Fungicide containing arbuscular mycorrhizal fungi and preparation method and application thereof

Abstract

The invention belongs to the technical field of fungal inoculants, and particularly relates to an arbuscular mycorrhizal fungus-containing inoculant and a preparation method and application thereof.The preparation method comprises the following steps: smashing tomato stems and leaves to obtain tomato stem powder; performing acid treatment on the tomato stem powder to obtain inactivated tomato stems; taking the tomato stem powder, and sintering and carbonizing to obtain tomato stem carbon; taking the inactivated tomato stems, the tomato stem charcoal and nutrient substances to prepare a substrate; tomato seeds are taken and placed in a culture medium containing tomato stem powder to grow hairy roots; and culturing the arbuscular mycorrhizal fungus spores and hairy roots in a substrate together to obtain the fungicide containing the arbuscular mycorrhizal fungi. The fungicide containing the arbuscular mycorrhizal fungi is applied when tomatoes grow, the dip dyeing rate of the arbuscular mycorrhizal fungi can be increased, the growth of the tomatoes is promoted, and the yield is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of fungal agents, and particularly relates to a method for producing an agent containing arbuscular mycorrhizal fungi. Background Art

[0002] Arbuscular mycorrhizal fungi (AMF) are symbiotic microorganisms that form specialized vesicles and arbuscules within root cells through the exchange of signaling molecules, forming a mutually beneficial symbiotic relationship with plants. 80% of terrestrial plants can form arbuscular mycorrhizal (AM) networks with these fungi. Once established, these networks significantly enhance plant absorption of mineral nutrients, increase plant resistance, and alter plant secondary metabolism. Tomatoes, eggplants, cucumbers, cowpeas, potatoes, strawberries, and watermelons are also susceptible to mycorrhizae. However, vegetables from the cruciferous, Chenopodiaceae, and sedge families are less susceptible. AMF absorb various mineral nutrients from the soil through their extraradical hyphae and transfer them to the plant to promote growth. AMF also obtains carbohydrates and lipids from the plant itself, forming a mutually beneficial symbiotic relationship with the host plant. AMF have been used to improve plant photosynthesis and promote plant growth and development. AMF regulates peanut growth primarily by affecting root morphology, particularly improving root volume and length. AMF may also alleviate stress by regulating biological processes such as photosynthesis, redox processes, carbohydrate metabolism, cell wall biogenesis, and cell growth.

[0003] However, the growth of AMF and plants is also subject to environmental constraints. Large amounts of organic pollutants and heavy metals remain in the soil, affecting not only its normal structure and function but also inhibiting plant growth and development. Furthermore, when fungi are exposed to various polluted soils, the pollutants can affect fungal activity, spore germination, hyphal elongation, and the reproduction and changes of fungal communities. Therefore, organic and inorganic pollutants in the environment inevitably affect the formation, structure, and function of arbuscular mycorrhizae, thereby compromising the effectiveness of inoculation. Given these environmental constraints, mitigating these adverse effects by improving AMF's resistance to the environment or preventing pollutants from affecting them is crucial to better utilize arbuscular mycorrhizae to regulate plant growth. Summary of the Invention

[0004] The present invention mainly provides a microbial agent containing arbuscular mycorrhizal fungi, which can reduce the influence of the environment on the growth of plants and arbuscular mycorrhizal fungi in the early growth stage and has a high infection rate, and a preparation method of the microbial agent:

[0005] A method for preparing an arbuscular mycorrhizal fungus-containing inoculum comprises the following steps: 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 charcoal; preparing a matrix using the inactivated tomato stems, the tomato stem charcoal, and nutrients; placing tomato seeds in a culture medium containing the tomato stem powder to grow hairy roots; and culturing arbuscular mycorrhizal fungus spores and the hairy roots together in the matrix to obtain an inoculum containing the arbuscular mycorrhizal fungus.

