A mycorrhizal fungus-containing microbial agent, a preparation method and application thereof
By preparing an inoculant containing arbuscular mycorrhizal fungi and using a matrix of tomato stem charcoal and magnesium carbonate, the problem of limited fungal growth in polluted soil was solved, the infection rate and tomato growth were improved, and a highly efficient plant growth promotion effect and cost savings were achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGSU VOCATION & TECHNICAL COLLEGE OF FINANCE & ECONOMICS
- Filing Date
- 2025-05-12
- Publication Date
- 2026-06-26
AI Technical Summary
Environmental pollutants and heavy metal residues affect the activity and infection rate of arbuscular mycorrhizal fungi, leading to limited plant growth. Existing technologies are insufficient to effectively improve the growth and infection rate of arbuscular mycorrhizal fungi in polluted soil.
By using an inoculant containing arbuscular mycorrhizal fungi, a suitable growth environment is provided by preparing a matrix containing tomato stem charcoal and magnesium carbonate, which promotes fungal colonization and spore protection. Combined with the nutrients and antibacterial effects of tomato stems and leaves, the infection rate and plant growth are improved.
It improved the infection rate of arbuscular mycorrhizal fungi and the growth of tomatoes, reduced the cost of nutrient use, saved the cost of drying fungicide, and utilized agricultural and forestry waste to promote plant growth and yield.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of fungal inoculants technology, specifically relating to a method for developing an inoculant containing arbuscular mycorrhizal fungi. Background Technology
[0002] Arbuscular mycorrhizal fungi (AMF) are symbiotic microorganisms that form vesicles and arbuscular structures within root cells through hyphae, enabling them to establish a mutually beneficial symbiotic system with plants via signaling molecules. 80% of terrestrial plants can form arbuscular mycorrhizal (AM) networks with AMF. Once the hyphal network is formed, it significantly enhances the plant's absorption of mineral nutrients, increases plant resistance, and alters secondary metabolism. Tomatoes, eggplants, cucumbers, cowpeas, potatoes, strawberries, and watermelons readily form mycorrhizae. However, cruciferous, chenopodiaceous, and sedge vegetables are less susceptible to infection. AMF absorbs various mineral nutrients from the soil through its extraroot hyphae and transports them to the plant to promote growth. Simultaneously, it obtains carbohydrates and lipids necessary for its own growth from the plant, thus forming a mutually beneficial symbiotic relationship with the host plant. AMF has been used to improve plant photosynthesis and promote plant growth and development. AMF primarily regulates peanut growth by influencing root morphology, particularly improving root volume and length. Simultaneously, AMF may alleviate stress by regulating biological processes such as photosynthesis, redox processes, carbohydrate metabolism, cell wall biogenesis, and cell growth.
[0003] However, the growth of arbuscular mycorrhizae (AMF) and plants is also limited by the environment. Large amounts of organic pollutants and heavy metals remain in the soil, affecting not only the normal structure and function of the soil but also inhibiting plant growth and development. Furthermore, when fungi are exposed to various polluted soils, the pollutants also affect fungal activity, spore germination, hyphal elongation, and community reproduction and changes. Therefore, organic and inorganic pollutants in the environment inevitably affect the formation, structure, and function of arbuscular mycorrhizae, thus impacting inoculation effectiveness. Due to environmental limitations, it is necessary to reduce these adverse effects by improving the resistance of AMF to the environment or by hindering the impact of pollutants on AMF, in order to better utilize arbuscular mycorrhizae for regulating plant growth. Summary of the Invention
[0004] This invention mainly provides a fungal agent containing arbuscular mycorrhizal fungi that can reduce the impact of the environment on the growth of plants and arbuscular mycorrhizae in the early stage of growth, and has a high infection rate, as well as a method for preparing the fungal agent:
[0005] A method for preparing an inoculant containing arbuscular mycorrhizal fungi involves: crushing tomato stems and leaves to obtain tomato stem powder; subjecting the tomato stem powder to acid treatment to obtain inactivated tomato stems; sintering and carbonizing the tomato stem powder to obtain tomato stem char; preparing a substrate using the inactivated tomato stems, tomato stem char, and nutrients; placing tomato seeds in a culture medium containing tomato stem powder to grow hairy roots; and culturing arbuscular mycorrhizal fungal spores and hairy roots together in the substrate to obtain an inoculant containing arbuscular mycorrhizal fungi.
