A method for planting concrete with dark-colored, septate endophytic fungal agents

Abstract

The present invention provides a method for preparing dark-colored septate endophytic fungal inoculant vegetation concrete. The preparation method of the inoculant of the present invention comprises fermenting and culturing glucose, corn steep liquor powder, and soybean cake powder, and preparing the inoculant by freeze-spray drying. The method of the present invention for improving plant drought resistance in ecological restoration of vegetation concrete 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, so that urease induces urea and calcium chloride to produce calcium carbonate precipitation, which serves as reinforcement; the middle layer additionally adds additives such as lignocellulose, biochar, and ceramsite to improve soil water retention, porosity, and fertility, and enhance plant rooting and branching; and the surface layer adds inoculants and plant seeds, so that the inoculants can promote seed germination and plant growth.

Description

Technical Field

[0001] This invention relates to the fields of ecological restoration and microbial technology, and in particular to a method for preparing a dark-colored, septate endophytic fungal inoculant-based vegetation concrete. Background Technology

[0002] Vegetated concrete ecological protection technology, which combines engineering protection and ecological greening functions, has become one of the effective means of implementing ecological restoration. By creating a vegetation growth environment, it promotes the formation and succession of vegetation communities and is currently being applied in more than 20 provinces and cities in my country.

[0003] With the widespread application of this technology, it also faces numerous challenges, especially in the early stages of vegetation restoration. Due to the steep terrain, surface water does not infiltrate sufficiently before surface runoff, and the substrate evapotranspiration effect places plants under drought stress, hindering the restoration process. Later input-based control methods, such as permanent irrigation systems and replanting, can temporarily alleviate these stresses, but they do not provide long-term improvement or restoration of plant nutrient utilization or the ecosystem's self-sustaining capacity, leading to continued ecosystem deterioration. Microbial remediation technology, applied to disturbed ecological environments, is a sustainable approach from the perspective of restoring the function and structure of the ecosystem. This method can be combined with vegetated concrete slope ecological restoration technology to improve slope restoration efficiency.

[0004] Dark septate endophytes (DSEs) are a class of beneficial microorganisms living in the rhizosphere of plants. They can form a mutualistic symbiotic relationship with most terrestrial plants, significantly improving plant stress resistance and nutrient utilization, stimulating soil potential fertility, and maintaining ecological balance and sustainable productivity. Furthermore, DSEs can be cultured in pure form, and the use of DSEs and other beneficial microorganisms for the ecological restoration of disturbed habitats has significant potential for widespread application, fundamentally enhancing ecosystem function and promoting sustainable ecosystem development. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned shortcomings and provide a method for preparing dark-colored septate endophytic fungal inoculant-based vegetation concrete. This method promotes plant growth under drought stress by inoculating with dark-colored septate endophytic fungal inoculant.

[0006] To achieve the above-mentioned technical features, the objective of this invention is as follows: A method for preparing dark-colored, septate endophytic fungal inoculant-based vegetation concrete, comprising the following steps:

[0007] S1. Based on the colonization characteristics of root fungi of dominant plants in the vegetation concrete sample plot, dark-colored septate endophytic fungi were selected and fermentation culture experiments were conducted. Finally, the fresh mycelium was prepared into a fungal agent using the freeze spray drying method.

[0008] S2, Vegetation Concrete Configuration:

[0009] 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 conditioner, 2-3 parts of organic fertilizer, and 2-3 parts of habitat substrate organic material to a mixer according to the above weight ratio, mix for 10-15 minutes, and mix evenly to obtain the bottom layer, which is a reinforcement layer with a thickness of 2-3.5 cm;

[0010] Add 83-86 parts soil, 5-7 parts cement, 2-3 parts habitat substrate conditioner, 2-3 parts organic fertilizer, 2-3 parts habitat substrate organic material, 3-5 parts lignocellulose, 3-5 parts biochar, and 3-5 parts ceramsite to a mixer according to the above weight ratio, mix for 10-15 minutes, and mix evenly to obtain a middle layer with a thickness of 7-9 cm;

[0011] Add 83-86 parts soil, 5-7 parts cement, 2-3 parts habitat substrate conditioner, 2-3 parts organic fertilizer, 2-3 parts habitat substrate organic material, 5-7 parts tall fescue seeds, and 10-15 parts microbial agent to a mixer according to the above weight ratio. Mix for 10-15 minutes until a uniform surface layer with a thickness of 1.5-2 cm is obtained.

