A method for segmented propagation of arbuscular mycorrhizal fungi

Abstract

This application belongs to the technical field of fungal propagation, and specifically relates to a method for segmented propagation of arbuscular mycorrhizal fungi. In the segmented propagation method described in this application, zeolite and phosphate rock powder are used as culture substrates, significantly reducing the preparation cost of the culture substrates for arbuscular mycorrhizal fungi. Different exogenous substances are added during the growth stage of the host plant, the arbuscular mycorrhizal fungi infection stage, and the arbuscular mycorrhizal fungi sporulation stage, or the humidity of each stage is further controlled to increase the root-shoot ratio of the host plant, promote the infection and sporulation of arbuscular mycorrhizal fungi, shorten the production cycle of arbuscular mycorrhizal fungi inoculants, and increase the propagules of arbuscular mycorrhizal fungi inoculants, thereby obtaining high-yield and high-quality arbuscular mycorrhizal fungi inoculants efficiently and at low cost.

Description

Technical Field

[0001] This application belongs to the technical field of fungal propagation, and specifically relates to a method for segmented propagation of arbuscular mycorrhizal fungi. Background Art

[0002] Mycorrhiza is a common plant symbiotic phenomenon in nature, which is a symbiotic body formed by mycorrhizal fungi in the soil and the roots of higher plants. Arbuscular mycorrhizal fungi (AMF) are widely distributed in nature, have a very wide range of hosts, can form mutually beneficial symbiotic bodies with the roots of most monocotyledonous and dicotyledonous plants, and are a type of symbiotic endophytic fungi that are widely colonized in plant roots and play an important role in plant growth and development. Arbuscular mycorrhizal fungi have functional diversity, can improve the absorption of mineral elements by host plants, promote the growth and development of host plants, improve the rhizosphere microbial environment of plants, improve the tolerance of host plants to drought, salinity, extreme temperature, humidity, heavy metals, and can enhance the stress resistance ability of host plants to pathogenic microorganisms such as bacteria, fungi, and nematodes, thereby improving the growth status of host plants. In addition, arbuscular mycorrhizal fungi can improve the soil while reducing the application of chemical fertilizers and pesticides and reducing environmental pollution, and play an important role in maintaining the agricultural ecological balance. In recent years, it has been reported that arbuscular mycorrhizal fungi play a significant role in improving salinity, disease resistance, and growth promotion, and some related mechanisms have been elucidated.

[0003] Therefore, standardizing and scaling up the production of arbuscular mycorrhizal fungi through an efficient propagation system has high application value as a new type of microbial fertilizer; making it into a microbial inoculant has become a research hotspot for people currently.

[0004] The method of propagation culture using greenhouse potting is currently the main method for large-scale production of arbuscular mycorrhizal fungi inoculants, with simple operation and reliable method. When using greenhouse potting culture, the growth of arbuscular mycorrhizal fungi is affected by various factors such as host plants, propagation substrates, humidity, temperature, and nutrients, and different hosts will produce different responses to the infection and sporulation of arbuscular mycorrhizal fungi. In addition, there are currently problems in the production of arbuscular mycorrhizal fungi such as limited production quantity of inoculants, small number of propagules, easy contamination during the propagation process, and unstable quality of inoculant products. Therefore, developing an efficient, simple, low-cost, scientific, and suitable propagation method for large-scale breeding has an important role in production practice. Summary of the Invention

[0005] The purpose of this application is to provide an efficient segmented propagation method that can greatly increase the propagules of arbuscular mycorrhizal fungi, standardize the large-scale production of arbuscular mycorrhizal fungi inoculants. This method has low cost and high yield, and can provide high-quality arbuscular mycorrhizal fungi inoculant products.

[0006] The present application provides a method for segmented propagation of arbuscular mycorrhizal fungi, which is characterized by including the following steps:

[0007] S1 - Host plant growth: Sow the seeds of the host plant into the culture substrate. After emergence for 5 - 10 days, irrigate with indole - 3 - acetic acid solution, mineral source fulvic acid potassium solution, and Hoagland nutrient solution.

[0008] S2 - Inoculation of arbuscular mycorrhizal fungi: After the host plant grows 2 - 5 true leaves, inoculate with arbuscular mycorrhizal fungal inoculum.

[0009] S3 - Arbuscular mycorrhizal fungi infection stage: After inoculation, irrigate with abscisic acid solution and phosphorus - deficient Hoagland nutrient solution.

[0010] S4 - Arbuscular mycorrhizal fungi spore - producing stage: 2 - 4 weeks after inoculation, irrigate with flavonoids and phosphorus - deficient Hoagland nutrient solution.

[0011] S5 - Harvest of arbuscular mycorrhizal fungi products: 10 - 16 weeks after inoculation, stop irrigation. After the propagation substrate dries, cut off the above - ground part to harvest the propagules of arbuscular mycorrhizal fungi.