[0006] Furthermore, per 1000 parts of the matrix, by mass, comprises 0.5-1.5 parts of inactivated tomato stems, 2-5 parts of tomato stem charcoal, 3-5 parts of sucrose, 0.3-0.6 parts of magnesium carbonate, 0.1-0.3 parts of magnesium sulfate, 3-5 parts of gel and the balance water.

[0007] Furthermore, the preparation of the matrix comprises the following steps:

[0008] a. inactivating the tomato stem end to obtain an inactivated tomato stem;

[0009] b. Disperse the tomato stem powder in water, add ammonium chloride, and hydrothermally react at 210-250°C for 2-4 hours. After venting the gas, a portion of the product is sulfonated and then added to the system. The reaction is continued in a water bath at 90-100°C for 1-2 hours. The solid product is collected and dried to obtain tomato stem charcoal.

[0010] c. According to the formula, magnesium carbonate, magnesium sulfate and tomato stem charcoal are fully mixed in water to obtain a tomato stem charcoal mixture; the remaining nutrients are dissolved in water, and then the inactivated tomato stems are added and mixed evenly. The tomato stem charcoal mixture is quickly and evenly spread on the surface and solidified to obtain a matrix.

[0011] Furthermore, the cleaned tomato stems and leaves are dried to a constant weight, and crushed to an average particle size of 0.1 to 1 mm to obtain tomato stem powder.

[0012] Furthermore, the tomato stem powder is placed in methanol and fully dispersed, formic acid is added and mixed evenly, and the system is refluxed for reaction for 0.5 to 2 hours under boiling conditions. After the solvent and formic acid are evaporated, the inactivated tomato stem is obtained; the mass ratio of the formic acid to the tomato stem powder is 0.5 to 2:1.

[0013] Furthermore, the mass ratio of the ammonium chloride in step b to the tomato stem end is 1.5-2.5:1; and the mass of the partial product in step b accounts for 5-15% of the total product.

[0014] Furthermore, the sulfonation is performed by mixing part of the product with concentrated sulfuric acid and reacting at 80-110° C. for 2-4 hours.

[0015] Furthermore, the method comprises the following steps: mixing tomato stem ends with agar to prepare a culture medium; sterilizing tomato seeds and inoculating them on the culture medium for cultivation until hairy roots grow; transferring the hairy roots to a substrate, inoculating a bacterial agent next to the isolated hairy roots, and cultivating in the dark for 15 to 25 days; after cultivating until a large number of spores are produced, cutting the tomato stem charcoal on the surface of the substrate; spraying a sodium alginate solution on the surface, and dividing it after solidification to obtain the bacterial agent.

[0016] Furthermore, 15 to 30 spores are inoculated on the edge of every 0.1 to 0.15 g of hairy roots; the distance between the spores and the hairy roots is no more than 2 cm.

[0017] An application of the above-mentioned bacterial agent containing arbuscular mycorrhizal fungi in agricultural planting is applied when planting tomatoes.

[0018] By adopting the above scheme, the method of the present invention has the following advantages:

[0019] The microbial agent of the present 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 little external influence. It forms protection in the early stage of fungal germination and does not affect its extension and growth into the external soil in the later stage. When applied during tomato growth, it can increase the infection rate of arbuscular mycorrhizal fungi, promote tomato growth, and increase yield.

[0020] The microbial agent of the present invention contains biochar that can promote the colonization and growth of arbuscular mycorrhizal fungi, and has suitable porosity for inactivating tomato stems and a large amount of nutrients such as nitrogen, phosphorus and potassium. It can not only reduce the use of nutrients and growth hormones and save costs, but also is conducive to the full utilization of agricultural and forestry wastes such as tomato stems and leaves, thereby reducing the disposal costs of agricultural and forestry wastes.

[0021] This invention carbonizes powder made from tomato stems and leaves to produce a high-porosity tomato stem charcoal, which facilitates the colonization and growth of arbuscular mycorrhizal fungi. The tomato stem charcoal floats on the surface of the substrate, easily separating from the higher-moisture substrate below. This reduces the moisture content of the inoculum and saves drying costs. The large number of pores protects the generated spores, minimizing spore loss.

