Method for preparing deep-color separated endophytic fungus fungicide vegetation concrete

Abstract

The invention provides a preparation method of dark isolated endophytic fungus inoculant vegetation concrete. The preparation method of the inoculant comprises the following steps: carrying out fermentation culture on glucose, corn steep liquor dry powder and soybean cake powder, and preparing into the inoculant by using a freeze spray drying method. The method for improving the drought resistance of the plants in ecological restoration of the vegetation concrete comprises the following steps: layering the vegetation concrete: increasing the cement proportion in the base material proportion of a bottom layer, additionally adding urea, calcium chloride and urease, and inducing the urea and the calcium chloride to generate calcium carbonate precipitate through the urease to play a reinforcing role; additives such as lignocellulose, biochar and ceramsite are additionally added to the middle layer, so that the water-retaining property, porosity and fertility of soil can be improved, and the effect of enhancing plant rooting and branching is achieved; the fungicide and the plant seeds are added into the surface layer, and the fungicide can promote seed germination and plant growth.

Description

Technical Field

[0001] The invention relates to the field of ecological restoration and microbial technology, and in particular to a method for preparing dark-colored septate endophytic fungus agent vegetation concrete. Background Art

[0002] The massive infrastructure construction and energy development in my country inevitably cause great disturbance to the local ecological environment, especially the slope wounds formed by earthwork excavation, which seriously disturb the ecosystem, leading to a series of problems such as reduced biodiversity, soil erosion, and frequent geological disasters. Vegetation concrete ecological protection technology, which has both engineering protection and ecological green functions, has become one of the effective means of implementing ecological restoration. It promotes the formation and succession of vegetation communities by creating a vegetation growth environment. It is currently used in more than 20 provinces and cities in my country.

[0003] With the widespread application of this technology, it also faces many challenges, especially in the early stage of vegetation restoration. Due to the high and steep terrain, the slope water does not fully infiltrate and forms surface runoff. At the same time, due to the evaporation effect of the substrate, plants are easily in a drought stress environment, which affects the plant recovery process. The input control methods adopted in the later stage, such as the establishment of a permanent irrigation system and plant replanting measures, can temporarily alleviate the above stress, but there is no long-term improvement and restoration effect on plant nutrient utilization and the self-sustaining ability of the ecosystem, resulting in the continued deterioration of the ecosystem. The application of microbial remediation technology in disturbed ecological environments is a sustainable method from the perspective of repairing the function and structure of the ecosystem. This method can be used in conjunction with the ecological restoration technology of vegetation concrete slopes to improve the efficiency of slope restoration.

[0004] Dark septate endophytes (DSE) are a type of beneficial microorganisms that live in the rhizosphere of plants and can form a mutualistic symbiotic relationship with most terrestrial plants. They play a significant role in improving plant stress resistance and nutrient utilization, stimulating soil potential fertility, and maintaining ecological balance and sustainable productivity. In addition, DSE can be cultivated in pure culture, and the use of DSE and other beneficial microorganisms to restore the disturbed habitat has promotional significance, which can fundamentally improve the function of the ecosystem and promote the sustainable development of the ecosystem. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned shortcomings and provide a method for preparing dark septate endophytic fungus agent vegetation concrete, which promotes the growth of plants under drought stress by inoculating dark septate endophytic fungus agent.

