Alkali-resistant vegetation cement soil preparation and ecological restoration method based on compound microbial agent

Abstract

The invention provides a preparation and ecological restoration method of alkali-resistant plant-growing cement soil based on a compound microbial agent, and relates to the technical field of plant-growing cement soil. The compound microbial agent is prepared by synergistic propagation of arbuscular mycorrhizal fungi and dark partitioned endophytic fungi domesticated in an alkaline environment; and a trehalose-soluble starch composite protective agent is adopted to improve the activity and stability of the microbial inoculum through a vacuum freeze-drying process. The microbial agent disclosed by the invention has good environmental adaptability, can effectively promote root development, nutrient absorption and drought resistance of plants in plant-growing cement soil, and remarkably improves the survival rate and sustainability of slope ecological restoration. The method is simple in process, low in cost, green, environment-friendly and suitable for large-scale popularization and application.

Description

Technical Field

[0001] This invention relates to the field of vegetated cement soil technology, and in particular to a method for preparing and ecologically restoring alkali-resistant vegetated cement soil based on composite microbial agents. Background Technology

[0002] Vegetated cement-soil technology is an important means of slope ecological restoration, but its site conditions are poor, and the initial survival and growth of plants are often limited by nutrient deficiency, water stress, and high alkalinity of the substrate. Arbuscular mycorrhizal fungi can effectively expand the absorption range of plant roots and promote nutrient and water absorption, but their infection efficiency and effectiveness are often limited under alkaline stress conditions.

[0003] Dark-colored septate endophytic fungi are an important class of beneficial plant endophytes. Studies have shown that they can not only enhance the drought resistance of host plants, but also promote the spore germination, hyphal growth, and infection of plant roots by producing specific metabolites or altering the rhizosphere microenvironment.

[0004] Existing technologies often prepare arbuscular mycorrhizal fungi and dark-colored septate endophytic fungi separately and then mix them together for later use, failing to fully utilize their synergistic promoting effect in the early stages of life activities. Furthermore, existing fermentation processes for dark-colored septate endophytic fungi do not consider the alkaline environment of vegetated cement soil, resulting in low colonization efficiency after application of the fungal agents. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned shortcomings and provide a method for preparing and ecologically restoring alkali-resistant vegetated cement soil based on composite microbial agents. This alkali-resistant vegetated concrete can give full play to the synergistic effect of arbuscular mycorrhizal fungi and dark-colored septate endophytic fungi, effectively promoting the root growth, nutrient absorption and drought resistance of plants in the vegetated cement soil, which has important theoretical significance and practical application value; at the same time, it has the advantages of simple preparation, significant synergistic effect and strong environmental adaptability.

[0006] To achieve the above-mentioned technical features, the objective of this invention is as follows: A method for preparing alkali-resistant vegetated cement soil based on composite microbial agents, comprising the following steps: S1, Preparation of dark-colored septate endophytic fungal inoculum; S2, the arbuscular mycorrhizal fungal inoculum and the dark-colored septate endophytic fungal inoculum are co-cultured in a propagation substrate, and the basic complex is obtained after harvest; S3, the basic complex is mixed with an additional dark-colored septate endophytic fungal agent to prepare the composite microbial agent; S4 is a mixture of vegetated cement and soil.

[0007] Preferably, the dark-colored septate endophytic fungus is Alternaria alternata (…). Alternaria alternataFurthermore, this strain was independently isolated and identified from the root system of dominant plants on a vegetated cement-soil sample after 5 years of vegetation cover.

[0008] Preferably, step S1 specifically includes: Alternaria alternata was inoculated into a liquid culture medium with a pH of 7.8–8.2 and cultured with shaking at 25–28°C and 150–180 rpm for 2–4 days to obtain a seed culture. The seed culture was then transferred to a fermenter at an inoculation rate of 10%–15% for propagation. The aeration rate of the fermenter was 2–3 m³ / h, and dissolved oxygen was controlled by adjusting the stirring speed. After cultivation, the mycelium was collected by filtration. The wet mycelium was mixed with a composite protectant at a mass ratio of 1:1.5–2.0 to form a slurry, which was then freeze-dried under vacuum to produce a powdered, dark-colored, septate endophytic fungal agent.

