environmental changes.

[0093] Regarding nutrient stress, 7 days of nutrient stress was the optimal treatment. Substrate composition, host combination, and stress conditions all influenced the number of mycorrhizal fungal spores, mycorrhizal infection, and the growth and development of arbuscular mycorrhizal branches to varying degrees. The combination of ZP and ac was most beneficial to host growth, while the combination of ZP and dc was most beneficial to sporulation and infection rate. 7 days of nutrient stress was the optimal treatment, significantly affecting the number of mycorrhizal fungal spores, mycorrhizal infection, and the growth and development of arbuscular mycorrhizal branches. Nutrient stress also significantly affected spore density and infection intensity; the pairwise and triadic interactions among substrate, host, and stress treatment all significantly influenced spore density and infection intensity. Furthermore, compared to drought stress, plant biomass was relatively better under nutrient stress, indicating that nutrient stress had less disturbance to plant growth, and plants may be able to adapt to nutrient stress environments to some extent through their own regulatory mechanisms. A complex synergistic effect exists between the substrate and the host. This synergistic effect may be achieved by the substrate providing a suitable growth environment for the host, while the host's characteristics affect the efficiency of nutrient utilization in the substrate.

[0094] Different substrates significantly affected the aboveground and underground biomass of pumpkin. RV (radical violet) showed the highest value among all substrates and was significantly higher than other substrates. This indicates that RV substrate performed optimally in supporting the growth of the aboveground parts of pumpkin, possibly due to its unique nutrient content, water and fertilizer retention capacity, or aeration, better meeting the material and environmental needs of the aboveground parts. The average fresh weight of the underground parts in ZV substrate was 7.11 g, significantly higher than other substrates. The combination of zeolite, vermiculite, and perlite better promoted root growth. ZP substrate had the highest spore density, with a propagation effect of 26.67 times. This result provides supporting evidence for further research on the propagation of endophytic arbuscular mycorrhizal fungi using pumpkin as a host.

[0095] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for promoting the sporulation and propagation of arbuscular mycorrhizal fungi, characterized in that, Arbuscular mycorrhizal fungi were inoculated into the substrate, host seeds were sown in the substrate, and normal care was carried out. Stress treatment was carried out 5-7 weeks after the host seeds were sown, and samples were harvested after the culture period was completed. The stress treatment is drought stress treatment and / or nutrient stress treatment.

2. The method for promoting sporulation and propagation of arbuscular mycorrhizal fungi according to claim 1, characterized in that, The drought stress treatment involves reducing watering for 3-8 days, preferably not watering for 5-7 days. And / or, the nutritional stress treatment involves reducing the addition of nutrients for 3-8 days, preferably 3-7 days.

3. The method for promoting sporulation and propagation of arbuscular mycorrhizal fungi according to claim 1 or 2, characterized in that, The normal maintenance involves watering once a day, and applying Hoagland nutrient solution every three days starting from the 7th day after sowing. And / or, the nutrient stress treatment uses Hoagland nutrient solution diluted 3-7 times.

4. The method for promoting sporulation and propagation of arbuscular mycorrhizal fungi according to claim 3, characterized in that, The above-mentioned method of promoting spore production and propagation of arbuscular mycorrhizal fungi was carried out by pot cultivation. And / or, the arbuscular mycorrhizal fungus is *M. moses*.

5. The method for promoting sporulation and propagation of arbuscular mycorrhizal fungi according to claim 4, characterized in that, The matrix is ​​composed of multiple materials including zeolite, river sand, vermiculite, perlite, and peat moss. And / or, the host seed is one or more seeds selected from oats, pumpkin, clover and sorghum.

6. The method for promoting sporulation and propagation of arbuscular mycorrhizal fungi according to claim 5, characterized in that, The matrix is ​​one of the following: zeolite + river sand, river sand + vermiculite + peat moss, zeolite + vermiculite + perlite, or zeolite + peat moss + vermiculite. And / or, the host seed is oat, oat + clover, pumpkin, or sorghum + clover.

7. The method for promoting spore production and propagation of arbuscular mycorrhizal fungi according to claim 6, characterized in that, In the zeolite + river sand matrix, the mass ratio of zeolite to river sand is (1-2):(1-2), preferably 1:1; in the river sand + vermiculite + peat moss matrix, the mass ratio of river sand, vermiculite, and peat moss is (1-2):(3-5):(1-2), preferably 1:4:1; in the zeolite + vermiculite + perlite matrix, the mass ratio of zeolite, vermiculite, and perlite is (1-3):(1-2)(1-2), preferably 2:1:1; in the zeolite + peat moss + vermiculite matrix, the mass ratio of zeolite, peat moss, and vermiculite is (1-2):(1-3)(1-2), preferably 1:2:

1.

8. The method for promoting sporulation and propagation of arbuscular mycorrhizal fungi according to claim 7, characterized in that, The specific method is as follows: (1) Mix river sand, vermiculite and peat moss in a mass ratio of (1-2):(3-5):(1-2), sterilize the mixed substrate and place it in a flowerpot; preferably, the mass ratio of river sand, vermiculite and peat moss is 1:4:

1. (2) Inoculate the arbuscular mycorrhizal fungal agent into the substrate of the flowerpot; the inoculation amount of the arbuscular mycorrhizal fungus in the substrate is 245-300 spores / L substrate; (3) Mix sterilized oat seeds and clover seeds and sow them into the substrate. Water once a day after sowing. Starting from the 7th day after sowing, water with Hoagland nutrient solution every three days. After 6 weeks of sowing, subject the plants to drought stress treatment of 5 or 7 days without watering. (4) Samples were collected after the 12th week of cultivation following sowing.

9. The method for promoting spore production and propagation of arbuscular mycorrhizal fungi according to claim 7, characterized in that, The specific method is as follows: (1) Mix zeolite and river sand in a mass ratio of (1-2):(1-2), sterilize the mixed substrate, and place it in a flowerpot; preferably, the mass ratio of zeolite and river sand is 1:

1. (2) Inoculate the arbuscular mycorrhizal fungal agent into the substrate of the flowerpot; the inoculation amount of the arbuscular mycorrhizal fungus in the substrate is 245-300 spores / L substrate; (3) Mix sterilized sorghum seeds and clover seeds and sow them into the substrate. Water once a day after sowing. Starting from the 7th day after sowing, water with Hoagland nutrient solution every three days. Six weeks after sowing, dilute Hoagland nutrient solution 5 times and perform nutrient stress treatment for 3 or 7 days. (4) Samples were collected after the 12-week cultivation period following sowing.

10. The method for promoting sporulation and propagation of arbuscular mycorrhizal fungi according to claim 7, characterized in that, The specific method is as follows: (1) Mix zeolite, peat moss and vermiculite in a mass ratio of (1-2):(1-3)(1-2), sterilize the mixed substrate and place it in a flowerpot; preferably, the mass ratio of zeolite, peat moss and vermiculite is 1:2:

1. (2) Inoculate the arbuscular mycorrhizal fungal agent into the substrate of the flowerpot; the inoculation amount of the arbuscular mycorrhizal fungus in the substrate is 245-300 spores / L substrate; (3) After sterilizing the pumpkin seeds, germinate them and sow them into the substrate. Water once a day after sowing. Starting from the 7th day after sowing, water with Hoagland nutrient solution every three days. Six to seven weeks after sowing, dilute Hoagland nutrient solution 5 times and perform nutrient stress treatment for 3 or 7 days. (4) Samples were collected after the 12-week cultivation period following sowing.

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