[0006] In order to achieve the above technical features, the purpose of the present invention is achieved as follows: a method for preparing a dark septate endophytic fungus agent vegetation concrete comprises the following steps: S1, according to the colonization characteristics of dominant plant root fungi in the vegetation concrete plot, dark septate endophytic fungi were selected, and fermentation culture experiments were carried out. Finally, fresh mycelium was prepared into inoculants by freeze-spray drying method; S2, configuration of vegetated concrete: Add 83-86 parts of soil, 10-12 parts of cement, 5-8 parts of 1.0 mol / L calcium chloride, 5-8 parts of 1.0 mol / L urea, 1-2 parts of 4 g / L urease, 2-3 parts of habitat substrate improver, 2-3 parts of organic fertilizer, and 2-3 parts of habitat substrate organic material into a mixer according to the above weight ratio, stir for 10-15 minutes, and mix evenly to obtain a bottom layer, which is a reinforcement layer with a thickness of 2-3.5 cm; Add 83-86 parts of soil, 5-7 parts of cement, 2-3 parts of habitat substrate improver, 2-3 parts of organic fertilizer, 2-3 parts of habitat substrate organic material, 3-5 parts of lignocellulose, 3-5 parts of biochar, and 3-5 parts of ceramsite into a mixer according to the above weight ratio, stir for 10-15 minutes, and mix evenly to obtain a middle layer with a thickness of 7-9 cm; Add 83-86 parts of soil, 5-7 parts of cement, 2-3 parts of habitat substrate improver, 2-3 parts of organic fertilizer, 2-3 parts of habitat substrate organic material, 5-7 parts of tall fescue seeds, and 10-15 parts of bacterial agent into a mixer according to the above weight ratio, stir for 10-15 minutes, and mix evenly to obtain a surface layer with a thickness of 1.5-2 cm.

[0007] Preferably, the dark septate endophytic fungal species are Alternaria alternata and Cladosporium.

[0008] Preferably, during fermentation in S1, the mycelium on the potato dextrose agar solid culture medium is inoculated into a sterilized matrix, and then cultured in a shaker at 25-28°C and 150-180 rpm, wherein the shaker matrix is ​​prepared with a mass ratio of glucose: soybean cake powder: corn steep powder: pure water of 3-5:1-2:1-2:400-450; after culture for 2-4 days, the mycelium and the matrix in the shake flask are transferred to a 5 L fermenter at a ratio of 10%-15% for culture, wherein the feed matrix in the fermenter is glucose: soybean cake powder: corn steep powder: water in a mass ratio of 6-8:1-3:1-3:67-72, and the fermentation matrix is ​​sterilized, wherein the sterilization method is 115-121°C, high temperature steam sterilization for 20-30 min.

[0009] Preferably, during fermentation in S1, the ventilation rate of the fermenter is constant at 2-3 m 3 / h, the initial stirring speed is 180~200 rpm, when the dissolved oxygen drops to 8%~10%, the stirring speed is manually adjusted, increasing by 30~50 rpm each time, and the maximum stirring speed is 300~330 rpm. When the dissolved oxygen shows an upward trend during the fermentation process and the dissolved oxygen increases to 30~40%, automatic feeding is used for feeding. Feeding is stopped when the dissolved oxygen drops to 10%, and the total volume of the feeding liquid is 1 L.

[0010] Preferably, before freeze spray drying in S1, the substrate after fermentation is filtered with a 1000-mesh filter, and the filtered mycelium is mixed with maltodextrin to prepare a slurry, wherein the mycelium: maltodextrin = 1:1-1.2, and after the slurry preparation is completed, freeze spray drying is performed to prepare a powdered bacterial agent.

[0011] Preferably, calcium chloride, urea and urease are added to the vegetation concrete base in S2 by utilizing urease to promote the formation of calcium carbonate precipitation by the carbonate ions generated by the hydrolysis of calcium ions in calcium chloride and urea, so as to cement the soil. Increasing the cement ratio combined with calcium carbonate precipitation can improve the strength, stability and anti-scour performance of the substrate base while maintaining the interaction between the middle layer and the ground.

[0012] Preferably, the wood cellulose in the middle layer of the vegetation concrete in S2 has good porosity and water retention, and while releasing nutrients, it can also increase soil aeration and promote the growth and reproduction of soil microorganisms; the biochar in the middle layer of the vegetation concrete in S2 has a porous structure and a large surface area, which can increase the porosity of the soil, improve the air permeability and water retention of the soil, absorb nutrients, and maintain soil fertility; the expanded clay in the middle layer of the vegetation concrete in S2 has different levels of porosity and pore size, which can increase the permeability and aeration inside the soil and improve the soil's water retention capacity.