[0012] The propagules obtained by the present application include plant roots, hyphae, spores, and culture substrate.

[0013] In the propagation method of the present application, in S1 and S2 segments, the relative humidity of the culture substrate is 40 - 60%; in S3 and S4 segments, the relative humidity of the culture substrate is 15 - 30%.

[0014] The propagation method of the present application preferably uses greenhouse pot cultivation; preferably, the culture container is disinfected before use, and more preferably, it is disinfected with a disinfectant solution. A rectangular plastic basket can be used for propagation, preferably with a bottom length of 45 cm, a basket width of 32 cm, and a height of 15 cm. The propagation temperature of the present application is the suitable growth temperature of corn, and natural light is used for illumination or fluorescent lamps (such as Philips 36W) are used to supplement natural light; preferably, the propagation daytime temperature is 20 - 30 °C, the nighttime temperature is 15 - 25 °C, and the daily light - cultivation time is 12 - 15 h; more preferably, the propagation daytime temperature is 25 - 30 °C, and the nighttime temperature is 20 - 25 °C.

[0015] The host plant of the present application is preferably corn or sorghum. The seeding density of the host plant seeds is 40 - 60 cm 2 / seed, preferably 45 - 55 cm 2 / seed, and more preferably 50 cm 2per plant to establish a high-density host plant body and improve space utilization. Before sowing, the seeds are disinfected, cleaned with clean water, and then soaked in clean water for germination treatment; preferably, they are disinfected with 10% H2O2 for 10 minutes, cleaned with clean water, and soaked in clean water for 12 h for germination treatment.

[0016] The arbuscular mycorrhizal fungi propagated in this application are preferably Rhizophagus irregulris, Rhizophagus intraradice, Glomus mosseae, and Glomus etunicatum.

[0017] The culture medium in this application is a mixed matrix of zeolite and phosphate rock powder, preferably a mixed matrix of zeolite and phosphate rock powder in a volume ratio of 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, preferably a mixed matrix of zeolite and phosphate rock powder with different mesh numbers. In a specific embodiment, the culture medium is a mixed matrix of 20-40 mesh zeolite, 40-60 mesh zeolite, 60-80 mesh zeolite, and phosphate rock powder, and the volume ratio is preferably (1-3):(4-6):(1-3):1, more preferably 2:5:2:1. The mixed matrix in this application has a low cost, and can improve the water holding capacity of the culture medium, alleviate the severity of humidity changes, extend the watering cycle of the matrix and other physical properties, which is beneficial to the growth and extension of hyphae, and is beneficial to water and gas exchange, thus providing a good microenvironment for the reproduction of arbuscular mycorrhizal fungi. Preferably, the culture medium is sterilized before use, and more preferably, it is sterilized with a disinfectant solution and dried for use.

[0018] In the S1 stage, irrigating indoleacetic acid solution and mineral source fulvic acid potassium solution can promote the growth of the roots of host plants and increase the root-shoot ratio. The indoleacetic acid solution, mineral source fulvic acid potassium solution, and Hoagland nutrient solution refer to an aqueous solution containing 50 μmol / L - 100 μmol / L indoleacetic acid, 0.3% - 2 wt% mineral source fulvic acid potassium, and 20 wt% Hoagland nutrient solution with a weight of 10-20% of the culture medium.

[0019] Preferably, an aqueous solution with a weight of 15% of the culture medium is irrigated. The concentration of indoleacetic acid in the irrigated aqueous solution is preferably 50, 60, 70, 80, 90, 100 μmol / L, and the concentration of mineral source fulvic acid potassium is preferably 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2 wt%.

[0020] In the S1 stage, the relative humidity of the culture medium is controlled at 40-60%, preferably 40-50%.

[0021] The specific composition of the Hoagland nutrient solution is as follows: potassium sulfate 607 mg / L, diammonium hydrogen phosphate 115 mg / L, magnesium sulfate 493 mg / L, sodium iron EDTA 20 mg / L, ferrous sulfate 2.86 mg / L, borax 4.5 mg / L, manganese sulfate 2.13 mg / L, copper sulfate 0.05 mg / L, zinc sulfate 0.22 mg / L, ammonium sulfate 0.02 mg / L, calcium nitrate 0.945 g / L, and the remaining component is water, with a total volume of 1 L.

[0022] In the S1 stage, the irrigation means: (1) Irrigate indole acetic acid solution, mineral source fulvic acid potassium solution and Hoagland nutrient solution, and supplement water as needed to control the relative humidity of the culture medium to 40 - 60%; (2) After the relative humidity of the culture medium is lower than 40%, irrigate indole acetic acid solution, mineral source fulvic acid potassium solution and Hoagland nutrient solution again, and supplement water as needed to control the relative humidity of the culture medium to 40 - 60%; (3) Repeat step (2) as needed.