[0009] In this study, Alternaria alternata was fermented in a weakly alkaline liquid culture medium (pH 7.8~8.2). The alkaline environment was conducive to the survival and infection of the strain in the alkaline environment.

[0010] Preferably, the dark-colored septate endophytic fungal agent in S1, S2, and S3 is prepared by mixing collected mycelia with a trehalose-soluble starch composite protectant and then freeze-drying it under vacuum to produce a powdered agent.

[0011] Preferably, the liquid fermentation culture medium in the fermenter comprises glucose, soybean meal powder, yeast extract powder and pure water, in a mass ratio of 7~8 : 1.5~2 : 0.5~1 : 68~70; The composite protective agent is composed of trehalose and soluble starch in a mass ratio of 1:1 to 1:1.5.

[0012] Preferably, in S2: The arbuscular mycorrhizal fungus is *M. moses* (…). Funneliformismosseae The strain is a fungal strain that was isolated and identified autonomously from the root system of dominant plants on a cement-soil sample after 5 years of vegetation cover; the amount of the dark-colored septate endophytic fungal agent added is 8% to 12% of the dry weight of the arbuscular mycorrhizal fungal agent; the propagation substrate is composed of peat moss, vermiculite, and zeolite in a mass ratio of 2.5 to 3: 2 to 2.5: 1 to 1.5.

[0013] Preferably, the peat moss has a particle size of 5-15 mm, the vermiculite has a particle size of 1-3 mm, and the zeolite has a particle size of 0.5-1 mm.

[0014] Preferably, the co-culture of arbuscular mycorrhizal fungi and dark-colored septate endophytic fungi in S2 specifically involves: After the propagation substrate is mixed evenly according to the ratio, it is placed in a breathable sterile bag and then sterilized by autoclaving at 121~124℃ for 25~35 minutes. After sterilization, the substrate is transferred to a sterilized culture container. First, two-thirds of the substrate is spread evenly in the container. The initial inoculum of *Polygonum mossioides* and the powdered dark-colored septate endophytic fungus inoculum are evenly mixed and spread evenly on the surface of the substrate. Then, the remaining one-third of the substrate is covered with the substrate, with a covering thickness of 2~3 cm. Sorghum seeds, the host plant, are sown and cultured in a greenhouse. During the culture period, low phosphorus nutrient solution is applied every 7~10 days. The culture period is 5~6 months. After the culture is completed, the above-ground parts of the potted plant are cut off, and all the culture in the non-woven fabric in the pot is harvested, crushed, and the propagated complex is obtained.

[0015] Preferably, in step S3, the mass ratio of the additional dark-colored septate endophytic fungal agent to the basic complex is 1:15 to 1:20.

[0016] Preferably, the preparation of the base course of the vegetated cement-soil in S4 includes two parts: the base course and the surface course. The base layer preparation contains the following components by weight: 90-110 parts soil, 7-9 parts cement, 3-5 parts habitat substrate conditioner, 3-5 parts sawdust, 1-3 parts sodium carboxymethyl cellulose, and 18-22 parts compound microbial agent. The surface layer formulation comprises the following components by weight: 90-110 parts soil, 7-9 parts cement, 3-5 parts habitat substrate conditioner, 3-5 parts sawdust, 1-2 parts sodium carboxymethyl cellulose, 18-22 parts compound microbial inoculant, and 3-6 parts pioneer plant seeds.

[0017] Preferably, in the surface substrate configuration of S4, the inoculant and the seeds are mixed first, which is beneficial to the early germination of plant seeds.

[0018] Preferably, in the S4 composite microbial agent for vegetated cement soil surface layer, the dark-colored septate endophytic fungal agent is in powder form. After being dissolved in water, the protective agent is rapidly decomposed by soil microorganisms and converted into readily available nutrients to meet the needs of plant growth.

[0019] Preferably, the alkali-resistant vegetated cement soil is used in ecological restoration.

[0020] The present invention has the following beneficial effects: 1. The arbuscular mycorrhizal fungi and dark septate endophytic fungi strains used in this invention are both derived from the target vegetated cement soil sample plots. The dark septate endophytic fungi undergo directional acclimatization in a weakly alkaline environment (pH 7.8~8.2) during the fermentation stage, which makes them highly adaptable to the high alkaline environment of the vegetated cement soil, significantly improving the colonization efficiency and survival rate after application.