[0006] Furthermore, by weight, every 1000 parts of the matrix comprises 0.5 to 1.5 parts of inactivated tomato stem, 2 to 5 parts of tomato stem charcoal, 3 to 5 parts of sucrose, 0.3 to 0.6 parts of magnesium carbonate, 0.1 to 0.3 parts of magnesium sulfate, 3 to 5 parts of gel, and the balance being water.
[0007] Furthermore, the preparation of the matrix includes the following steps:
[0008] a. Deactivate the tomato stem powder to obtain deactivated tomato stems;
[0009] b. Disperse tomato stem powder in water, add ammonium chloride, and hydrothermally react at 210-250℃ for 2-4 hours; after venting the gas, take a portion of the product, sulfonate it, and then add it to the system, and continue the water bath reaction at 90-100℃ for 1-2 hours; collect the solid product, dry the obtained product, and obtain tomato stem char.
[0010] c. Mix magnesium carbonate, magnesium sulfate and tomato stem charcoal thoroughly in water according to the formula to obtain tomato stem charcoal mixture; dissolve the remaining nutrients in water, then add inactivated tomato stems, mix evenly, and quickly and evenly sprinkle the tomato stem charcoal mixture onto the surface. After solidification, the matrix is obtained.
[0011] Furthermore, the clean tomato stems and leaves are dried to constant weight and then crushed to an average particle size of 0.1–1 mm to obtain tomato stem powder.
[0012] Furthermore, the tomato stem powder was fully dispersed in methanol, formic acid was added and mixed evenly, and the mixture was refluxed for 0.5 to 2 hours under boiling conditions. After removing the solvent and formic acid by evaporation, inactivated tomato stems were obtained; the mass ratio of formic acid to tomato stem powder was 0.5 to 2:1.
[0013] Furthermore, the mass ratio of ammonium chloride to tomato stem powder in step b is 1.5 to 2.5:1; the mass of the partial product in step b accounts for 5 to 15% of the total product.
[0014] Furthermore, the sulfonation involves mixing a portion of the product with concentrated sulfuric acid and reacting it at 80–110°C for 2–4 hours.
[0015] Further steps include: mixing tomato stem powder with agar to prepare a culture medium; disinfecting tomato seeds and inoculating them onto the culture medium for cultivation until hairy roots grow; transferring the hairy roots to the substrate, inoculating the detached hairy roots with the inoculum, and culturing in the dark for 15-25 days; after the production of a large number of spores, cutting off the tomato stem charcoal from the surface of the substrate; spraying sodium alginate solution onto the surface, and dividing it after solidification to obtain the inoculum.
[0016] Furthermore, 15 to 30 spores are inoculated at the edge of every 0.1 to 0.15 g of hairy root; the distance between the spores and the hairy root is no more than 2 cm.
[0017] The application of the above-mentioned inoculant containing arbuscular mycorrhizal fungi in agricultural planting, specifically in the cultivation of tomatoes.
[0018] By adopting the above scheme, the method of the present invention has the following advantages:
[0019] The fungal agent of this invention contains tomato stem charcoal made from tomato stems and leaves, which can provide a growth environment that is conducive to the colonization and growth of fungi and has minimal external influence. It forms a protective barrier in the early stage of fungal germination and does not affect its later extension and growth into the external soil. When applied during tomato growth, it can increase the infection rate of arbuscular mycorrhizal fungi, promote tomato growth, and increase yield.