[0012] Preferred dark-colored septate endophytic fungal species are Alternaria alternata and Cladosporium.

[0013] Preferably, during fermentation in S1, mycelia from potato dextrose agar solid medium are inoculated into a sterilized substrate and then cultured in a shaker at 25-28 ℃ and 150-180 rpm. The shaker substrate is prepared with a glucose:soybean meal:corn steep liquor powder:pure water mass ratio of 3-5:1-2:1-2:400-450. After culturing for 2-4 days, the mycelia and substrate in the shake flask are transferred to a 5 L fermenter at a ratio of 10%-15%. The feed substrate in the fermenter is prepared with a glucose:soybean meal:corn steep liquor powder:water mass ratio of 6-8:1-3:1-3:67-72. The fermentation substrate is sterilized by high-temperature steam sterilization at 115-121 ℃ for 20-30 min.

[0014] Preferably, during fermentation in S1, the aeration rate in the fermenter stage is kept constant at 2~3 m. 3The initial stirring speed is 180~200 rpm. When the dissolved oxygen decreases to 8%~10%, the stirring speed is manually adjusted, increasing by 30~50 rpm each time, with a maximum stirring speed of 300~330 rpm. When the dissolved oxygen shows an upward trend during fermentation and increases to 30~40%, automatic feeding is used. Feeding is stopped when the dissolved oxygen decreases to 10%. The total volume of the feeding liquid is 1 L.

[0015] Preferably, before freeze spray drying in S1, the substrate after fermentation is filtered through a 1000-mesh filter, and the filtered mycelium is mixed with maltodextrin to form a slurry, wherein the ratio of mycelium to maltodextrin is 1:1~1.2. After the slurry is formed, freeze spray drying is performed to produce a powdered inoculum.

[0016] Preferably, the addition of calcium chloride, urea, and urease to the vegetation concrete base layer in S2 utilizes urease to promote the formation of calcium carbonate precipitates from the hydrolysis of calcium ions in calcium chloride and urea, thereby cementing the soil. Increasing the cement ratio and combining it with calcium carbonate precipitation can improve the strength, stability, and erosion resistance of the base layer while maintaining the interaction between the intermediate layer and the ground.

[0017] Preferably, the lignocellulose in the middle layer of the vegetation concrete in S2 has good porosity and water retention, which can increase soil aeration and promote the growth and reproduction of soil microorganisms while releasing nutrients; the biochar in the middle layer of the vegetation concrete in S2 has a porous structure and a large surface area, which can increase soil porosity, improve soil aeration and water retention, adsorb 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 of the soil interior and improve the soil water retention capacity.

[0018] Preferably, 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 damage to the seeds from the substrate.

[0019] The vegetation concrete containing the dark-colored, septate endophytic fungal agent is used in ecological restoration to improve the drought resistance of plants.

[0020] The present invention has the following beneficial effects:

[0021] 1. This invention utilizes microbial technology and ecological restoration technology to achieve plant growth promotion. Experiments have shown that dark-colored endophytic fungal agents can effectively improve the drought resistance of plants in vegetated concrete, which has important theoretical significance and application value.

[0022] 2. The dark-colored, septate endophytic fungal hyphae of this invention can help host plants absorb nutrients and water, regulate the balance of plant hormones in the host and the synthesis of osmotic regulators, thereby improving the drought resistance and growth efficiency of host plants, promoting vegetation community succession, and ensuring the ecological restoration process.

[0023] 3. The microbial agent of this invention, when mixed with seeds during sowing, improves the pH of the environment surrounding plant seeds and increases the infection rate of dark-colored septate endophytic fungi in the root system of host plants. After the two form a symbiotic relationship, the dark-colored septate endophytic fungi can secrete hydrolytic enzymes and other substances to improve the plant's availability of nutrients, improve the nutrient structure and permeability of the soil, increase soil microbial richness, enhance soil vitality, and stimulate the potential fertility of the soil.