[0021] 2. During the propagation stage of arbuscular mycorrhizal fungi, a dark-colored septate endophytic fungus agent acclimated to an alkaline environment was introduced. The dark-colored septate endophytic fungi effectively stimulated the spore germination and mycelial growth of arbuscular mycorrhizal fungi in a slightly alkaline rhizosphere environment through their metabolic activities. This dual optimization significantly improved the infection rate of arbuscular mycorrhizal fungi, thereby preparing a basic compound fungal agent with higher spore concentration, stronger activity, and better environmental adaptability.

[0022] 3. A composite protective agent composed of trehalose and soluble starch is used to replace traditional maltodextrin. Trehalose can form a stable glassy protective layer on the surface of mycelium, while soluble starch acts as a filler and dispersant. The synergistic effect of the two, combined with vacuum freeze-drying technology, greatly improves the survival rate of the inoculum during the drying process and its long-term storage stability.

[0023] 4. The final product is a physical mixture of arbuscular mycorrhizal fungi and dark-colored septate endophytic fungi. The dark-colored septate endophytic fungi, which have already established a synergistic relationship with arbuscular mycorrhizal fungi during the propagation stage, and the additional dark-colored septate endophytic fungi that have been acclimated to alkalinity before application, work together to achieve a dual promoting effect on the target plant.

[0024] 5. The preparation method of the microbial agent of this invention is simple, inexpensive, green, non-toxic, and can be stored at room temperature; it is environmentally friendly and highly sustainable. This method makes full use of the mutualistic relationship between microorganisms and natural materials, reducing the dependence on chemical fertilizers and pesticides, and conforms to the green and sustainable development concept of ecological restoration. Detailed Implementation

[0025] The embodiments of the present invention will be further described below.

[0026] 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.

[0027] Example 1: Preparation of Dark-colored Septate Endophytic Fungal Inoculant Strain activation: *Alternaria alternata* isolated from the vegetated cement soil sample was inoculated onto PDA plates and incubated at 28°C for 5 days for activation. Shaking substrate preparation: 40g glucose, 15g soybean meal powder, 8g yeast extract, and 2000g purified water were used, and the pH was adjusted to 8.0 using 1mol / L dipotassium hydrogen phosphate-sodium hydroxide buffer. The activated strain was inoculated into a 500mL Erlenmeyer flask containing 200mL of sterilized shaking substrate and incubated at 28°C and 170rpm for 3 days to obtain the seed culture. Fermentation substrate preparation: 75g glucose, 18g soybean meal powder, 8g yeast extract, and 690g purified water were used, and the initial pH was adjusted to 8.0 using 1mol / L dipotassium hydrogen phosphate-sodium hydroxide buffer. The culture was sterilized at 121°C for 2 days. 5 minutes; transfer the seed liquid to a 5L fermenter at an inoculation rate of 13% and ferment at 28℃; fermentation control: the aeration rate is kept constant at 2.8 m³ / h, and the initial stirring speed is 195 rpm; when the dissolved oxygen drops to 9%, adjust the stirring speed to 240 rpm; when the dissolved oxygen rises to 38%, start the automatic feeding system to feed, and stop feeding when the dissolved oxygen drops to 10%; inoculum formation: after 4 days of fermentation, filter the fermentation broth through a 1000-mesh filter and collect the mycelium; mix the wet mycelium with a compound protectant (trehalose: soluble starch = 1:1.2) at a mass ratio of 1:1.8 to make a slurry, and then freeze-dry under vacuum to produce a powdered, dark-colored, septate endophytic fungal inoculum.

