Method for promoting growth of festuca arundinacea in vegetation concrete

Abstract

The invention provides a method for promoting the growth of festuca arundinacea in vegetation concrete, which comprises the following steps: selecting dark partitioned endophytic fungi according to the characteristics of a vegetation concrete base material and the plant festuca arundinacea, preparing a fungicide through propagation, preparing the vegetation concrete, disinfecting the vegetation concrete, soaking festuca arundinacea seeds, and performing high-temperature treatment on the festuca arundinacea seeds. Finally, the fungicide and the festuca arundinacea seeds subjected to seed soaking treatment are mixed and sown into a flowerpot. According to the invention, a microbial technology is introduced in the field of ecological restoration, and the growth of festuca arundinacea is promoted by utilizing the characteristic of symbiosis of dark partitioned endophytic fungi and plants. According to the method, plant growth is promoted by using a microbial technology, the method has positive ecological significance in promoting rapid regreening of vegetation on the wounded side slope, and a microbial technical support is provided for ecological restoration of the side slope.

Description

Technical Field

[0001] The invention relates to the field of ecological restoration and microbial utilization, and in particular to a method for promoting the growth of tall fescue in vegetation concrete. Background Art

[0002] While promoting local economic development, the construction of transportation, water conservancy and other projects inevitably causes strong interference to the ecological environment of the project site, forming many exposed slopes, which in turn cause a series of problems such as soil erosion, ecological damage, and frequent geological disasters, seriously endangering the safety of the project and the environment, landscape and sustainable development around the disturbed area. The ecological restoration of disturbed slopes has become a topic of focus for scholars around the world. Vegetation concrete ecological restoration technology is a slope ecological restoration technology that can have both engineering protection and ecological restoration functions. It achieves the purpose of ecological restoration by constructing a habitat substrate for plant growth.

[0003] With the widespread application of this technology, it also faces many challenges. When applied to arid areas, 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 prone to drought stress, affecting plant growth. When applied to saline-alkali areas, vegetation is prone to salt stress due to the influence of saline soil; salt stress can cause plant water loss, osmotic imbalance, and decreased photosynthetic rate, seriously restricting plant growth. 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 endophytic fungi are a type of fungi that live in the rhizosphere of plants. They usually form a symbiotic relationship with the roots of plants. They can promote plant growth and enhance plant resistance by exchanging substances with plants through contact with the roots of plants, making them more adaptable to various environmental stresses such as drought, salinity and heavy metals. Dark-septate endophytic fungi form an association with plant roots to increase the contact area between plant roots and soil, thereby increasing the plant's ability to absorb water and nutrients. At the same time, dark-septate endophytic fungi can improve soil fertility, increase soil aeration, live together with other microorganisms to form complex microbial communities, and promote soil biodiversity, effectively promoting plant growth. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for promoting the growth of tall fescue in vegetation concrete, which promotes the growth of tall fescue in vegetation concrete by inoculating dark septate endophytic fungi.

[0006] In order to achieve the above technical features, the purpose of the present invention is achieved as follows: a method for promoting the growth of tall fescue in vegetation concrete, comprising the following steps: Step 1: Seed selection: Tall fescue was selected based on the characteristics of vegetation concrete substrate and its easy survival, salt-alkali resistance, drought resistance, strong regeneration ability and suitability for a variety of climate environments and soil conditions; Step 2, culture medium preparation: Prepare PDA medium and MMN agar medium; Step 3: Activation treatment of dark septate endophytic fungi: The dark septate endophytic fungi were transferred to PDA medium containing antibiotics and cultured in an inverted culture chamber at a constant temperature for 7-8 days; Step 4: Proliferation and culture of dark septate endophytic fungi: The colonies in the PDA medium in step 3 were made into 5 mm to 6 mm cakes and inoculated into MMN medium, and placed in a constant temperature shaker for dark shaking culture for 14 days to 15 days; Step 5, preparation of dark septate endophytic fungal agent: The bacterial solution obtained in step 4 is filtered through multi-layer sterile gauze to obtain fresh mycelium, which is then crushed at low temperature to obtain a bacterial agent; Step 6: Sowing seeds: Prepare vegetation concrete, then disinfect the vegetation concrete, soak the tall fescue seeds, and finally mix the bacterial agent with the soaked tall fescue seeds for sowing.