[0023] In the S2 stage, after the host plant grows 3 - 4 true leaves, inoculate with arbuscular mycorrhizal fungal inoculant. Preferably, inoculate 1 - 2 wt% of the arbuscular mycorrhizal fungal inoculant in the culture medium, and the propagule number of the inoculant is greater than 70 per mL.

[0024] In the S3 stage, irrigating abscisic acid solution can promote the infection rate of arbuscular mycorrhizal fungi. The abscisic acid solution and phosphorus - deficient Hoagland nutrient solution refer to an aqueous solution containing 50 μmol / L - 100 μmol / L abscisic acid and 20% phosphorus - deficient Hoagland nutrient solution, which is 10 - 20% of the weight of the culture medium.

[0025] Preferably, irrigate an aqueous solution with a weight of 15% of the culture medium. The concentration of abscisic acid in the irrigated aqueous solution is preferably 50, 60, 70, 80, 90, 100 μmol / L.

[0026] In the S3 stage, the relative humidity of the culture medium is controlled at 15 - 30%, preferably 15 - 25%.

[0027] In the S3 stage, the irrigation means: (1) Irrigate abscisic acid solution and phosphorus - deficient Hoagland nutrient solution, and supplement water as needed to control the relative humidity of the culture medium to 15 - 30%; (2) When the relative humidity of the culture medium is lower than 15%, irrigate abscisic acid solution and phosphorus - deficient Hoagland nutrient solution again, and supplement water as needed to control the relative humidity of the culture medium to 15 - 30%; (3) Repeat step (2) as needed.

[0028] In the S4 stage, irrigating with flavonoids can promote the production of arbuscular mycorrhizal fungal spores. The flavonoids and the phosphorus-deficient Hoagland nutrient solution refer to an aqueous solution containing 100 nM - 200 nM flavonoids and 20% phosphorus-deficient Hoagland nutrient solution, which is 10 - 20% by weight of the culture substrate.

[0029] Preferably, an aqueous solution with a weight of 15% of the culture substrate is irrigated. The concentration of flavonoids in the irrigated aqueous solution is preferably 100, 120, 150, 180, 200 nM.

[0030] In the S4 stage, the relative humidity of the culture substrate is controlled at 15 - 30%, preferably 15 - 25%.

[0031] In the S4 stage, the irrigation means: (1) Irrigating with flavonoids and the phosphorus-deficient Hoagland nutrient solution, and supplementing water as needed to control the relative humidity of the culture substrate at 15 - 30%; (2) When the relative humidity of the culture substrate is lower than 15%, irrigating again with flavonoids and the phosphorus-deficient Hoagland nutrient solution, and supplementing water as needed to control the relative humidity of the culture substrate at 15 - 30%; (3) Repeating step (2) as needed.

[0032] In the S3 and S4 stages, the composition of the phosphorus-deficient Hoagland nutrient solution is: potassium sulfate 607 mg / L, diammonium hydrogen phosphate 115 mg / L, magnesium sulfate 493 mg / L, sodium ferric EDTA 20 mg / L, ferrous sulfate 2.86 mg / L, borax 4.5 mg / L, manganese sulfate 2.13 mg / L, copper sulfate 0.05 mg / L, zinc sulfate 0.22 mg / L, ammonium sulfate 0.02 mg / L, calcium nitrate 0.945 g / L, and the remaining component is water, with a total volume of 1 L.

[0033] In the S5 stage, after stopping irrigation, it is placed in an environment with relatively stable temperature and humidity for drying for 1 - 2 weeks. After the propagation substrate is completely dry, the above-ground part is removed, and the propagules of arbuscular mycorrhiza are harvested.

[0034] In one embodiment, the step-by-step propagation method described in this application includes the following steps:

[0035] S1 stage - Host plant growth: Seeds of the host plant maize are placed at 40 - 60 cm 2Sown into the culture medium at a seeding density of [X] seeds per plant. One week after emergence, (1) irrigate with an aqueous solution containing 50 μmol / L - 100 μmol / L indoleacetic acid, 0.3% - 2 wt% mineral source fulvic acid potassium, and 20 wt% Hoagland nutrient solution, accounting for 15% of the weight of the culture medium, and supplement water as needed to control the relative humidity of the culture medium at 40 - 60%; (2) after the relative humidity of the culture medium is lower than 40%, irrigate again with an aqueous solution containing 50 μmol / L - 100 μmol / L indoleacetic acid, 0.3% - 2 wt% mineral source fulvic acid potassium, and 20 wt% Hoagland nutrient solution, accounting for 15% of the weight of the culture medium, and supplement water as needed to control the relative humidity of the culture medium at 40 - 60%; (3) repeat step (2) as needed;

[0036] S2 - inoculating arbuscular mycorrhizal fungi: After the host plant grows 3 - 4 true leaves, inoculate with arbuscular mycorrhizal fungal inoculum;