[0020] The inoculant of the present invention contains biochar that can promote the colonization and growth of arbuscular mycorrhizal fungi, and inactivates the suitable porosity and large amounts of nutrients such as nitrogen, phosphorus and potassium in tomato stems. This not only reduces the use of nutrients and growth hormones, saving costs, but also facilitates the full utilization of agricultural and forestry waste such as tomato stems and leaves, reducing the disposal costs of agricultural and forestry waste.
[0021] This invention carbonizes powder made from tomato stems and leaves to obtain highly porosity tomato stem charcoal, which facilitates the colonization and growth of arbuscular mycorrhizal fungi. The tomato stem charcoal floats on the substrate surface, easily separating from the higher moisture content of the substrate below, reducing the moisture content of the fungal agent, saving on drying costs, and the numerous pores protect the generated spores, minimizing spore loss.
[0022] In this invention, tomato stem charcoal is added later during the preparation of the substrate, so that the tomato stem charcoal floats on the surface of the substrate, which can directly adsorb spores and facilitates separation from the substrate below in the later stage.
[0023] This invention involves hydrothermal carbonization of tomato stems and leaves, followed by sterilization. This process creates more pores while retaining a large amount of active substances and lipids, providing energy for the germination and growth of arbuscular mycorrhizal fungi.
[0024] This invention utilizes carbonized tomato stem charcoal, which, after sulfonation, serves as a catalyst for esterification. The carbonized product is then catalytically esterified to increase its lipid content. Before being used for the growth of arbuscular mycorrhizal fungi, it is mixed with magnesium carbonate. On the one hand, magnesium ions can act as nutrients to promote fungal reproduction; on the other hand, magnesium carbonate consumes the acidity of sulfonic acid groups, causing them to form salts and preventing the sulfonic acid groups from affecting the growth of the fungi.
[0025] This invention utilizes the rich nitrogen, phosphorus, potassium and other nutrients and growth factors naturally contained in tomato stems and leaves, which are more suitable for tomato growth. The tomato stems and leaves are used to participate in the germination and growth of tomatoes, and the antibacterial effect of tomatine can also be used to prevent the tomatoes from being contaminated by miscellaneous bacteria during the growth process.
[0026] This invention inactivates tomato stems and leaves, degrading the relatively high content of solanine in tomato stems and leaves, thereby reducing the impact of solanine on the growth of arbuscular mycorrhizal fungi. Detailed Implementation
[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0028] Example 1:
[0029] (1) Dry the clean tomato stems and leaves to constant weight, crush them to an average particle size of 0.5 mm to obtain tomato stem powder; take the tomato stem powder in methanol at a mass ratio of 1:1 and disperse it fully, add formic acid and mix evenly, reflux the reaction for 1.5 h under boiling state, and after evaporating the solvent and formic acid, obtain inactivated tomato stems.
[0030] (2) Disperse tomato stem powder in water, add ammonium chloride twice the mass of tomato stem powder, and hydrothermally react at 230℃ for 3h; after the gas is released, take 10% of the product by mass and mix it with concentrated sulfuric acid, react at 100℃ for 3h for sulfonation, then add it to the system, and continue the water bath reaction at 95℃ for 1.5h; collect the solid product, dry the obtained product, and obtain tomato stem char.
[0031] (3) By weight, take 0.5 parts magnesium carbonate, 0.2 parts magnesium sulfate and 4 parts tomato stem charcoal and mix them thoroughly in 200 parts water to obtain tomato stem charcoal mixture; dissolve 4 parts sucrose and 4 parts agar in 790 parts water, then add 1 part inactivated tomato stem, mix evenly, and quickly sprinkle the tomato stem charcoal mixture evenly on the surface. After solidification, the matrix is obtained.
[0032] (4) Mix tomato stem powder with agar to prepare a culture medium; disinfect tomato seeds and inoculate them on the culture medium for dark culture until hairy roots grow; transfer the hairy roots to the substrate, inoculate the inoculum next to the detached hairy roots, inoculate 25 spores within 2 cm of each 0.1-0.15 g hairy root, and culture in the dark; culture for 1-2 months until the spore density is 30-50 spores / g, then cut the tomato stem charcoal on the surface of the substrate; fix it on the surface with sodium alginate solution, and divide it after solidification to obtain the inoculum.