[0024] 4. The preparation method of the microbial agent of this invention is simple, inexpensive, green and non-toxic. The propagation and microbial agent production process only takes 7 days and can be stored at room temperature. It can meet the large-scale application of dark-colored endophytic fungal agents in the ecological restoration of vegetation concrete, and can reduce the use of chemical fertilizers and other substances, reduce environmental pollution, improve the ecological environment, and promote green and sustainable development. Attached Figure Description

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0026] Figure 1 It is a dark-colored, septate endophytic fungal inoculant.

[0027] Figure 2 This is a schematic diagram showing the diameter of dark-colored, septate endophytic fungal colonies.

[0028] Figure 3 The changes in the diameter of dark-colored septate endophytic fungal colonies at different PEG concentrations. Detailed Implementation

[0029] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0030] Example 1:

[0031] Screening for drought-resistant, dark-colored, septate endophytic fungi:

[0032] Different concentrations of sterilized polyethylene glycol (PEG) solution (0 g / L, 6 g / L, 66 g / L, 120 g / L, and 188 g / L) were added to PDA plates. After standing for 24 hours, the plates were poured off. Dark-colored, septate endophytic fungi cultured for 7 days were then collected. Cladosporium colombiae, Alternaria alternata, Cladosporium sp, AcrocalymmavagumUsing a punch, 0.5 cm diameter mycelial discs were inoculated onto PDA solid culture media containing different concentrations of PEG along the edge of the colony surface towards the center of the medium using a punch. The media were incubated at 28 ℃ for 7 days, and the changes in colony diameter were observed. The experimental results showed... Alternaria alternata, Cladosporium sp. It has the best drought resistance.

[0033] Example 2:

[0034] Preparation of a drought-resistant, dark-colored, septate endophytic fungal inoculant:

[0035] After inoculation with Alternaria alternifolia ( Alternaria alternata ), Cladosporium ( Cladosporium sp. Using a punch, inoculate 0.5 cm diameter mycelial discs onto the center of potato dextrose agar (PDA) solid medium along the edge of the colony surface towards the center. Incubate at 28°C for 5 days. Prepare a 1×10⁻⁶ concentration solution from the mycelial discs using physiological saline. 9 pcs·mL -1 The spore suspensions yielded Alternaria alternifolia (Alternaria alternifolia) Alternaria alternata ) and Cladosporium ( Cladosporium sp. )Spore suspension;

[0036] Alternaria alternifolia ( Alternaria alternata ) and Cladosporium ( Cladosporium sp. The spore suspension was inoculated into 3.25 L of sterile glucose, soybean meal, and corn steep liquor mixed substrate in a fermenter at a volume ratio (volume of mixed suspension / volume of glucose, soybean meal, and corn steep liquor mixed substrate, mL / mL) of 15%. The mixture was cultured at 28℃ and pH 6 for 4 days with the rotation speed controlled according to dissolved oxygen to obtain the fermentation mixture.

[0037] After fermentation, the mycelium is filtered through a 1000-mesh filter. The filtered mycelium is then mixed with maltodextrin to form a slurry, with the ratio of mycelium to maltodextrin being 1:1 to 1.2. After the slurry is prepared, it is freeze-spray dried to produce a powdered inoculum.

[0038] The PDA solid culture medium formula is 200 g potato, 20 g glucose, and natural pH;

[0039] Example 3:

[0040] (1) Pot experiment simulating drought stress:

[0041] Configuration of vegetation concrete: in accordance with the technical specifications for ecological restoration of steep slopes with vegetation concrete in hydropower projects;

[0042] Inoculation treatment: Soak tall fescue seeds in clean water for 24 hours. After thoroughly watering each flowerpot, evenly sow about 1000 tall fescue seeds and 0.42 g of dark-colored septate endophytic fungal agent on the surface, and finally cover with soil.

[0043] One month after the equal-height fescue began to grow, drought stress was introduced, with drought levels ranging from normal water availability to moderate and severe drought. Plant physiological and biochemical indicators were measured in the second month.