[0022] In the present invention, when preparing the matrix, the tomato stem charcoal is added later, so that the tomato stem charcoal floats on the surface of the matrix, can directly adsorb spores, and is also convenient for separation from the matrix part below in the later stage.

[0023] The present invention hydrothermally carbonizes tomato stems and leaves and performs sterilization treatment, thereby obtaining more pores while retaining a large amount of active substances and lipids, thereby providing energy for the germination and growth of arbuscular mycorrhizal fungi.

[0024] The present invention utilizes carbonized tomato stem charcoal, which is used as an esterification catalyst after sulfonation. The carbonized product is catalytically esterified to increase the lipid content. The product is mixed with magnesium carbonate before being used for the growth of arbuscular mycorrhizal fungi. On the one hand, magnesium ions can be used as nutrients to promote the reproduction of fungi. On the other hand, magnesium carbonate is used to consume the acidity of sulfonic acid groups to form salts, thereby preventing the sulfonic acid groups from affecting the growth of fungi.

[0025] The present invention utilizes the characteristics that tomato stems and leaves contain rich nutrients such as nitrogen, phosphorus, potassium and growth hormones that are more suitable for tomato growth, uses tomato stems and leaves to participate in the germination and growth of tomatoes, and can also utilize the antibacterial effect of tomatine to prevent tomatoes from being contaminated by miscellaneous bacteria during growth.

[0026] The invention inactivates tomato stems and leaves, degrades solanine with a relatively high content in the stems and leaves, and reduces the influence of solanine on the growth of arbuscular mycorrhizal fungi. DETAILED DESCRIPTION

[0027] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0028] Example 1:

[0029] (1) The cleaned tomato stems and leaves were dried to a constant weight and crushed to an average particle size of 0.5 mm to obtain tomato stem powder; the tomato stem powder was fully dispersed in methanol at a mass ratio of 1:1, formic acid was added and mixed evenly, and the mixture was refluxed for 1.5 hours under boiling conditions. After evaporating the solvent and formic acid, the inactivated tomato stem was obtained;

[0030] (2) Dispersing the tomato stems in water, adding ammonium chloride twice the mass of the tomato stems, and hydrothermally reacting at 230°C for 3 hours; after exhausting the gas, taking 10% of the total mass of the product and mixing it with concentrated sulfuric acid, reacting it at 100°C for 3 hours for sulfonation, and then adding it to the system, and continuing the water bath reaction at 95°C for 1.5 hours; collecting the solid product, and drying the obtained product to obtain tomato stem charcoal;

[0031] (3) By weight, 0.5 parts of magnesium carbonate, 0.2 parts of magnesium sulfate, and 4 parts of tomato stem charcoal were mixed thoroughly in 200 parts of water to obtain a tomato stem charcoal mixture; 4 parts of sucrose and 4 parts of agar were dissolved in 790 parts of water, and then 1 part of inactivated tomato stem was added. After mixing well, the tomato stem charcoal mixture was quickly and evenly spread on the surface and solidified to obtain a matrix;

[0032] (4) Mixing the tomato stem end with agar to prepare a culture medium; sterilizing tomato seeds and inoculating them on the culture medium for dark culture until hairy roots grow; transferring the hairy roots to a substrate, inoculating the bacterial agent next to the isolated hairy roots, inoculating 25 spores within 2 cm of each 0.1-0.15 g of hairy roots, and dark culture; after culturing for 1-2 months until the spore density reaches 30-50 spores / g, cutting the tomato stem charcoal on the surface of the substrate; fixing it with a sodium alginate solution on the surface, and dividing it after solidification to obtain the bacterial agent.

[0033] Example 2: The difference from Example 1 is that:

[0034] (1) The cleaned tomato stems and leaves were dried to a constant weight and crushed to an average particle size of 0.5 mm to obtain tomato stem powder; the tomato stem powder was taken in methanol at a mass ratio of 1:1 and fully dispersed, formic acid was added and mixed evenly, and the mixture was refluxed for 0.5 h under boiling conditions, and the solvent and formic acid were evaporated to obtain inactivated tomato stems.