[0033] Example 2: The difference from Example 1 is as follows:
[0034] (1) Dry the clean tomato stems and leaves to constant weight, crush them to an average particle size of 0.5 mm to obtain tomato stem powder; take the tomato stem powder in methanol at a mass ratio of 1:1 and disperse it fully, add formic acid and mix evenly, reflux the reaction for 0.5 h under boiling state, and after evaporating the solvent and formic acid, obtain inactivated tomato stems.
[0035] Example 3: The difference from Example 1 is as follows:
[0036] (2) Disperse tomato stem powder in water, add ammonium chloride twice the mass of tomato stem powder, and hydrothermally react at 230℃ for 3h; after the gas is released, take 5% of the product by mass and mix it with concentrated sulfuric acid, react at 100℃ for 3h to sulfonate, then add it to the system, and continue the water bath reaction at 95℃ for 1.5h; collect the solid product, dry the obtained product, and obtain tomato stem charcoal.
[0037] Example 4: The difference from Example 1 is as follows:
[0038] (3) By mass, take 0.5 parts magnesium carbonate, 0.2 parts magnesium sulfate and 2 parts tomato stem charcoal and mix them thoroughly in 200 parts water to obtain tomato stem charcoal mixture; dissolve 4 parts sucrose and 4 parts agar in 790 parts water, then add 1 part inactivated tomato stem, mix evenly, and quickly sprinkle the tomato stem charcoal mixture evenly on the surface. After solidification, the matrix is obtained.
[0039] Comparative Example 1: The difference from Example 1 is that:
[0040] (1) Dry the clean tomato stems and leaves to constant weight, and crush them to an average particle size of 0.5 mm to obtain tomato stem powder.
[0041] Comparative Example 2: The difference from Example 1 is that:
[0042] (2) Disperse tomato stem powder in water, add ammonium chloride twice the mass of tomato stem powder, and hydrothermally react at 230℃ for 3 hours; after the gas is released, collect the solid product, and dry the product to obtain tomato stem char.
[0043] Comparative Example 3: The difference from Example 1 is that:
[0044] (3) By weight, take 4 parts sucrose, 4 parts agar, 1 part inactivated tomato stem, 0.5 parts magnesium carbonate, 0.2 parts magnesium sulfate and 4 parts tomato stem charcoal and mix them thoroughly in 990 parts water. After solidification, the matrix is obtained.
[0045] Comparative Example 4: The difference from Example 1 is that:
[0046] (3) By mass, take 0.5 parts magnesium carbonate, 0.2 parts magnesium sulfate, 4 parts sucrose and 4 parts agar and dissolve them in 990 parts water. Then add 1 part inactivated tomato stem, mix well and solidify to obtain the matrix.
[0047] Comparative Example 5: The difference from Example 1 is that:
[0048] (4) Prepare 1 / 2MS medium containing antibiotics; disinfect tomato seeds and inoculate them on the medium for dark culture until hairy roots grow; transfer the hairy roots to the substrate, inoculate the inoculum next to the detached hairy roots, inoculate 25 spores within 5cm of each 0.1-0.15g hairy root, and culture in the dark; culture for 1-2 months until the spore density is 30-50 spores / g, then cut the tomato stem charcoal from the surface of the substrate; fix the charcoal on the surface with sodium alginate solution, and divide it after solidification to obtain the inoculum.
[0049] Example Sample Testing:
[0050] Tomato: Omanda No. 3; Arbuscular mycorrhizal fungus strain: Rhizocystis ulmoides;
[0051] A commercially available rhizocyst fungicide with a spore density of 45 spores / g was used as a control. A pot experiment was conducted; seeds were disinfected, germinated, and inoculated at sowing. Each treatment was repeated three times. Normal field management was maintained, with Hogrange nutrient solution (1 / 4 phosphorus) applied weekly. Infection rate was measured 30 days after inoculation, and morphological and physiological indicators were measured after tomato ripening.