[0044] Biomass determination: The aboveground and underground biomass of the plant were determined 30 days after re-inoculation. The aboveground parts of tall fescue were harvested separately from the underground parts by cutting them from the base. After removing impurities, the plants were cleaned and then blanched in an oven at 105 ℃ for 30 min. They were then dried at 75 ℃ to constant weight. The dry weight of each part was recorded as the biomass of tall fescue.

[0045] Determination of chlorophyll a and chlorophyll b content: ① Pigment extraction: Wash the plant leaves and blot dry with filter paper. Accurately weigh 0.1 g of the leaves, add a small amount of CaCO3, quartz sand, and 3 mL of 80% acetone, grind thoroughly, transfer to a centrifuge tube, and extract the residue again with 3 mL of 80% acetone. Combine the extracts, and finally wash the mortar with 10 mL of 80% acetone. Transfer to a centrifuge tube, centrifuge at 5000xg for 15 min, and make up to 25 mL with the supernatant. Store in the dark for later use. ② Pigment determination: Measure chlorophyll a and chlorophyll b content separately using a 1 cm cuvette. 663 、A 645 and A 470 The absorbance should be controlled between 0.1 and 0.8.

[0046] Determination of superoxide dismutase (SOD) 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 slurry with 2 mL of enzyme extract and combine with it. Centrifuge at 10000~15000 xg for 20 min at 4 ℃, and take 5 mL of the supernatant. ② Determination: Take 2.85 mL of SOD reaction mixture (add 0.5 μL of PBS buffer to the maximum photoreduction tube without enzyme solution; the blank tube is the same as the maximum photoreduction tube, but wrapped with black cloth), add 50 μL of sample enzyme solution. Then add 100 μL of riboflavin solution, with a final volume of 3 mL. Place the sample in a photochemical reduction incubator (light intensity approximately 4000 lx) and react at 25 ℃ for 20 min.

[0047] (2) Experimental results:

[0048] Biomass: On day 60 of tall fescue growth, different drought levels (CK) and the effects of added microbial agents were measured. Alternaria alternate (Represented by Aa in the table) Adding microbial agents Cladosporium sp. (Represented as Cs in the table) Tall fescue biomass. The results are shown in Table 1. Both DSEs increased tall fescue biomass under drought stress compared to the CK group. Cs showed a better effect under moderate drought, while Aa showed a more significant effect under severe drought.

[0049] Table 1. Effects of different drought levels and microbial inoculant treatments on tall fescue biomass.

[0050] Chlorophyll a and chlorophyll b contents were measured at 60 days of growth of tall fescue under different drought conditions (CK) and with added microbial agents. Alternaria alternata Adding microbial agents Cladosporium sp. The contents of photosynthetic pigments chlorophyll a and chlorophyll b in tall fescue were measured. The results are shown in Table 2. Under drought stress, the contents of chlorophyll a and chlorophyll b in tall fescue increased compared with the control group, with chlorophyll a showing a more significant effect under normal water conditions, moderate drought conditions, and severe drought conditions.

[0051] Table 2. Effects of different drought levels and microbial treatments on photosynthetic pigments in tall fescue.

[0052] Superoxide dismutase activity: At day 60 of tall fescue growth, activity was measured under different drought conditions (CK) and with added microbial agents. Alternaria alternata Adding microbial agents Cladosporium sp. The superoxide dismutase (SOD) activity of tall fescue was measured. The results are shown in Table 3. Under drought stress, the activity of both DSEs was increased compared with that of the control group, with the Aa effect being more significant under normal water conditions, moderate drought conditions, and severe drought conditions.

[0053] Table 3. Effects of different drought levels and microbial treatments on the superoxide dismutase activity of tall fescue.