[0013] Preferably, the bacterial agent used in the surface layer of S2 is in powder form, which can be activated by adding water to adjust the pH around the seeds and reduce the damage of the seeds to the substrate.

[0014] The dark-colored vegetation concrete containing endophytic fungi is used in ecological restoration to improve the drought resistance of plants.

[0015] The present invention has the following beneficial effects: 1. The present invention utilizes microbial technology and ecological restoration technology to achieve plant growth promotion effects. Experiments have shown that dark-colored septate endophytic fungi agents can effectively improve the drought resistance of plants in vegetation concrete, which has important theoretical significance and application value.

[0016] 2. The dark septate endophytic fungal hyphae of the present invention can help the host plant absorb nutrients and water, regulate the balance of plant hormones in the host body and the synthesis of osmotic regulating substances, thereby improving the drought resistance and growth efficiency of the host plant, promoting the succession of vegetation communities, and ensuring the ecological restoration process.

[0017] 3. The microbial agent of the present invention improves the pH of the environment around the plant seeds and can increase the infection rate of dark septate endophytic fungi in the root system of the host plant by mixing the microbial agent with seeds for sowing. After the two form a symbiotic body, the dark septate endophytic fungi can secrete substances such as hydrolases to improve the availability of nutrients to plants, improve the nutritional structure and permeability in the soil, increase the richness of soil microorganisms, improve soil vitality, and stimulate the potential fertility of the soil.

[0018] 4. The preparation method of the microbial agent of the present invention is simple, low-cost, green and non-toxic. The propagation and preparation process of the microbial agent only takes 7 days and can be stored at room temperature. It can meet the large-scale application of dark-septate endophytic fungal agents in the ecological restoration of vegetation concrete and can reduce the use of fertilizers and other substances, reduce environmental pollution, improve the ecological environment, and promote green and sustainable development. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0020] Figure 1 It is a dark-colored septate endophytic fungal agent.

[0021] Figure 2 Schematic diagram of the diameter of dark septate endophytic fungal colonies.

[0022] Figure 3 The changes in the diameters of dark septate endophytic fungal colonies at different PEG concentrations. DETAILED DESCRIPTION

[0023] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0024] Embodiment 1: Screening of dominant drought-resistant dark-colored septate endophytic fungi: Different concentrations of sterilized polyethylene glycol (PEG) solution were added to the PDA plate, with the concentrations being 0 g / L, 6 g / L, 66 g / L, 120 g / L, and 188 g / L, respectively, and then poured out after standing for 24 h. Cladosporium colombiae, Alternaria alternata, Cladosporium sp, Acrocalymmavagum The PDA plates were inoculated with a puncher along the edge of the colony on the surface of the culture medium toward the center, with the diameter of 0.5 cm large and small bacterial cakes inoculated into the center of the PDA solid culture medium containing different concentrations of PEG. The culture was carried out at 28 °C for 7 days, and the changes in the colony diameter of the bacterial cakes were observed. Alternaria alternata, Cladosporium sp. The best drought resistance.

[0025] Embodiment 2: Preparation of a drought-resistant dark-colored septate endophytic fungal agent: Inoculated with Alternaria alternifolia ( Alternaria alternata ), Cladosporium ( Cladosporium sp. ) plates were inoculated with a hole puncher along the edge of the colony on the surface of the culture medium toward the center of the 0.5 cm diameter bacterial cakes, respectively, into the center of the potato dextrose agar (PDA) solid culture medium and cultured at 28°C for 5 days. The spores on the bacterial cakes were prepared with physiological saline to a concentration of 1×10 9 pc·mL -1 spore suspension, namely, Alternaria alternata ( Alternaria alternata ) and Cladosporium ( Cladosporium sp. ) spore suspension; Alternaria alternifolia Alternaria alternata ) and Cladosporium ( Cladosporium sp. ) The spore suspension was inoculated into 3.25 L of sterilized mixed matrix of glucose, soybean cake powder and corn steep liquor in a fermentation tank at a volume ratio of 15% (mixed suspension volume / mixed matrix volume of glucose, soybean cake powder and corn steep liquor, mL / mL), at 28°C, pH 6, and the rotation speed was controlled according to dissolved oxygen, and the culture was carried out for 4 days to obtain a fermentation mixture; After fermentation, filter with a 1000 mesh filter, mix the filtered mycelium with maltodextrin to prepare a slurry, wherein the mycelium: maltodextrin = 1:1~1.2. After the slurry is prepared, freeze-spray dry it to make a powdered bacterial agent.