[0052] Determination of morphological indicators: Cut off the above-ground parts, weigh the fresh weight of the above-ground parts, blanch at 95℃ for 15 minutes, and then dry at 60℃ to constant weight to determine the dry weight of the above-ground parts; carefully remove the root system, rinse it clean, use a millimeter ruler to measure the distance from the stem base to the root tip as the root length, measure the fresh weight of the roots, blanch at 95℃ for 15 minutes, and then dry at 60℃ to constant weight to determine the dry weight of the underground parts.
[0053] Mycorrhizal infection rate: The classic trichomoniasis blue staining method was used for observation. Roots were randomly selected from each symbiotic tomato plant. The washed roots were cut into approximately 1 cm segments and placed in an Erlenmeyer flask. 10% KOH was added, and the flask was incubated in a 90℃ water bath for 45–60 min. The alkali solution was discarded, and the roots were washed 3–5 times with water. 2% hydrochloric acid was added, and the roots were acidified at room temperature for 5 min. 0.05% trichomoniasis blue was added, and the roots were stained in a 90℃ water bath for 30 min. After removing the trichomoniasis blue, the solution was discarded, and the roots were rinsed with tap water. Lactic acid glycerol solution (1:1:1) was added, and the roots were decolorized at room temperature for 24 h. Ten mycorrhizal infection segments were arranged on a glass slide (30 segments per sample, 3 slides in total) using tweezers and examined under a microscope (10×10). Infection rate (%) = (Number of mycorrhizally infected root segments / Total number of root segments examined) × 100%. The results are shown in the table below.
[0054]
[0055] Comparing Examples 1 and 2, Example 2, which underwent a shorter inactivation process, showed a slightly lower infection rate. This may be because the shorter treatment time was insufficient to fully inactivate antibacterial components such as tomatine in tomato stems and leaves, and also hindered further porosification of the tomato stems and leaves, resulting in a reduced spore content in the prepared fungal agent and affecting the infection rate. In contrast, Comparative Example 1, which did not undergo inactivation, showed a significant decrease in infection rate, demonstrating that inactivation treatment of tomato stems and leaves plays a crucial role in ensuring the quality of the fungal agent. Example 3 showed less sulfonated product, a lower infection rate, and lower tomato plant biomass compared to Example 1. Furthermore, Comparative Example 2, which did not undergo sulfonation treatment, showed a significant decrease in infection rate, and tomato plant growth was also affected. This indicates that the sulfonation step is beneficial to the growth of arbuscular mycorrhizal fungi, promoting spore production. Sulfonation can catalyze esterification, providing more nutrients to arbuscular mycorrhizal fungi. Moreover, the sulfur in the fungal agent's groups after application also benefits plant growth.
[0056] The tomato stem charcoal content in Example 4 was low, and the growth of tomato plants after application was not as good as in Example 1, especially the weight of the above-ground parts was significantly reduced. This indicates that tomato stem charcoal is beneficial to the growth of tomatoes, especially to increasing tomato yield.
[0057] Comparative Example 3 directly mixed all raw materials to prepare the substrate simultaneously. After application, the infection rate and tomato biomass both decreased. Comparative Example 4, without the addition of tomato stem charcoal, showed a decreased infection rate and a significant decrease in the fresh weight of the above-ground parts of the tomato, indicating that tomato stem charcoal is beneficial for improving the infection rate and tomato yield. Comparative Example 5 did not add tomato stem powder during tomato germination and hairy root preparation. After application of the fungal agent, the infection rate and tomato biomass were similar to, or even lower than, those in Example 1, indicating that tomato stem powder can replace some nutrients and antibiotics in conventional culture media, reducing production costs. Furthermore, it can produce a long-lasting antibacterial effect during growth and avoids the absorption of antibiotics by the hairy roots, thus preventing interference with the growth of arbuscular mycorrhizal fungi.