[0037] S3 - arbuscular mycorrhizal fungi infection stage: After inoculation, (1) irrigate with an aqueous solution containing 50 μmol / L - 100 μmol / L abscisic acid and 20% phosphorus - deficient Hoagland nutrient solution, accounting for 15% of the weight of the culture medium, and supplement water as needed to control the relative humidity of the culture medium at 15 - 30%; (2) when the relative humidity of the culture medium is lower than 15%, irrigate again with an aqueous solution containing 50 μmol / L - 100 μmol / L abscisic acid and 20% phosphorus - deficient Hoagland nutrient solution, accounting for 15% of the weight of the culture medium, and supplement water as needed to control the relative humidity of the culture medium at 15 - 30%; (3) repeat step (2) as needed;

[0038] S4 - arbuscular mycorrhizal fungi spore - producing stage: 2 - 4 weeks after inoculation, (1) irrigate with an aqueous solution containing 1 nM - 200 nM flavonoids and 20% phosphorus - deficient Hoagland nutrient solution, accounting for 15% of the weight of the culture medium, and supplement water as needed to control the relative humidity of the culture medium at 15 - 30%; (2) when the relative humidity of the culture medium is lower than 15%, irrigate again with an aqueous solution containing 1 nM - 200 nM flavonoids and 20% phosphorus - deficient Hoagland nutrient solution, accounting for 15% of the weight of the culture medium, and supplement water as needed to control the relative humidity of the culture medium at 15 - 30%; (3) repeat step (2) as needed;

[0039] S5 - product harvest: 10 - 16 weeks after inoculating the arbuscular mycorrhizal fungal strain, stop irrigation, place it in an environment with relatively stable temperature and humidity for drying for 1 - 2 weeks. After the propagation substrate is dry, cut off the above - ground part to harvest the propagules of arbuscular mycorrhizal fungi;

[0040] The propagation temperature of the method is the suitable growth temperature of corn, and natural light irradiation or fluorescent lamps are used to supplement natural light; preferably, the daytime temperature during propagation is 20-30°C, the nighttime temperature is 15-25°C, and the daily light culture time is 12-15h; more preferably, the daytime temperature is 25-30°C and the nighttime temperature is 20-25°C.

[0041] The culture medium is a mixed substrate with a volume ratio of 2:5:2:1 of 20-40 mesh zeolite, 40-60 mesh zeolite, 60-80 mesh zeolite, and phosphate rock powder.

[0042] In the propagation method described in this application, when the host plant grows to the flowering and grain-filling stage, decapitation treatment is carried out, which can change the root-shoot ratio of the plant, inhibit the height of the host plant, regulate vegetative growth, inhibit reproductive growth, and reduce the consumption of plant nutrients by reproductive growth.

[0043] The irrigation described in this application is preferably irrigation by the root application method.

[0044] In the propagation method described in this application, the host plant roots and the mixed propagation substrate can be randomly taken in the spore-producing section to detect the infection and spore-producing effect of arbuscular mycorrhizal fungi. For example, the host plant roots and the mixed propagation substrate are randomly taken at the 6th week, 10th week, and 15th week after inoculating the arbuscular mycorrhizal fungal strain to detect the infection and spore-producing effect of arbuscular mycorrhizal fungi.

[0045] In this application, the Phillips and Haymann trypan blue staining method is used to determine the infection rate; the wet sieving and decantation technique combined with the sugar centrifugation method is used to determine the spore yield.

[0046] In this application, the aqueous solution of the irrigation and the inoculation amount of the arbuscular mycorrhizal fungal agent both refer to the ratio to the culture medium before sowing.

[0047] Compared with the current technology, this application has the following beneficial effects:

[0048] The segmented propagation method described in this application shortens the production cycle of the fungal agent, and can obtain high-yield and high-quality arbuscular mycorrhizal fungal agent products at low cost and high efficiency. The propagules in the fungal agent can be as high as more than 1000 / mL.

[0049] 1. This application adopts a segmented propagation method, adding different exogenous substances in the host plant growth stage, arbuscular mycorrhizal fungi infection stage, and spore-producing stage to promote the growth of host plant roots, the infection and spore production of arbuscular mycorrhizal fungi in each stage, increase the spore content in the fungal agent, and improve the product quality.

[0050] 2. This application uses a mixed substrate of zeolite and phosphate rock powder. On the one hand, the cost is low. On the other hand, it can improve the water holding capacity, alleviate the severity of drastic humidity changes, extend the watering cycle, and is beneficial to the growth and extension of hyphae.

[0051] 3. The present application further controls the humidity in each stage, such as the host plant growth stage, the arbuscular mycorrhizal fungus infection stage, and the spore production stage, etc., which can further promote the growth of the host plant roots, as well as the infection and spore production of the arbuscular mycorrhizal fungus, and is beneficial to efficiently obtaining high-yield and high-quality inoculant products. Detailed Embodiments

[0052] The following provides a detailed description of the specific embodiments of the present application. It should be understood that the specific embodiments described herein are only for the purpose of illustrating and explaining the present application, and are not used to limit the present application.