[0035] Example 3: The difference from Example 1 is that:

[0036] (2) The tomato stems were dispersed in water, and ammonium chloride twice the mass of the tomato stems was added, and the mixture was hydrothermally reacted at 230°C for 3 hours. After the gas was exhausted, 5% of the product was mixed with concentrated sulfuric acid, and the mixture was sulfonated at 100°C for 3 hours. The mixture was then added to the system and the reaction was continued in a water bath at 95°C for 1.5 hours. The solid product was collected and dried to obtain tomato stem charcoal.

[0037] Example 4: The difference from Example 1 is that:

[0038] (3) By mass, 0.5 parts of magnesium carbonate, 0.2 parts of magnesium sulfate, and 2 parts of tomato stem charcoal were mixed thoroughly in 200 parts of water to obtain a tomato stem charcoal mixture; 4 parts of sucrose and 4 parts of agar were dissolved in 790 parts of water, and then 1 part of inactivated tomato stem was added. After mixing well, the tomato stem charcoal mixture was quickly and evenly spread on the surface and solidified to obtain a matrix.

[0039] Comparative Example 1: The difference from Example 1 is:

[0040] (1) The cleaned tomato stems and leaves were dried to a constant weight and crushed to an average particle size of 0.5 mm to obtain tomato stem powder.

[0041] Comparative Example 2: The difference from Example 1 is:

[0042] (2) The tomato stems were dispersed in water, and ammonium chloride twice the mass of the tomato stems was added, and the mixture was hydrothermally reacted at 230°C for 3 hours. After the gas was exhausted, the solid product was collected and dried to obtain tomato stem charcoal.

[0043] Comparative Example 3: The difference from Example 1 is:

[0044] (3) By weight, 4 parts of sucrose, 4 parts of agar, 1 part of inactivated tomato stem, 0.5 parts of magnesium carbonate, 0.2 parts of magnesium sulfate, and 4 parts of tomato stem charcoal were thoroughly mixed in 990 parts of water and solidified to obtain a matrix.

[0045] Comparative Example 4: The difference from Example 1 is:

[0046] (3) By mass, 0.5 parts of magnesium carbonate, 0.2 parts of magnesium sulfate, 4 parts of sucrose, and 4 parts of agar were dissolved in 990 parts of water, and then 1 part of inactivated tomato stem was added, mixed evenly, and solidified to obtain a matrix.

[0047] Comparative Example 5: The difference from Example 1 is:

[0048] (4) Prepare 1 / 2MS culture medium containing antibiotics; sterilize 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 bacterial agent next to the isolated hairy roots, inoculate 25 spores within 5 cm of each 0.1-0.15 g of hairy roots, and culture in the dark; after culturing for 1-2 months until the spore density reaches 30-50 / g, cut the tomato stem charcoal on the surface of the substrate; fix it with sodium alginate solution on the surface, and divide it after solidification to obtain the bacterial agent.

[0049] Example sample test:

[0050] Tomato: Omanda No. 3; Arbuscular mycorrhizal fungus: Rhizocystis endoradiata;

[0051] A commercially available root cyst inoculant with a spore density of 45 spores / g was used as a control. Potted seeds were disinfected, germinated, and inoculated at sowing. Each treatment was replicated three times. Normal field management was followed, with Hoagland's nutrient solution containing 1 / 4 phosphorus added once a week. The infection rate was measured 30 days after inoculation, and morphological and physiological parameters were measured after the tomatoes matured.

[0052] Determination of morphological indicators: cut off the aboveground part, weigh the fresh weight of the aboveground part, after killing the green at 95℃ for 15 minutes, dry it at 60℃ to constant weight to measure the dry weight of the aboveground part; carefully remove the root system, rinse it clean, use a millimeter ruler to measure the distance from the base of the stem to the root tip as the root length, measure the fresh weight of the root, after killing the green at 95℃ for 15 minutes, dry it at 60℃ to constant weight to measure the dry weight of the underground part.