[0058] For those skilled in the art, various other corresponding changes and modifications can be made based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of this invention.
Claims
1. A method for preparing an inoculant containing arbuscular mycorrhizal fungi, characterized in that, Preparation of the substrate: Tomato seeds are placed in a culture medium containing tomato stem fragments to grow hairy roots; arbuscular mycorrhizal fungal spores and hairy roots are cultured together in the substrate to obtain an inoculum containing arbuscular mycorrhizal fungi; the preparation of the substrate includes the following steps: a. Deactivate the tomato stem powder to obtain deactivated tomato stems; b. Disperse tomato stem powder in water, add ammonium chloride, and hydrothermally react at 210~250℃ for 2~4h; after venting the gas, take a portion of the product, sulfonate it, and then add it to the system, and continue the water bath reaction at 90~100℃ for 1~2h; collect the solid product, and dry the obtained product to obtain tomato stem charcoal; c. Mix magnesium carbonate, magnesium sulfate and tomato stem charcoal thoroughly in water according to the formula to obtain tomato stem charcoal mixture; dissolve the remaining nutrients in water, then add inactivated tomato stems, mix evenly, and quickly and evenly sprinkle the tomato stem charcoal mixture onto the surface. After solidification, the matrix is obtained.
2. The method for preparing the inoculant containing arbuscular mycorrhizal fungi according to claim 1, characterized in that, By weight, every 1000 parts of the matrix comprises 0.5 to 1.5 parts of inactivated tomato stem, 2 to 5 parts of tomato stem charcoal, 3 to 5 parts of sucrose, 0.3 to 0.6 parts of magnesium carbonate, 0.1 to 0.3 parts of magnesium sulfate, 3 to 5 parts of gel, and the balance being water.
3. The method for preparing the inoculant containing arbuscular mycorrhizal fungi according to claim 1, characterized in that, Clean tomato stems and leaves are dried to constant weight and then crushed to an average particle size of 0.1-1 mm to obtain tomato stem powder.
4. The method for preparing the inoculant containing arbuscular mycorrhizal fungi according to claim 1, characterized in that, Tomato stem powder was fully dispersed in methanol, formic acid was added and mixed evenly, and the mixture was refluxed for 0.5-2 hours under boiling conditions. After removing the solvent and formic acid by evaporation, inactivated tomato stems were obtained. The mass ratio of formic acid to tomato stem powder was 0.5-2:
1.
5. The method for preparing the inoculant containing arbuscular mycorrhizal fungi according to claim 1, characterized in that, The mass ratio of ammonium chloride to tomato stem powder in step b is 1.5~2.5:1; the mass of the partial product in step b accounts for 5~15% of the total product.
6. The method for preparing the inoculant containing arbuscular mycorrhizal fungi according to claim 1, characterized in that, The sulfonation process involves mixing a portion of the product with concentrated sulfuric acid and reacting it at 80-110°C for 2-4 hours.
7. The method for preparing the inoculant containing arbuscular mycorrhizal fungi according to claim 1, characterized in that, Includes the following steps: A culture medium was prepared by mixing tomato stem fragments with agar. Tomato seeds were disinfected and inoculated onto the culture medium and cultured until hairy roots grew. The hairy roots were transferred to the substrate, and the inoculum was inoculated next to the detached hairy roots and cultured in the dark for 15-25 days. After a large number of spores were produced, the tomato stem charcoal on the surface of the substrate was cut off. The charcoal was fixed on the surface with sodium alginate solution, and after solidification, it was divided to obtain the inoculum.
8. The method for preparing the inoculant containing arbuscular mycorrhizal fungi according to claim 7, characterized in that, For every 0.1 to 0.15 g of hairy root, 15 to 30 spores are inoculated at the edge; the distance between the spores and the hairy root is no more than 2 cm.
9. The application of a fungal agent containing arbuscular mycorrhizal fungi as described in any one of claims 1 to 7 in agricultural planting, characterized in that, Apply when planting tomatoes.