[0053] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0054] The terms "comprise" and "include" in the present application and any variations thereof are intended to cover non-exclusive inclusion.

[0055] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0056] Example 1:

[0057] Greenhouse pot culture was used for propagation. The culture temperature was 25°C - 30°C during the day and 20 - 25°C at night. During the entire culture period, natural light was used or fluorescent lamps (Philips 36W) were supplemented as needed. The daily light culture time was 12 - 15h.

[0058] Before use, the culture containers were disinfected with a disinfectant. The culture containers were rectangular plastic baskets with a bottom length of 45cm, a width of 32cm, and a height of 15cm.

[0059] The culture substrate was a mixed substrate obtained by mixing zeolites with particle sizes of 20 - 40 mesh, 40 - 60 mesh, 60 - 80 mesh, and phosphate rock powder in a volume ratio of 2:5:2:1. Before use, it was disinfected with a disinfectant and dried. 2 / 3 of the volume of the culture substrate was filled into the culture containers.

[0060] Before sowing, the corn seeds were disinfected with 10% H2O2 for 10 minutes, then washed clean with clear water, and soaked in clear water for 12 hours for later use.

[0061] S1 - Host plant growth stage: Sow 28 corn seeds evenly into the culture medium. One week after emergence, (1) irrigate with an aqueous solution containing 50 μmol / L - 100 μmol / L indole acetic acid, 0.3% - 2 wt% mineral source fulvic acid potassium, and 20 wt% Hoagland nutrient solution, with a weight of 15% of the culture medium, and supplement water as needed to control the relative humidity of the culture medium at 40 - 60%; (2) after the relative humidity of the culture medium is lower than 40%, irrigate again with an aqueous solution containing 50 μmol / L - 100 μmol / L indole acetic acid, 0.3% - 2 wt% mineral source fulvic acid potassium, and 20 wt% Hoagland nutrient solution, with a weight of 15% of the culture medium, and supplement water as needed to control the relative humidity of the culture medium at 40 - 60%; (3) repeat step (2) as needed;

[0062] S2 - Inoculation of arbuscular mycorrhizal fungal strain: After the corn has grown 3 - 4 true leaves, inoculate with a commercial inoculant of Rhizophagus irregulris, with an inoculation amount of 1 wt% of the culture medium, and the number of propagules of the inoculant is greater than 70 per mL;

[0063] S3 - Arbuscular mycorrhizal fungal infection stage: After inoculation, (1) irrigate with an aqueous solution containing 50 μmol / L - 100 μmol / L abscisic acid and 20% phosphorus - deficient Hoagland nutrient solution, with a weight of 15% of the culture medium, and supplement water as needed to control the relative humidity of the culture medium at 15 - 30%; (2) when the relative humidity of the culture medium is lower than 15%, irrigate again with an aqueous solution containing 50 μmol / L - 100 μmol / L abscisic acid and 20% phosphorus - deficient Hoagland nutrient solution, with a weight of 15% of the culture medium, and supplement water as needed to control the relative humidity of the culture medium at 15 - 30%; (3) repeat step (2) as needed;

[0064] S4 - Arbuscular mycorrhizal fungal spore - producing stage: Two weeks after inoculation, (1) irrigate with an aqueous solution containing 100 nM - 200 nM flavonoids and 20% phosphorus - deficient Hoagland nutrient solution, with a weight of 15% of the culture medium, and supplement water as needed to control the relative humidity of the culture medium at 15 - 30%; (2) when the relative humidity of the culture medium is lower than 15%, irrigate again with an aqueous solution containing 100 nM - 200 nM flavonoids and 20% phosphorus - deficient Hoagland nutrient solution, with a weight of 15% of the culture medium, and supplement water as needed to control the relative humidity of the culture medium at 15 - 30%; (3) repeat step (2) as needed;

[0065] Harvest of arbuscular mycorrhizal fungal products: After 15 weeks of maize growth, irrigation was stopped. After the propagation substrate dried, the above-ground parts were cut off. Approximately 1 week later, propagules of arbuscular mycorrhizal fungi (including plant roots, hyphae, spores, and substrate) were harvested.

[0066] Product quality inspection: At the 6th week, 10th week, and 15th week after inoculating the arbuscular mycorrhizal fungal strains, host plant roots and mixed propagation substrates were randomly sampled to detect the infection and sporulation effects of arbuscular mycorrhizal fungi. The Phillips and Haymann trypan blue staining method was used to determine the infection rate; the wet sieving decantation technique combined with sugar centrifugation was used to determine the spore production.

[0067] Example 2: Rhizophagus intraradice and Glomus mosseae were propagated in the greenhouse. The specific implementation method of propagation was the same as that in Example 1.