[0054] The mechanism of action of this invention is as follows:

[0055] This invention discloses a method for preparing a dark-colored, septate endophytic fungal inoculant and a method for improving the drought resistance of plants in vegetated concrete ecological restoration using the inoculant. The preparation method involves fermenting and culturing glucose, corn steep liquor powder, and soybean meal powder, followed by freeze-spray drying. The method for improving the drought resistance of plants in vegetated concrete ecological restoration involves layering the vegetated concrete: the bottom layer has a higher cement ratio and additional urea, calcium chloride, and urease are added to the substrate mix; urease induces urea and calcium chloride to precipitate calcium carbonate, providing reinforcement; the middle layer adds lignocellulose, biochar, and ceramsite to improve soil water retention, porosity, and fertility, and enhances rooting and branching; the top layer contains the inoculant and plant seeds, with the inoculant promoting seed germination and plant growth. This invention utilizes microbial and ecological restoration technologies to promote plant growth. Experiments have shown that the dark-colored, septate endophytic fungal inoculant can effectively improve the drought resistance of plants in vegetated concrete, demonstrating significant theoretical and practical value.

[0056] In addition, the habitat substrate modifier and habitat substrate organic material used in the preparation of vegetation concrete are provided by Hubei Runzhi Ecological Technology Co., Ltd., which are products of patent achievement transfer from Three Gorges University, patent number: 01138343.7.

Claims

1. A method for preparing a dark-colored, septate endophytic fungal inoculant-based vegetation concrete, characterized in that, Includes the following steps: S1. Based on the colonization characteristics of root fungi of dominant plants in the vegetation concrete sample plot, dark-colored septate endophytic fungi were selected and fermentation culture experiments were conducted. Finally, the fresh mycelium was prepared into a fungal agent using the freeze spray drying method. S2, Vegetation Concrete Configuration: 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 conditioner, 2-3 parts of organic fertilizer, and 2-3 parts of habitat substrate organic material to a mixer according to the above weight ratio, mix for 10-15 minutes, and mix evenly to obtain the bottom layer, which is a reinforcement layer with a thickness of 2-3.5 cm; Add 83-86 parts soil, 5-7 parts cement, 2-3 parts habitat substrate conditioner, 2-3 parts organic fertilizer, 2-3 parts habitat substrate organic material, 3-5 parts lignocellulose, 3-5 parts biochar, and 3-5 parts ceramsite to a mixer according to the above weight ratio, mix for 10-15 minutes, and mix evenly to obtain a middle layer with a thickness of 7-9 cm; Add 83-86 parts soil, 5-7 parts cement, 2-3 parts habitat substrate conditioner, 2-3 parts organic fertilizer, 2-3 parts habitat substrate organic material, 5-7 parts tall fescue seeds, and 10-15 parts microbial agent to a mixer according to the above weight ratio. Mix for 10-15 minutes until a uniform surface layer with a thickness of 1.5-2 cm is obtained.

2. The method for preparing dark-colored, septate endophytic fungal inoculant-based vegetation concrete according to claim 1, characterized in that, The dark-colored, septate endophytic fungi are Alternaria alternata and Cladosporium.

3. The method for preparing dark-colored, septate endophytic fungal inoculant-based vegetation concrete according to claim 1, characterized in that, During fermentation in S1, mycelia from potato dextrose agar solid medium were inoculated into a sterilized substrate and then cultured in a shaker at 25-28 ℃ and 150-180 rpm. The shaker substrate was prepared with a glucose:soybean meal:corn steep liquor powder:pure water mass ratio of 3-5:1-2:1-2:400-450. After culturing for 2-4 days, the mycelia and substrate in the shake flasks were transferred to a 5 L fermenter at a ratio of 10%-15%. The feed substrate in the fermenter was prepared with a glucose:soybean meal:corn steep liquor powder:water mass ratio of 6-8:1-3:1-3:67-72. The fermentation substrate was sterilized by high-temperature steam sterilization at 115-121 ℃ for 20-30 min.

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

5. The method for preparing dark-colored, septate endophytic fungal inoculant-based vegetation concrete according to claim 1, characterized in that, Before freeze spray drying in S1, the substrate after fermentation is filtered through a 1000-mesh filter. The filtered mycelium is mixed with maltodextrin to form a slurry, wherein the ratio of mycelium to maltodextrin is 1:1~1.

2. After the slurry is formed, freeze spray drying is performed to produce a powdered inoculum.

6. The method for preparing dark-colored, septate endophytic fungal inoculant-based 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 damage to the seeds from the substrate.

7. Applying the dark-colored, septate endophytic fungal agent described in any one of claims 1-6 to vegetation concrete in ecological restoration, thereby improving the drought resistance of plants.

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