[0053] Mycorrhizal infection rate: The classic phenanthenic blue staining method was used. Roots were randomly selected from each symbiotic tomato plant, cleaned, cut into approximately 1 cm segments, and placed in a conical flask. Roots were then incubated in a 90°C waterbath for 45–60 minutes in 10% KOH. The caustic soda was discarded, and the roots were washed 3–5 times with clean water. The caustic soda was then acidified with 2% hydrochloric acid at room temperature for 5 minutes. 0.05% phenanthenic blue was added and the roots were stained in a 90°C waterbath for 30 minutes. After removing the phenanthenic blue, the solution was discarded, rinsed with tap water, and decolorized with a lactic acid–glycerol solution (1:1:1) at room temperature for 24 hours. Ten root segments were then picked up with tweezers and arranged on a glass slide. Three slides of 30 root segments were prepared for each sample and examined under a microscope (10 × 10). Infection rate (%) = number of root segments infested with mycorrhizae / total number of root segments examined × 100%. The results are shown in the table below.

[0054]

[0055] Comparing Examples 1 and 2, the infection rate of Example 2, which underwent a shorter inactivation operation, decreased slightly. This may be because the shorter treatment time prevented the full inactivation of antibacterial components such as tomatidine in the tomato stems and leaves, hindered further porosification of the tomato stems and leaves, and reduced the spore content in the prepared inoculum, affecting the infection rate. However, the infection rate of Comparative Example 1, which did not undergo inactivation, decreased significantly, demonstrating that inactivating tomato stems and leaves plays a significant role in ensuring the quality of the inoculum. The sulfonated product in Example 3 was less abundant, resulting in a decreased infection rate and a lower biomass of the tomato plants. Furthermore, the infection rate of Comparative Example 2, which did not undergo sulfonation, decreased significantly, and the growth of the tomato plants was also affected. This indicates that the sulfonation step is beneficial for the growth of arbuscular mycorrhizal fungi, promoting spore production. Sulfonation can also catalyze esterification, providing more nutrients for the fungi. Furthermore, after application of the inoculum, the sulfur in the radical also contributes to plant growth.

[0056] The tomato stem charcoal content in Example 4 is low, and the growth of the tomato plants after application is not as good as in Example 1, especially the weight of the aboveground part is significantly reduced, indicating that the tomato stem charcoal is beneficial to the growth of tomatoes, especially to increasing tomato yield.

[0057] In Comparative Example 3, all raw materials were directly mixed and the matrix was prepared simultaneously. After application, the infection rate and tomato biomass decreased. In contrast, in Comparative Example 4, where no tomato stem charcoal was added, the infection rate decreased, and the fresh weight of the above-ground tomatoes decreased significantly, indicating that tomato stem charcoal is beneficial for increasing the infection rate and yield. In Comparative Example 5, tomato stem charcoal was not added during tomato germination, but during the preparation of hairy roots. After application of the inoculum, the infection rate and tomato biomass were similar to those in Example 1, or even lower than those in Example 1, indicating that the tomato stem charcoal can replace some nutrients and antibiotics in conventional culture media, reducing production costs. Furthermore, during the growth process, it produced a long-lasting antibacterial effect and prevented antibiotics from being absorbed by the hairy roots, thereby preventing the growth of arbuscular mycorrhizal fungi.

[0058] Those skilled in the art can make various other corresponding changes and deformations based on the technical solutions and concepts described above, and all of these changes and deformations should fall within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a microbial agent containing arbuscular mycorrhizal fungi, characterized in that: The tomato stems and leaves are crushed to obtain tomato stem powder; the tomato stem powder is treated with acid to obtain inactivated tomato stems; the tomato stem powder is sintered and carbonized to obtain tomato stem charcoal; a matrix is prepared using the inactivated tomato stems, tomato stem charcoal and nutrients; tomato seeds are placed in a culture medium containing the tomato stem powder to grow hairy roots; arbuscular mycorrhizal fungal spores and hairy roots are cultured together in the matrix to obtain an inoculum containing arbuscular mycorrhizal fungi.