[0026] The formula of PDA solid medium is 200 g potato, 20 g glucose, natural pH; Embodiment 3: (1) Pot experiment with simulated drought stress: Configuration of vegetation concrete: in accordance with the technical specifications for ecological restoration of vegetation concrete on steep slopes of hydropower projects; Inoculation treatment: Soak tall fescue seeds in clean water for 24 h, water each flowerpot thoroughly, and then evenly spread about 1,000 tall fescue seeds and 0.42 g of dark septate endophytic fungus agent on the surface, and finally cover with soil.

[0027] After one month of growth, the tall fescue began to experience drought stress, with the drought levels being normal water, moderate drought, and severe drought. In the second month, plant physiological and biochemical indicators were measured.

[0028] Determination of biomass: The aboveground and underground biomass of the plants were determined 30 days after re-grafting. The aboveground part of tall fescue was cut from the base and harvested separately from the underground part. After removing impurities, it was cleaned and dried in an oven at 105°C for 30 min, and then dried at 75°C to constant weight. The dry weight was weighed separately and recorded as tall fescue biomass.

[0029] Determination of chlorophyll a and chlorophyll b content: ① Pigment extraction: Wash the plant leaves and dry them with filter paper, accurately weigh 0.1 g of leaves, add a little CaCO3 and quartz sand and 3 mL of 80% acetone, grind them thoroughly, transfer them to a centrifuge tube, extract the residue with 3 mL of 80% acetone, combine the extracts, and finally wash the mortar with 10 mL of 80% acetone, transfer them to a centrifuge tube, centrifuge at 5000xg for 15 min, make up the supernatant to 25 mL, and place it in a dark place for later use. ② Pigment determination: Use 1 cm colorimetric dishes to determine A, B, C, D and E respectively. 663 , A 645 and A 470 . Control the absorbance between 0.1 and 0.8.

[0030] Determination of superoxide dismutase enzyme activity: ① Extraction: Weigh 0.5 g of fresh plant leaves, add 3 mL of pre-cooled enzyme extract and a small amount of quartz sand, grind thoroughly in an ice bath, and transfer to a centrifuge tube. Rinse the mortar with 2 mL of enzyme extract and combine with it. Centrifuge at 4 °C, 10000~15000 xg for 20 min and take 5 mL of supernatant. ② Determination: Take 2.85 mL of SOD reaction mixture (add 0.5 μL PBS buffer to the maximum light reduction tube without enzyme solution; the blank tube is the same as the maximum light reduction tube, but wrapped with black cloth), add 50 μL sample enzyme solution. Then add 100 μL of riboflavin solution, and the final volume is 3 mL. Place the sample in a photochemical reduction reaction in a light incubator (light intensity of about 4000 lx) and react at 25 °C for 20 minutes.

[0031] (2) Test results: Biomass: On the 60th day of growth, the CK of different drought levels and the addition of bacterial agents were measured. Alternaria alternate , (indicated by Aa in the table), adding bacterial agents Cladosporium sp. The results are shown in Table 1. The biomass of tall fescue in the two DSE groups increased compared with that in the CK group under drought stress. Among them, Cs had a better effect under moderate drought, and Aa had a more significant effect under severe drought.