[0068] Comparative Example 1-1:

[0069] Rhizophagus irregulris was propagated in the greenhouse. In the S1 segment - host plant growth segment different from Example 1, indole-3-acetic acid was not added, and the remaining propagation and management methods were the same as those in Example 1.

[0070] Comparative Example 1-2:

[0071] Rhizophagus irregulris was propagated in the greenhouse. In the S3 segment - infection segment different from Example 1, abscisic acid solution was not added, and the remaining propagation and management methods were the same as those in Example 1.

[0072] Comparative Example 1-3:

[0073] Rhizophagus irregulris was propagated in the greenhouse. Different from Example 1, flavonoids were not added in the S4 segment - sporulation segment, and the remaining propagation and management methods were the same as those in Example 1.

[0074] Comparative Example 1-4:

[0075] Rhizophagus irregulris was propagated in the greenhouse. Different from Example 1, the relative humidity of the culture medium substrate in the S1 segment - host plant growth segment was controlled at 15% - 20%, and the remaining propagation and management methods were the same as those in Example 1.

[0076] Result analysis:

[0077] Effect of different treatments in each segment on the infection of arbuscular mycorrhizal fungi:

[0078] After culturing for 6 weeks, 10 weeks, and 15 weeks respectively according to the methods of Example 1, Comparative Example 1-1, Comparative Example 1-2, Comparative Example 1-3, and Comparative Example 1-4, corn roots and the propagation substrate were taken to detect the infection and sporulation effects. The results of the infection effect determination of Rhizophagus irregularis are shown in Table 1. The results of the sporulation effect determination of Rhizophagus irregularis are shown in Table 2.

[0079] Table 1: Results of the infection effect determination of Rhizophagus irregularis

[0080]

[0081] From the data in Table 1 above, at the 6th week, the infection rates of Example 1, Comparative Example 1-1, Comparative Example 1-2, Comparative Example 1-3, and Comparative Example 1-4 were 48.78%, 33.13%, 28.56%, 38.77%, and 31.27 respectively; at the 10th week, the infection rates reached 76.87%, 57.37, 48.44, 55.22, and 50.89 respectively, and at the 15th week, the infection rates reached 91.62%, 72.43%, 58.74%, 69.62, and 61.82% respectively. There were significant differences between Example 1 and other comparative examples.

[0082] Effect of each different treatment on the number of arbuscular mycorrhizal fungi spores:

[0083] Table 2: Results of the determination of the number of spores in the propagation substrate of Rhizophagus irregularis

[0084]

[0085] From the data in Table 2 above, at the 6th week, the number of spores in the propagation substrate of Example 1, Comparative Example 1-1, Comparative Example 1-2, Comparative Example 1-3, and Comparative Example 1-4 were 234, 119, 108, 98, and 102 respectively; at the 10th week, the number of spores in the propagation substrate were 508, 234, 255, 212, and 187 respectively, and at the 15th week, the number of spores in the propagation substrate were 982, 327, 301, 289, and 277 respectively. There were significant differences between Example 1 and other comparative examples.

[0086] Effect of the same conditions on the growth of different arbuscular mycorrhizal fungi

[0087] Table 3: Results of the determination of the infection rate and spore production of different arbuscular mycorrhizal fungi under the same conditions

[0088]

[0089] From the data in Table 3 above, it can be obtained that by using the segmented propagation method provided by the present invention to culture Rhizophagus irregulris, Rhizophagus intraradice, and Glomus mosseae, the infection rate and spore production of each strain are very high.

[0090] From the above results, it can be seen that Example 1 is the best segmented propagation method provided by the present invention. However, the implementation mode of the present invention is not limited by the above embodiments. It should be noted that without departing from the principle of the present invention, several improvements, substitutions, and combinations can be made, and these improvements, substitutions, and combinations should also be regarded as the protection scope of the present invention.

[0091] In addition, it should be noted that among the various specific technical features described in the above specific implementation manners, they can be combined in any appropriate manner without conflict. To avoid unnecessary repetition, this application will not separately describe various possible combination manners.

[0092] In addition, any combination can be made among various different implementation manners of this application, as long as it does not violate the idea of this application, it should also be regarded as the content disclosed in this application.