[0028] Example 2: Co-culture of arbuscular mycorrhizal fungi and dark-colored septate endophytic fungi Preparation of propagation substrate: Weigh 2.8 kg of peat moss with a particle size of 5-15 mm, 2.2 kg of vermiculite with a particle size of 1-3 mm, and 1 kg of zeolite with a particle size of 0.5-1 mm. Mix them evenly and place them into a breathable non-woven fabric bag. Place the bag containing the substrate in an autoclave and sterilize it at 123°C for 30 minutes. After sterilization, transfer the substrate to a clean bench and spread it out to cool naturally to room temperature. Take a sterilized plastic basin (30 cm in diameter) and spread about two-thirds of the cooled substrate evenly on the bottom of the basin. Mix 50 g of *Aureobasidium mossense* initial inoculum with 5 g (10% of the mass of the arbuscular mycorrhizal fungus inoculum) of the dark-colored septate endophytic fungal powder inoculum prepared in Example 1 thoroughly in a sterile container. Evenly spread the mixed inoculant on the substrate surface; then cover it with the remaining one-third of the substrate, controlling the covering thickness to about 2.0 cm; sow 25 sorghum seeds on the surface and gently compact them; move the cultivation pots to a greenhouse with controlled temperature and light, and water regularly to keep the substrate moist; starting from the second week after sowing, water with low-phosphorus Hoaglands nutrient solution every 7 days, 100 mL per pot each time; after 5 months of cultivation, cut off the above-ground parts of the sorghum. Remove the entire contents of the pot (including roots, mycorrhizae, spores, and all substrate), air dry at 40℃ for 48 hours, and then grind it into powder through a 2 mm sieve to obtain the basic arbuscular mycorrhizal fungi-dark-colored septate endophytic fungi compound inoculant.

[0029] Example 3: Preparation of Compound Microbial Agent Take 1000g of the basic arbuscular mycorrhizal fungi-dark-colored septate endophytic fungi composite inoculant prepared in Example 2, add 60g of the powdered dark-colored septate endophytic fungi inoculant prepared in Example 1 (mass ratio 1:16.7), mix thoroughly in a sterile bag to obtain the final composite microbial inoculant.

[0030] Example 4: Preparation of Vegetated Cement Soil Base layer preparation: Take 100kg of soil, 8kg of cement, 4kg of habitat substrate conditioner, 2.5kg of organic fertilizer, 4kg of sawdust, and 1.5kg of sodium carboxymethyl cellulose.

[0031] Surface layer preparation: Take 100kg of soil, 6kg of cement, 4kg of habitat substrate conditioner, 2.5kg of organic fertilizer, 20kg of compound microbial inoculant, and 5kg of seeds. Add all materials to a mixer and stir for 12 minutes until evenly mixed.

[0032] Spray the prepared vegetated cement soil base layer onto the slope to a thickness of about 8cm; finally, spray the prepared vegetated cement soil surface layer onto the base layer to a thickness of about 2cm.

[0033] Example 5: (1) Simulated drought stress experiment: After the hydroseeding of the vegetated cement soil, normal watering and maintenance were carried out for 30 days. Subsequently, two water treatments were set up: normal water (WW, maintaining soil field capacity of 75%~80%) and drought stress (DS, maintaining soil field capacity of 35%~40%). Under each water condition, two treatment groups were set up: control group (CK, without inoculation with inoculant) and inoculated group (FJ, inoculated with the compound microbial inoculant of this invention).

[0034] Sampling was conducted 30 days after drought stress treatment to determine the following indicators: Plant carbon, nitrogen, and phosphorus content: determined using an elemental analyzer. Biomass: determined by drying and weighing method. Photosynthetic parameters: net photosynthetic rate (Pn) was determined using a portable photosynthesis system. Photosynthetic pigments: chlorophyll a (Chla), chlorophyll b (Chlb), and total chlorophyll (Chl) were determined by acetone extraction method. Stress resistance indicators: malondialdehyde (MDA) was determined by the thiobarbituric acid method, proline (Pro) by the ninhydrin method, superoxide dismutase (SOD) by the nitroblue tetrazolium method, and catalase (CAT) by ultraviolet absorption method.

[0035] (2) Experimental results: As shown in Table 1, under normal moisture conditions, the carbon content, nitrogen content, phosphorus content, and biomass of the inoculated group (FJ) were increased by 11.6%, 64.5%, 4.5%, and 28.4% respectively compared with the control group (CK). Under drought stress, the inoculated group showed increases of 44.4%, 163.4%, 3.6%, and 10.7% respectively compared with the control group. This indicates that the compound microbial agent of the present invention can significantly promote the absorption and accumulation of nutrients by plants, especially under drought stress, the effect of promoting carbon and nitrogen absorption is more prominent.