[0032] Table 1 Effects of different drought levels and bacterial agent treatments on biomass of tall fescue

[0033] Chlorophyll a and chlorophyll b content: On the 60th day of growth, the CK of different drought levels and the addition of bacterial agents were measured. Alternaria alternata , add bacterial agents Cladosporium sp.The results are shown in Table 2. The chlorophyll a and chlorophyll b contents of tall fescue in the two DSE groups increased compared with those in the CK group under drought stress, and the effect of Aa was more significant under normal water content, moderate and severe drought.

[0034] Table 2 Effects of different drought levels and bacterial agent treatments on photosynthetic pigments of tall fescue

[0035] Superoxide dismutase activity: On the 60th day of growth, the activity of superoxide dismutase in tall fescue was measured at different drought levels and after adding bacterial agents. Alternaria alternata , add bacterial agents Cladosporium sp. The results are shown in Table 3. The superoxide dismutase activity of tall fescue in the two DSE groups increased compared with that in the CK group under drought stress, and the effect of Aa was more significant under normal water content, moderate and severe drought.

[0036] Table 3 Effects of different drought levels and bacterial agent treatments on superoxide dismutase activity in tall fescue

[0037] The mechanism of action of the present invention is: Disclosed are a method for preparing a dark septate endophytic fungus agent and a method for improving the drought resistance of plants in the ecological restoration of vegetation concrete using the agent. The preparation method of the agent of the invention comprises fermenting and culturing glucose, corn syrup powder and soybean cake powder and preparing the agent using a freeze spray drying method. The method for improving the drought resistance of plants in the ecological restoration of vegetation concrete of the invention comprises configuring the vegetation concrete in layers: the bottom layer increases the cement ratio in the base material ratio and additionally adds urea, calcium chloride and urease, and the urease induces urea and calcium chloride to produce calcium carbonate precipitation, which plays a reinforcing role; the middle layer additionally adds additives such as lignocellulose, biochar, and ceramsite to improve the water retention, porosity, and fertility of the soil and has the effect of enhancing plant rooting and branching; the surface layer adds the agent and plant seeds, and the agent can promote seed germination and plant growth. The invention uses microbial technology and ecological restoration technology to achieve plant growth promotion effects, and experiments prove that the dark septate endophytic fungus agent can effectively improve the drought resistance of plants in vegetation concrete, which has important theoretical significance and application value.

[0038] In addition, during the preparation of vegetation concrete, the habitat substrate modifier and habitat substrate organic material used are provided by Hubei Runzhi Ecological Technology Co., Ltd., which is a patent transfer product of the Three Gorges University, patent number: 01138343.7.

Claims

1. A method for preparing dark septate endophytic fungus agent vegetation concrete, characterized in that: The following steps are involved: S1, according to the colonization characteristics of dominant plant root fungi in the vegetation concrete plot, dark septate endophytic fungi were selected, and fermentation culture experiments were carried out. Finally, fresh mycelium was prepared into inoculants by freeze-spray drying method; S2, configuration of vegetated concrete: Add 83-86 parts of soil, 10-12 parts of cement, 5-8 parts of 1.0 mol / L calcium chloride, 5-8 parts of 1.0 mol / L urea, 1-2 parts of 4 g / L urease, 2-3 parts of habitat substrate improver, 2-3 parts of organic fertilizer, and 2-3 parts of habitat substrate organic material into a mixer according to the above weight ratio, stir for 10-15 minutes, and mix evenly to obtain a bottom layer, which is a reinforcement layer with a thickness of 2-3.5 cm; Add 83-86 parts of soil, 5-7 parts of cement, 2-3 parts of habitat substrate improver, 2-3 parts of organic fertilizer, 2-3 parts of habitat substrate organic material, 3-5 parts of lignocellulose, 3-5 parts of biochar, and 3-5 parts of ceramsite into a mixer according to the above weight ratio, stir for 10-15 minutes, and mix evenly to obtain a middle layer with a thickness of 7-9 cm; Add 83-86 parts of soil, 5-7 parts of cement, 2-3 parts of habitat substrate improver, 2-3 parts of organic fertilizer, 2-3 parts of habitat substrate organic material, 5-7 parts of tall fescue seeds, and 10-15 parts of bacterial agent into a mixer according to the above weight ratio, stir for 10-15 minutes, and mix evenly to obtain a surface layer with a thickness of 1.5-2 cm.