[0007] In the step 1, high-quality and full tall fescue seeds are selected, the seeds are soaked in sterile water at 65° C. to 70° C., and naturally cooled for 24 h to 26 h.

[0008] The specific preparation process of the PDA culture medium in step 2 is: 6.0-7.0 g / L of potato starch, 20.0-22.0 g / L of glucose, and 20.0-22.0 g / L of agar are mixed, and then sterilized by high-pressure steam sterilization for 30 min-35 min.

[0009] The specific preparation process of the MMN agar medium in step 2 is as follows: 0.05-0.06 g / L of calcium chloride, 0.025-0.056 g / L of sodium chloride, 0.01-0.02 g / L of ferric chloride, 0.15-0.17 g / L of magnesium sulfate, 0.5-0.6 g / L of potassium dihydrogen phosphate, 0.25-0.27 g / L of diammonium hydrogen phosphate, 1-1.1 ug / L of vitamin B1, 15-17 g / L of glucose, and 0.2-0.22 g / L of citric acid are mixed, and then the pH is adjusted to 5.5 with HCL or NaOH, and sterilized by high-pressure steam sterilization for 30 min to 35 min.

[0010] In step 3, the antibiotics used are 50-52 mg / L of ampicillin and 50-52 mg / L of streptomycin sulfate.

[0011] The culture conditions in the constant temperature incubator in step 3 are 28° C. to 30° C. and dark culture.

[0012] In the step 4, a sterile puncher is used to punch a bacterial cake with a diameter of 5 mm at a distance of 1 to 1.5 cm from the edge of the culture dish, and two bacterial cakes are placed in each MMN culture medium.

[0013] In the step 5, the mycelium is crushed at low temperature to ensure the activity of the bacterial agent.

[0014] In step 6, the vegetation concrete is made by mixing planting soil, cement, sawdust and habitat substrate improver, wherein the weight ratio of each component is: planting soil: cement: sawdust: habitat substrate improver = 95-105: 6-9: 5-7: 3.5-4.5; The planting soil is sterilized by exposing it to sunlight for 3-5 days until the soil is dry, loose and free of lumps.

[0015] In the step 6, the tall fescue seeds need to be soaked in 65° C. constant temperature water for 20 to 30 minutes, the thickness of the mixture of the bacterial agent and the tall fescue seeds is 1 to 1.5 cm, and the thickness of the surface layer is 3 to 3.5 cm.

[0016] The present invention has the following beneficial effects: 1. The strains selected by the present invention are highly targeted, and dark-colored septate endophytic fungi have multiple advantages and can form endophytic symbiotic relationships with plants. When symbiotic with plants, they can produce complex carbon, nitrogen, and phosphorus compounds in the soil, prompting plants to obtain more nutrients, maintain the exchange of water and nutrients between the soil and plant roots, promote plant growth, and improve nutrient absorption efficiency; and enhance the stress resistance of plants by increasing the proline content of plants and reducing the malondialdehyde content in plants, making them more adaptable to different adverse environmental stresses and alleviating the inhibition of adverse environments on the growth of vegetation concrete plants.

[0017] 2. In the whole process of preparing fresh bacterial agents, the technical operations of the present invention are carried out in a clean bench and a sterile environment, which reduces the possibility of infection with foreign bacteria during the preparation of the bacterial agents, effectively improves the harvest efficiency of the bacterial agents, and reduces the loss of the bacterial agents. When filtering the mycelium and the bacterial solution, a four-layer gauze filtration method is used. Compared with the traditional high-speed centrifugation method and vacuum filtration method for separating the mycelium and the bacterial solution, it has the characteristics of being simple, efficient, low-cost, and having good collected mycelium.

[0018] 3. The dark septate endophytic fungi used in the present invention can effectively increase the plant height, root length, biomass and enhance the stress resistance of plants. The increase in plant root length can promote the plant to absorb water and nutrients in the soil, and can also help the plant to be better fixed in the soil; the increase in stress resistance can help plants better adapt to adverse environments and alleviate the impact of adverse environments on plant growth and development. At the same time, dark septate endophytic fungi can improve soil fertility, increase soil organic matter content, improve soil structure and physical properties, and improve the adaptability and competitiveness of plants in different growth environments.