Claims

1. A method for segmented propagation of arbuscular mycorrhizal fungi, characterized in that, It includes the following steps: Step S1 - Host plant growth: Sow the seeds of the host plant into the culture substrate. After emergence for 5 - 10 days, irrigate with indoleacetic acid solution, mineral source fulvic acid potassium solution, and Hoagland nutrient solution. Step S2 - Inoculation with arbuscular mycorrhizal fungi: After the host plant grows 2 - 5 true leaves, inoculate with arbuscular mycorrhizal fungal inoculum. Step S3 - Arbuscular mycorrhizal fungi infection stage: After inoculation, irrigate with abscisic acid solution and phosphorus-deficient Hoagland nutrient solution. Step S4 - Arbuscular mycorrhizal fungi spore production stage: 2 - 4 weeks after inoculation, irrigate with flavonoids and phosphorus-deficient Hoagland nutrient solution. Step S5 - Harvest of arbuscular mycorrhizal fungi products: Obtain the propagules of arbuscular mycorrhizal fungi. The culture substrate is a mixed substrate of zeolite and phosphate rock powder. In steps S3 and S4, the relative humidity of the culture substrate is 15 - 30%.

2. The stepwise propagation method according to claim 1, characterized in that In steps S1 and S2, the relative humidity of the culture substrate is 40 - 60%.

3. The stepwise propagation method according to claim 1 or 2, characterized in that The propagation temperature is the suitable growth temperature of corn, and natural light is used for irradiation or fluorescent lamps are used to supplement natural light.

4. The stepwise propagation method according to claim 3, wherein The propagation day temperature is 20 - 30 °C, the night temperature is 15 - 25 °C, and the daily light culture time is 12 - 15 h.

5. The segmented propagation method according to claim 1 or 2, characterized in that, The host plants are corn and sorghum.

6. The stepwise propagation method according to claim 5, characterized in that The seeding density of seeds in the S1 segment is 40 - 60 cm 2 / seed.

7. The stepwise propagation method according to claim 6, wherein The seeding density of the seeds in the S1 section is 45 - 55 cm 2 / seed.

8. The stepwise propagation method according to claim 7, characterized in that, The seeding density of the seeds in section S1 is 50 cm 2 / seed.

9. The stepwise propagation method according to claim 1 or 2, wherein The arbuscular mycorrhizal fungi are Rhizophagus irregulris, Rhizophagus intraradice, Glomus mosseae, or Glomus etunicatum.

10. The stepwise propagation method according to claim 1 or 2, characterized in that, The culture substrate is a mixed substrate of zeolites with different mesh numbers and phosphate rock powder.

11. The stepwise propagation method according to claim 10, wherein The culture substrate is a mixed substrate of 20 - 40 mesh zeolite, 40 - 60 mesh zeolite, 60 - 80 mesh zeolite, and phosphate rock powder.

12. The stepwise propagation method according to claim 11, wherein The volume ratio of 20 - 40 mesh zeolite, 40 - 60 mesh zeolite, 60 - 80 mesh zeolite, and phosphate rock powder in the culture substrate is (1 - 3):(4 - 6):(1 - 3):

1.

13. The stepwise propagation method according to claim 12, wherein The volume ratio of 20 - 40 mesh zeolite, 40 - 60 mesh zeolite, 60 - 80 mesh zeolite, and phosphate rock powder is 2:5:2:

1.

14. The stepwise propagation method according to claim 1 or 2, characterized in that, In step S1, the indoleacetic acid solution, mineral source fulvic acid potassium solution, and Hoagland nutrient solution refer to an aqueous solution containing 50 μmol / L - 100 μmol / L indoleacetic acid, 0.3% - 2 wt% mineral source fulvic acid potassium, and 20 wt% Hoagland nutrient solution, with a weight of 10 - 20% of the culture substrate.

15. The stepwise propagation method according to claim 14, wherein In step S1, the irrigation means: (1) Irrigate with indoleacetic acid solution, mineral source fulvic acid potassium solution, and Hoagland nutrient solution, and supplement water as needed to control the relative humidity of the culture substrate at 40 - 60%; (2) After the relative humidity of the culture substrate is lower than 40%, irrigate with indoleacetic acid solution, mineral source fulvic acid potassium solution, and Hoagland nutrient solution again, and supplement water as needed to control the relative humidity of the culture substrate at 40 - 60%; (3) Repeat step (2) as needed.

16. The stepwise propagation method according to claim 1 or 2, characterized in that, In step S2, inoculate 1 - 2 wt% of the arbuscular mycorrhizal fungal inoculum into the culture substrate, and the number of propagules of the inoculum is greater than 17. The stepwise propagation method according to claim 1 or 2, characterized in that In step S3, the abscisic acid solution and the phosphorus-deficient Hoagland nutrient solution refer to an aqueous solution that is 10-20% by weight of the culture medium and contains 50 μmol / L - 100 μmol / L abscisic acid and 20% phosphorus-deficient Hoagland nutrient solution.

18. The stepwise propagation method according to claim 17, wherein, In step S3, the irrigation refers to: (1) Irrigate the abscisic acid solution and the phosphorus-deficient Hoagland nutrient solution, and supplement water as needed to control the relative humidity of the culture medium at 15-30%; (2) When the relative humidity of the culture medium is below 15%, irrigate the abscisic acid solution and the phosphorus-deficient Hoagland nutrient solution again, and supplement water as needed to control the relative humidity of the culture medium at 15-30%; (3) Repeat step (2) as needed.