2. The method for preparing the inoculum containing arbuscular mycorrhizal fungi according to claim 1, characterized in that: Calculated by mass, every 1000 parts of the matrix include 0.5-1.5 parts of inactivated tomato stems, 2-5 parts of tomato stem charcoal, 3-5 parts of sucrose, 0.3-0.6 parts of magnesium carbonate, 0.1-0.3 parts of magnesium sulfate, 3-5 parts of gel and the balance of water.

3. The method for preparing the inoculum containing arbuscular mycorrhizal fungi according to claim 1, characterized in that: The preparation of the matrix comprises the following steps: a. inactivating the tomato stem end to obtain an inactivated tomato stem; b. Disperse the tomato stem powder in water, add ammonium chloride, and hydrothermally react at 210-250°C for 2-4 hours. After venting the gas, a portion of the product is sulfonated and then added to the system. The reaction is continued in a water bath at 90-100°C for 1-2 hours. The solid product is collected and dried to obtain tomato stem charcoal. c. According to the formula, magnesium carbonate, magnesium sulfate and tomato stem charcoal are fully mixed in water to obtain a tomato stem charcoal mixture; the remaining nutrients are dissolved in water, and then the inactivated tomato stems are added and mixed evenly. The tomato stem charcoal mixture is quickly and evenly spread on the surface and solidified to obtain a matrix.

4. The method for preparing the inoculum containing arbuscular mycorrhizal fungi according to claim 3, characterized in that: The cleaned tomato stems and leaves are dried to a constant weight, and then crushed to an average particle size of 0.1 to 1 mm to obtain tomato stem powder.

5. The method for preparing the inoculum containing arbuscular mycorrhizal fungi according to claim 3, characterized in that: The tomato stem powder is placed in methanol and fully dispersed, formic acid is added and mixed evenly, and the system is refluxed for reaction for 0.5 to 2 hours under boiling conditions. After the solvent and formic acid are evaporated, an inactivated tomato stem is obtained; the mass ratio of the formic acid to the tomato stem powder is 0.5 to 2:

1.

6. The method for preparing the inoculum containing arbuscular mycorrhizal fungi according to claim 3, characterized in that: The mass ratio of the ammonium chloride in step b to the tomato stem end is 1.5-2.5:1; the mass of the partial product in step b accounts for 5-15% of the whole product.

7. The method for preparing the inoculum containing arbuscular mycorrhizal fungi according to claim 3, characterized in that: The sulfonation is carried out by mixing part of the product with concentrated sulfuric acid and reacting at 80-110° C. for 2-4 hours.

8. The method for preparing the inoculum containing arbuscular mycorrhizal fungi according to claim 1, characterized in that: The following steps are involved: The method comprises the following steps: mixing tomato stem ends with agar to prepare a culture medium; sterilizing tomato seeds and inoculating them on the culture medium for cultivation until hairy roots grow; transferring the hairy roots to a substrate, inoculating a microbial agent next to the isolated hairy roots, and culturing in the dark for 15 to 25 days; culturing until a large number of spores are produced, cutting the tomato stem charcoal on the surface of the substrate; fixing the surface with a sodium alginate solution, and dividing the microbial agent after solidification to obtain the microbial agent.

9. The bacterial agent containing arbuscular mycorrhizal fungi according to claim 8, characterized in that 15 to 30 spores are inoculated onto the edge of every 0.1 to 0.15 g of hairy roots; the distance between the spores and the hairy roots is no more than 2 cm.

10. Use of the inoculant containing arbuscular mycorrhizal fungi according to any one of claims 1 to 8 in agricultural planting, characterized in that: Apply when planting tomatoes.

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