[0019] 4. The present invention utilizes the technology of external microorganisms to improve the physiological characteristics of vegetation concrete plants, reduces the use of chemical fertilizers and other substances, and promotes green and sustainable development. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0021] Embodiment 1: Tall Fescue Growth Promotion and Drought Resistance Experiment Setup: A1: Normal moisture + no inoculation A2: Normal moisture + dark septate endophytic fungi ( Alternaria alternata ) A3: Moderate drought + no inoculation A4: Moderate drought + Alternaria alternata A5: Severe drought + no inoculation A6: Severe drought + Alternaria alternata A method for promoting the growth of tall fescue in vegetated concrete, the method comprising the following steps: Step 1: Culture medium preparation: PDA medium (Potato Dextrose Agar Medium), MMN medium (Modified Melin-Norkrans Agar); The preparation of PDA medium (Potato Dextrose Agar Medium) is as follows: 6.0 g / L potato starch, 20.0 g / L glucose, and 20.0 g / L agar, which are sterilized by high-pressure steam sterilization for 30 minutes; the preparation of MMN medium (Modified Melin-Norkrans Agar) in step 2 is as follows: 0.05 g / L calcium chloride, 0.025 g / L sodium chloride, 0.01 g / L ferric chloride, 0.15 g / L magnesium sulfate, 0.5 g / L potassium dihydrogen phosphate, 0.25 g / L diammonium hydrogen phosphate, 1ug / L vitamin B, 15 g / L glucose, and 0.2 g / L citric acid. The pH is adjusted to 5.5 with HCL or NaOH, and sterilized by high-pressure steam sterilization for 30 minutes.

[0022] Step 2: Activation treatment of dark septate endophytic fungi: transfer the dark septate endophytic fungi to PDA medium containing 50 mg / L ampicillin and 50 mg / L streptomycin sulfate for constant temperature inversion culture for 7 days; Step 3: Proliferation and culture of dark septate endophytic fungi: The colonies in the PDA medium were made into 5 mm cakes and inoculated into the MMN medium, and then placed in a constant temperature shaker (28°C, 120 r / min) for dark shaking culture for 14 days; Step 4: Preparation of dark septate endophytic fungal inoculant: Filter the bacterial solution obtained in step 4 through four layers of sterile gauze to obtain fresh mycelium, and then crush at low temperature to obtain the inoculant; Step 5: Seed treatment: Select high-quality, full tall fescue seeds, soak them in 65°C sterile water, and cool them naturally for 24 hours.

[0023] Step 6: Vegetation concrete configuration: The configuration is based on the vegetation concrete specification "Technical Specification for Ecological Restoration of Vegetation Concrete on Steep Slopes of Hydropower Projects" (NB / T35082-2016), and is configured in the proportion of planting soil: cement: sawdust: habitat substrate improver = 95~105:6~9:5~7:3.5~4.5; Among them, the habitat substrate improver is provided by Hubei Runzhi Ecological Technology Co., Ltd. and is a patent transfer product of the Three Gorges University, patent number: 01138343.7.

[0024] Step seven: Use potted plant experiments, and expose the vegetation concrete to sunlight for disinfection for 3-5 days before use. The thickness of the vegetation concrete base layer is about 8cm (excluding grass seeds and microbial agents), and the surface layer is about 2cm (including about 200 grass seeds). According to the experimental settings, the experimental group inoculated 0.5g of microbial agent per pot, and the control group did not inoculate microbial agents but used sterile water of the same mass instead, and finally covered with soil.

[0025] Step 8: After one month of normal growth, tall fescue was subjected to drought stress. Thirty days after the stress, the plant height, root length, biomass and stress resistance indexes (malondialdehyde, proline) of tall fescue were measured, with three repetitions.

[0026] Table 1 Inoculation Alternaria alternata Effects on Growth Promotion and Drought Resistance of Tall Fescue

[0027] The test results showed that vaccination Alternaria alternata Under different drought stresses, the growth of tall fescue was better than that of uninoculated conditions, the proline content was higher than that of uninoculated conditions, and the malondialdehyde content was lower than that of uninoculated conditions, indicating that Alternaria alternate It promotes the growth of tall fescue in vegetation concrete under drought stress, and at the same time promotes the resistance of tall fescue in vegetation concrete to drought.