2. The method for preparing a dark septate endophytic fungus agent vegetation concrete according to claim 1, characterized in that: The dark septate endophytic fungal species are Alternaria alternata and Cladosporium.

3. The method for preparing a dark septate endophytic fungus agent vegetation concrete according to claim 1, characterized in that: During fermentation in S1, the mycelium on the potato dextrose agar solid culture medium was inoculated into the sterilized matrix, and then cultured in a shaker at 25-28°C and 150-180 rpm, wherein the shaker matrix was prepared with glucose: soybean cake powder: corn syrup dry powder: pure water in a mass ratio of 3-5:1-2:1-2:400-450; after culture for 2-4 days, the mycelium and matrix in the shake flask were transferred to a 5 L fermenter at a ratio of 10%-15%, wherein the feed matrix in the fermenter was glucose: soybean cake powder: corn syrup dry powder: water in a mass ratio of 6-8:1-3:1-3:67-72, and the fermentation matrix was sterilized, wherein the sterilization method was 115-121°C, high temperature steam sterilization for 20-30 min.

4. The method for preparing a dark septate endophytic fungus agent vegetation concrete according to claim 1, characterized in that: During fermentation in S1, the ventilation rate of the fermenter is constant at 2~3 m 3 / h, the initial stirring speed is 180~200 rpm. When the dissolved oxygen drops to 8%~10%, the stirring speed is manually adjusted, increasing by 30~50 rpm each time. The maximum stirring speed is 300~330rpm. When the dissolved oxygen shows an upward trend during the fermentation process and the dissolved oxygen increases to 30~40%, automatic feeding is used for feeding. Feeding is stopped when the dissolved oxygen drops to 10%. The total volume of the feeding liquid is 1 L.

5. The method for preparing a dark septate endophytic fungus agent vegetation concrete according to claim 1, characterized in that: Before freeze spray drying in S1, the substrate after fermentation was filtered with a 1000-mesh filter, and the filtered mycelium was mixed with maltodextrin to prepare a slurry, wherein the mycelium: maltodextrin = 1:1-1.

2. After the slurry preparation was completed, freeze spray drying was performed to prepare a powdered bacterial agent.

6. The method for preparing a dark septate endophytic fungus agent vegetation concrete according to claim 1, characterized in that: The addition of calcium chloride, urea and urease to the vegetation concrete base in S2 utilizes urease to promote the formation of calcium carbonate precipitation by the carbonate ions generated by the hydrolysis of calcium ions in calcium chloride and urea, thereby cementing the soil. Increasing the cement ratio combined with calcium carbonate precipitation can improve the strength, stability and anti-scouring performance of the base layer of the substrate while maintaining the interaction between the middle layer and the ground.

7. The method for preparing a dark septate endophytic fungus agent vegetation concrete according to claim 1, characterized in that: The wood cellulose in the middle layer of S2 vegetation concrete has good porosity and water retention. While releasing nutrients, it can also increase soil aeration and promote the growth and reproduction of soil microorganisms. The biochar in the middle layer of S2 vegetation concrete has a porous structure and a large surface area, which can increase the porosity of the soil, improve the air permeability and water retention of the soil, absorb nutrients and maintain the fertility of the soil. The expanded clay in the middle layer of S2 vegetation concrete has different levels of porosity and pore size, which can increase the permeability and aeration inside the soil and improve the soil's water retention capacity.

8. The method for preparing a dark septate endophytic fungus agent vegetation concrete according to claim 1, characterized in that: The bacterial agent used in the surface layer of S2 is in powder form. After being activated by adding water, it can adjust the pH around the seeds and reduce the damage of the seeds to the substrate.

9. The dark-colored septate endophytic fungus agent-containing vegetation concrete according to any one of claims 1 to 8 is used in ecological restoration to thereby improve the drought resistance of plants.

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