19. The segmented propagation method according to claim 1 or 2, characterized in that, In step S4, the flavonoid substance and the phosphorus-deficient Hoagland nutrient solution refer to an aqueous solution that is 10-20% by weight of the culture medium and contains 100 nM - 200 nM flavonoid substance and 20% phosphorus-deficient Hoagland nutrient solution.

20. The stepwise propagation method according to claim 19, characterized in that In step S4, the irrigation refers to: (1) Irrigate the flavonoid substance and the phosphorus-deficient Hoagland nutrient solution, and supplement water as needed to control the relative humidity of the culture medium at 15-30%; (2) When the relative humidity of the culture medium is below 15%, irrigate the flavonoid substance and the phosphorus-deficient Hoagland nutrient solution again, and supplement water as needed to control the relative humidity of the culture medium at 15-30%; (3) Repeat step (2) as needed.

21. The stepwise propagation method according to claim 1 or 2, characterized in that, In step S5, after stopping irrigation, place it in an environment with relatively stable temperature and humidity and dry for 1-2 weeks.

22. The stepwise propagation method according to claim 1 or 2, characterized in that When the host plant grows to the flowering and grain-filling stage, perform the decapitation treatment.

23. The stepwise propagation method according to claim 1 or 2, characterized in that, It includes the following steps: Stage S1 - Host plant growth: Sow the seeds of the host plant maize into the culture medium at a seeding density of 40 - 60 cm 2 / seed, and after 1 week of emergence, (1) irrigate with an aqueous solution containing 50 μmol / L - 100 μmol / L indoleacetic acid, 0.3% - 2 wt% mineral source fulvic acid potassium, and 20 wt% Hoagland nutrient solution, which is 15% of the weight of the culture medium, and supplement water as needed to control the relative humidity of the culture medium at 40 - 60%; (2) after the relative humidity of the culture medium is lower than 40%, irrigate again with an aqueous solution containing 50 μmol / L - 100 μmol / L indoleacetic acid, 0.3% - 2 wt% mineral source fulvic acid potassium, and 20 wt% Hoagland nutrient solution, which is 15% of the weight of the culture medium, and supplement water as needed to control the relative humidity of the culture medium at 40 - 60%; (3) repeat step (2) as needed; Step S2 - Inoculate arbuscular mycorrhizal fungi: After the host plant grows 3-4 true leaves, inoculate the arbuscular mycorrhizal fungal inoculum. Step S3 - Arbuscular mycorrhizal fungi infection stage: After inoculation, (1) Irrigate an aqueous solution that is 15% by weight of the culture medium and contains 50 μmol / L - 100 μmol / L abscisic acid and 20% phosphorus-deficient Hoagland nutrient solution, and supplement water as needed to control the relative humidity of the culture medium at 15-30%; (2) When the relative humidity of the culture medium is below 15%, irrigate an aqueous solution that is 15% by weight of the culture medium and contains 50 μmol / L - 100 μmol / L abscisic acid and 20% phosphorus-deficient Hoagland nutrient solution again, and supplement water as needed to control the relative humidity of the culture medium at 15-30%; (3) Repeat step (2) as needed; S4 - Arbuscular mycorrhizal fungi spore - producing stage: After 2 - 4 weeks of inoculation, (1) irrigate with an aqueous solution containing 100 nM - 200 nM flavonoid substances and 20% phosphorus - deficient Hoagland nutrient solution, with a weight of 15% of the culture substrate, and supplement water as needed to control the relative humidity of the culture substrate at 15 - 30%; (2) when the relative humidity of the culture substrate is lower than 15%, irrigate again with an aqueous solution containing 100 nM - 200 nM flavonoid substances and 20% phosphorus - deficient Hoagland nutrient solution, with a weight of 15% of the culture substrate, and supplement water as needed to control the relative humidity of the culture substrate at 15 - 30%; (3) repeat step (2) as needed; S5 - Product harvest: After 10 - 16 weeks of inoculating the arbuscular mycorrhizal fungi strain, stop irrigation, place it in an environment with relatively stable temperature and humidity for drying for 1 - 2 weeks. After the propagation substrate is dry, cut off the above - ground part to harvest the propagules of arbuscular mycorrhizal fungi; The propagation temperature of the method is the suitable growth temperature of corn, using natural light irradiation or supplementing natural light with fluorescent lamps; The culture substrate is a mixed substrate with a volume ratio of 2:5:2:1 of 2,0 - 40 - mesh zeolite, 40 - 60 - mesh zeolite, 60 - 80 - mesh zeolite, and phosphate rock powder; 24. The stepwise propagation method according to claim 23, wherein, The propagation daytime temperature is 20 - 30 °C, the nighttime temperature is 15 - 25 °C, and the daily light - cultivation time is 12 - 15 h.

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