[0028] Embodiment 2: Tall Fescue Growth Promotion and Salt Tolerance Experiment Setup: B1: Normal salt + no inoculation B2: Normal salt + Alternaria alternata B3: Moderate salt stress + no inoculation B4: Moderate salt stress + Alternaria alternata B5: Severe salt stress + no inoculation B6: Severe salt stress + Alternaria alternata A method for promoting the growth of tall fescue in vegetated concrete, the method comprising the following steps: Step 1: Culture medium preparation: PDA medium (Potato Dextrose Agar Medium), MMN medium (Modified Melin-Norkrans Agar); Step 2: Activation treatment of dark septate endophytes: transfer the dark septate endophytes to PDA medium containing antibiotics (50 mg / L Ampicillin and 50 mg / L Streptomycin sulfate) and culture them at a constant temperature for 7 days; The preparation of PDA medium (Potato Dextrose Agar Medium) is 6.0 g / L potato starch, 20.0 g / L glucose, and 20.0 g / L agar, which is sterilized by high-pressure steam sterilization for 30 minutes; the preparation of MMN medium (Modified Melin-Norkrans Agar) in step 2 is 0.05 g / L calcium chloride, 0.025 g / L sodium chloride, 0.01 g / L ferric chloride, 0.15 g / L magnesium sulfate, 0.5 g / L potassium dihydrogen phosphate, 0.25 g / L diammonium hydrogen phosphate, 1ug / L vitamin B, 15 g / L glucose, and 0.2 g / L citric acid. The pH is adjusted to 5.5 with HCL or NaOH, and sterilized by high-pressure steam sterilization for 30 minutes.

[0029] Step 3: Proliferation and culture of dark septate endophytic fungi: The colonies in the PDA medium were made into 5 mm cakes and inoculated into the MMN medium, and then placed in a constant temperature shaker (28°C, 120 r / min) for dark shaking culture for 14 days; Step 4: Preparation of dark septate endophytic fungal inoculant: Filter the bacterial solution obtained in step 4 through four layers of sterile gauze to obtain fresh mycelium, and then crush at low temperature to obtain the inoculant; Step 5: Seed treatment: Select high-quality, full tall fescue seeds, soak them in 65°C sterile water, and cool them naturally for 24 hours.

[0030] Step 6: Vegetation concrete configuration: The configuration is based on the vegetation concrete specification "Technical Specification for Ecological Restoration of Vegetation Concrete on Steep Slopes of Hydropower Projects" (NB / T35082-2016), and is configured in the proportion of planting soil: cement: sawdust: habitat substrate improver = 95~105:6~9:5~7:3.5~4.5; Step seven: Use potted plant experiments. Expose the vegetation concrete to the sun for 3-5 days for disinfection before use. The thickness of the vegetation concrete base is about 8 cm (excluding grass seeds and bacterial agents). According to the different degrees of salt stress, the corresponding amount of NaCl is added (no salt is added for normal salt, 6g / kg for moderate salt stress, and 9g / kg for severe salt stress). The surface layer is about 2 cm (containing about 200 grass seeds). According to the experimental settings, 0.5g of bacterial agent is inoculated in each pot of the experimental group, and the control group is not inoculated with bacterial agent but is replaced by sterile water of the same mass, and finally covered with soil.

[0031] Step 8: After 60 days of growth, the plant height, root length, biomass and stress resistance indicators (malondialdehyde, proline) of tall fescue were measured, with three replicates.

[0032] Table 2 Inoculation Alternaria alternata Effects of salt tolerance on growth promotion of tall fescue

[0033] The test results showed that vaccination Alternaria alternata Under different salt stress levels, the growth of tall fescue was better than that of uninoculated conditions, the proline content was higher than that of uninoculated conditions, and the malondialdehyde content was lower than that of uninoculated conditions, indicating that Alternaria alternate It promotes the growth of tall fescue in vegetation concrete under salt stress, and at the same time promotes the resistance of tall fescue in vegetation concrete to salt stress.