[0036]

[0037] Note: In the table, WW represents normal moisture conditions, DS represents drought stress conditions; CK is the control treatment without inoculation with compound microbial agent, and FJ is the treatment with inoculation with compound microbial agent (the same applies below).

[0038] Table 2 shows that under normal moisture conditions, the net photosynthetic rate, chlorophyll a, chlorophyll b, and total chlorophyll content of the inoculated group increased by 75.2%, 90.6%, 143.5%, and 105.1%, respectively, compared to the control group; under drought stress conditions, these figures increased by 120.3%, 48.3%, 90.9%, and 59.7%, respectively. This indicates that the compound microbial agent of this invention can significantly enhance the photosynthetic capacity of plants, increase chlorophyll content, and maintain high photosynthetic efficiency even under drought conditions.

[0039]

[0040] As shown in Table 3, under normal moisture conditions, the MDA content in the inoculated group was 56.6% lower than that in the control group, while the activities of SOD and CAT increased by 56.9% and 38.3%, respectively. Under drought stress conditions, the MDA content in the inoculated group was 28.5% lower than that in the control group, while the activities of SOD and CAT increased by 110.7% and 184.6%, respectively. This indicates that the compound microbial agent of the present invention can effectively reduce the degree of cell membrane lipid peroxidation, enhance the activity of antioxidant enzyme systems, and improve the drought resistance of plants.

[0041]

[0042] The results of this embodiment demonstrate that the composite microbial agent prepared in this invention can significantly promote plant growth in vegetated cement soil, improve nutrient absorption efficiency, enhance photosynthetic capacity, and effectively alleviate oxidative damage caused by drought stress by activating the antioxidant defense system. Under drought stress conditions, the inoculated plants can maintain high growth and physiological activity, proving the significant effect of this invention in improving the drought resistance of plants in the ecological restoration of vegetated cement soil.

[0043] The mechanism of action of this invention is as follows: A method for preparing a composite microbial agent of arbuscular mycorrhizal fungi and dark-colored septate endophytic fungi and its application in the ecological restoration of vegetated cement-soil is disclosed. The preparation method of the composite microbial agent includes fermentation culture using glucose, soybean meal powder, and yeast extract under weakly alkaline conditions (pH 7.8-8.2). Alkaline acclimatization enhances the adaptability of the microbial agent to the vegetated cement-soil environment, and vacuum freeze-drying is used to prepare the microbial agent. Simultaneously, dark-colored septate endophytic fungi are introduced during the propagation stage of the arbuscular mycorrhizal fungi. The synergistic effect of the two significantly improves the infection efficiency of the arbuscular mycorrhizal fungi, and a highly active composite microbial agent is obtained through co-cultivation. The method of this invention to improve plant growth and stress resistance in the ecological restoration of vegetated cement-soil includes layering the vegetated cement-soil: adding the composite microbial agent and plant seeds to the surface layer. The microbial agent promotes seed germination and plant growth. This invention utilizes the synergistic effect of microorganisms and ecological restoration technology to achieve plant growth promotion. Experiments have shown that this compound microbial agent can effectively improve the drought resistance and growth of plants in vegetated cement soil, which has important theoretical significance and application value.

[0044] In addition, the habitat substrate improver used in the preparation of the vegetated cement soil was provided by Hubei Runzhi Ecological Technology Co., Ltd., which is a product of the patent achievement transfer of Three Gorges University, patent number: 01138343.7.

Claims

1. A method for preparing alkali-resistant vegetated cement soil based on composite microbial agents, characterized in that, Includes the following steps: S1, Preparation of dark-colored septate endophytic fungal inoculum; S2, the arbuscular mycorrhizal fungal inoculum and the dark-colored septate endophytic fungal inoculum are co-cultured in a propagation substrate, and the basic complex is obtained after harvest; S3, the basic complex is mixed with an additional dark-colored septate endophytic fungal agent to prepare the composite microbial agent; S4 is a mixture of vegetated cement and soil.

2. The method for preparing alkali-resistant vegetated cement soil based on composite microbial agents according to claim 1, characterized in that, The dark-colored, septate endophytic fungal agent is Alternaria alternifolia.