[0034] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for promoting the growth of tall fescue in vegetation concrete, characterized in that: The following steps are involved: Step 1: Seed selection: Tall fescue was selected based on the characteristics of vegetation concrete substrate and its easy survival, salt-alkali resistance, drought resistance, strong regeneration ability and suitability for a variety of climate environments and soil conditions; Step 2, culture medium preparation: Prepare PDA medium and MMN agar medium; Step 3: Activation treatment of dark septate endophytic fungi: The dark septate endophytic fungi were transferred to PDA medium containing antibiotics and cultured in an inverted culture chamber at a constant temperature for 7-8 days; Step 4: Proliferation and culture of dark septate endophytic fungi: The colonies in the PDA medium in step 3 were made into 5 mm to 6 mm cakes and inoculated into MMN medium, and placed in a constant temperature shaker for dark shaking culture for 14 days to 15 days; Step 5, preparation of dark septate endophytic fungal agent: The bacterial solution obtained in step 4 is filtered through multi-layer sterile gauze to obtain fresh mycelium, which is then crushed at low temperature to obtain a bacterial agent; Step 6: Sowing seeds: Prepare vegetation concrete, then disinfect the vegetation concrete, soak the tall fescue seeds, and finally mix the bacterial agent with the soaked tall fescue seeds for sowing.

2. A method for promoting the growth of tall fescue in vegetation concrete according to claim 1, characterized in that: In the step 1, high-quality and full tall fescue seeds are selected, the seeds are soaked in sterile water at 65° C. to 70° C., and naturally cooled for 24 h to 26 h.

3. A method for promoting the growth of tall fescue in vegetation concrete according to claim 1, characterized in that: The specific preparation process of the PDA culture medium in step 2 is: 6.0-7.0 g / L of potato starch, 20.0-22.0 g / L of glucose, and 20.0-22.0 g / L of agar are mixed, and then sterilized by high-pressure steam sterilization for 30 min-35 min.

4. A method for promoting the growth of tall fescue in vegetation concrete according to claim 3, characterized in that: The specific preparation process of the MMN agar medium in step 2 is as follows: 0.05-0.06 g / L of calcium chloride, 0.025-0.056 g / L of sodium chloride, 0.01-0.02 g / L of ferric chloride, 0.15-0.17 g / L of magnesium sulfate, 0.5-0.6 g / L of potassium dihydrogen phosphate, 0.25-0.27 g / L of diammonium hydrogen phosphate, 1-1.1 ug / L of vitamin B1, 15-17 g / L of glucose, and 0.2-0.22 g / L of citric acid are mixed, and then the pH is adjusted to 5.5 with HCL or NaOH, and sterilized by high-pressure steam sterilization for 30 min to 35 min.

5. A method for promoting the growth of tall fescue in vegetation concrete according to claim 3, characterized in that: In step 3, the antibiotics used are 50-52 mg / L of ampicillin and 50-52 mg / L of streptomycin sulfate.

6. A method for promoting the growth of tall fescue in vegetation concrete according to claim 5, characterized in that: The culture conditions in the constant temperature incubator in step 3 are 28° C. to 30° C. and dark culture.

7. A method for promoting the growth of tall fescue in vegetation concrete according to claim 5, characterized in that: In the step 4, a sterile puncher is used to punch a bacterial cake with a diameter of 5 mm at a distance of 1 to 1.5 cm from the edge of the culture dish, and two bacterial cakes are placed in each MMN culture medium.

8. A method for promoting the growth of tall fescue in vegetation concrete according to claim 5, characterized in that: In the step 5, the mycelium is crushed at low temperature to ensure the activity of the bacterial agent.

9. A method for promoting the growth of tall fescue in vegetation concrete according to claim 5, characterized in that: The vegetation concrete in step 6 is prepared by mixing planting soil, cement, sawdust and habitat substrate improver, wherein the weight ratio of each component is: planting soil: Cement: sawdust: habitat substrate improver = 95~105:6~9:5~7:3.5~4.5; The planting soil is sterilized by exposing it to sunlight for 3-5 days until the soil is dry, loose and free of lumps.

10. A method for promoting the growth of tall fescue in vegetation concrete according to claim 9, characterized in that: In the step 6, the tall fescue seeds need to be soaked in 65° C. constant temperature water for 20 to 30 minutes, the thickness of the mixture of the bacterial agent and the tall fescue seeds is 1 to 1.5 cm, and the thickness of the surface layer is 3 to 3.5 cm.

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