3. The method for preparing alkali-resistant vegetated cement soil based on composite microbial agents according to claim 2, characterized in that, The specific steps of S1 are as follows: Alternaria alternata was inoculated into a liquid culture medium with a pH of 7.8–8.2 and cultured with shaking at 25–28°C and 150–180 rpm for 2–4 days to obtain a seed culture. The seed culture was then transferred to a fermenter at an inoculation rate of 10%–15% for propagation. The aeration rate of the fermenter was 2–3 m³ / h, and dissolved oxygen was controlled by adjusting the stirring speed. After cultivation, the mycelium was collected by filtration. The wet mycelium was mixed with a composite protectant at a mass ratio of 1:1.5–2.0 to form a slurry, which was then freeze-dried under vacuum to produce a powdered, dark-colored, septate endophytic fungal agent.

4. The method for preparing alkali-resistant vegetated cement soil based on composite microbial agents according to claim 3, characterized in that, The liquid fermentation culture medium in the fermenter contains glucose, soybean meal powder, yeast extract powder and pure water, with a mass ratio of 7~8 : 1.5~2 : 0.5~1 : 68~70; The composite protective agent is composed of trehalose and soluble starch in a mass ratio of 1:1 to 1:1.

5.

5. The method for preparing alkali-resistant vegetated cement soil based on composite microbial agents according to claim 1, characterized in that, In S2: The arbuscular mycorrhizal fungus is *M. moses*; the amount of the dark-colored septate endophytic fungal agent added is 8% to 12% of the dry weight of the arbuscular mycorrhizal fungal agent; the propagation substrate is composed of peat moss, vermiculite, and zeolite in a mass ratio of 2.5 to 3: 2 to 2.5: 1 to 1.

5.

6. The method for preparing alkali-resistant vegetated cement soil based on composite microbial agents according to claim 5, characterized in that: The peat moss has a particle size of 5-15 mm, the vermiculite has a particle size of 1-3 mm, and the zeolite has a particle size of 0.5-1 mm.

7. The method for preparing alkali-resistant vegetated cement soil based on composite microbial agents according to claim 5, characterized in that, The specific details of the co-culture of arbuscular mycorrhizal fungi and dark-colored septate endophytic fungi in S2 are as follows: After the propagation substrate is mixed evenly according to the ratio, it is placed in a breathable sterile bag and then sterilized by autoclaving at 121~124℃ for 25~35 minutes. After sterilization, the substrate is transferred to a sterilized culture container. First, two-thirds of the substrate is spread evenly in the container. The initial inoculum of *Polygonum mossioides* and the powdered dark-colored septate endophytic fungus inoculum are evenly mixed and spread evenly on the surface of the substrate. Then, the remaining one-third of the substrate is covered with the substrate, with a covering thickness of 2~3 cm. Sorghum seeds, the host plant, are sown and cultured in a greenhouse. During the culture period, low phosphorus nutrient solution is applied every 7~10 days. The culture period is 5~6 months. After the culture is completed, the above-ground parts of the potted plant are cut off, and all the culture in the non-woven fabric in the pot is harvested, crushed, and the propagated complex is obtained.

8. The method for preparing alkali-resistant vegetated cement soil based on composite microbial agents according to claim 1, characterized in that, In S3, the mass ratio of the additional dark-colored septate endophytic fungal agent to the basic complex is 1:15 to 1:

20.

9. The method for preparing alkali-resistant vegetated cement soil based on composite microbial agents according to claim 1, characterized in that, The base course preparation of the vegetated cement soil in S4 includes two parts: the base course and the surface course. The base layer preparation contains the following components by weight: 90-110 parts soil, 7-9 parts cement, 3-5 parts habitat substrate conditioner, 3-5 parts sawdust, 1-3 parts sodium carboxymethyl cellulose, and 18-22 parts compound microbial agent. The surface layer formulation comprises the following components by weight: 90-110 parts soil, 7-9 parts cement, 3-5 parts habitat substrate conditioner, 3-5 parts sawdust, 1-2 parts sodium carboxymethyl cellulose, 18-22 parts compound microbial inoculant, and 3-6 parts pioneer plant seeds.

10. The alkali-resistant vegetated cement soil described in any one of claims 1 to 9 is used in ecological restoration.

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