Method for repairing saline-alkali soil through combination of microorganisms and plants

Abstract

The invention belongs to the technical field of soil remediation, and particularly relates to a method for remediating saline-alkali soil by combining microorganisms and plants, which comprises the following steps of: 1, selecting a preservation tube strain, inoculating the preservation tube strain into an LB liquid culture medium test tube and 5 mL / bottle, and carrying out shake culture overnight at the temperature of 30 DEG C and the speed of 200 rpm; inoculating the seed solution into 15 mL of an LB liquid culture solution test tube base according to the inoculum size of 1%, carrying out shake culture at 30 DEG C and 200 rpm for 24 h to prepare strain fermentation liquor, and then taking 15 mL of the strain fermentation liquor for each strain. The microbial strain used in the invention not only has saline-alkaline tolerance, but also can promote stress resistance and growth promotion of rice, and since rice is difficult to survive in saline-alkaline land, the yield is very low, and saline-alkaline tolerance rice is used in the experiment, so that the saline-alkaline degree of soil can be improved and the rice yield can be increased by applying the strain; the rice rhizomes can loosen the soil, improve the air permeability of the soil and further improve the living environment of the strains. The EC value of the soil subjected to combined remediation by the saline-alkaline tolerant microorganisms of the rice can be reduced by 78.9%.

Description

Technical Field

[0001] The present invention relates to the technical field of soil remediation, and particularly to a method for jointly remediating saline-alkali soil by microorganisms and plants. Background Art

[0002] Soil salinization is an ecological problem commonly existing in various countries around the world. So far in China, about 80% of saline-alkali soil has not been developed and utilized. With the continuous development of social economy and the increasing intensification of human activities, saline-alkali land resources have attracted more and more attention. In this regard, it is necessary to remediate saline-alkali soil to alleviate the increasingly tense land resources.

[0003] The existing improvement technologies in China include: physical engineering measures for improvement, using modifiers for improvement, etc. With the continuous progress of human society, engineering, chemical, biological and other measures will be comprehensively applied to improve saline-alkali land to varying degrees. Chemical improvement measures generally take effect quickly. The commonly used chemical substances include modifiers such as sulfuric acid, furfural residue, slag phosphogypsum and gypsum. However, this method has the disadvantages of high cost, difficult degradation and being unfavorable to sustainable development. Physical improvement measures mainly include those in water conservancy projects, as well as replacing soil, covering soil, etc. The problem with this method is that it requires additional sewage treatment, and moreover, it will cause a large amount of waste of water resources. In addition, this method will also generate high maintenance costs for water conservancy facilities. Ecological improvement methods mainly include halophyte measures and microbial measures. This method is green and environmentally friendly, and the improvement effect is lasting. In addition, the above-mentioned methods all have a certain improvement effect, but a single method can only reduce or mitigate the saline-alkali hazard within a certain range, and cannot fundamentally eliminate the saline-alkali hazard and solve the problem of soil salinization.

[0004] Currently, the existing patent publication number CN113105287A discloses a microbial soil remediation agent for improving saline-alkali land, which is composed of the following components: oxalic acid, organic fertilizer, seaweed, bacterial liquid, polyvinyl alcohol, litchi shell, corn residue, sugarcane residue, soybean residue, bentonite, microbial inoculant, chitosan, calcium sulfate, urea, livestock and poultry manure, molasses, straw, Pinellia ternata, Arisaema heterophyllum, pomegranate, apple peel. The raw materials of the microbial soil remediation agent are common and can effectively improve soil fertility; however, the components of the microbial soil remediation agent are complex, and its preparation requires complex operation methods such as mineralization and biological concentration, with a large workload and greatly increasing the remediation cost.

[0005] Currently, the existing patent publication number CN111117638A discloses a soda saline-alkali land improver and an improvement method, which is composed of iron ore tailings, organic fertilizer and microbial inoculant. The raw materials of the soda saline-alkali land improver are widely sourced, with low cost and simple production process, and can effectively reduce the soil salt content and promote plant growth, but this improvement method is very limited in reducing the pH value of saline-alkali land.

[0006] At present, the existing patent publication number CN111201854A discloses a method for improving saline-alkali land by using microorganisms, which consists of corn straw, soybean meal, tree leaves and a microbial compound bactericide. The microbial compound bactericide includes: lactic acid bacteria, Bacillus subtilis and yeast. This invention neutralizes excessive alkaline substances in the saline-alkali soil through the acidic substances produced by fermentation, effectively enhancing soil fertility and promoting crop growth. However, the growth period of the transitional plants is too long.

[0007] Therefore, it is necessary to provide a method for jointly repairing saline-alkali soil by microorganisms and plants to solve the above technical problems. Summary of the Invention

[0008] To solve the above technical problems, the present invention provides a method for jointly repairing saline-alkali soil by microorganisms and plants, which reduces the preparation cost, shortens the repair cycle, and effectively repairs the saline-alkali land.

[0009] A method for jointly repairing saline-alkali soil by microorganisms and plants provided by the present invention: Step 1: Pick the strains in the preservation tube and inoculate them into a test tube of LB liquid medium, 5 mL / bottle, and shake and culture overnight at 30 °C and 200 rpm; inoculate the seed liquid into a test tube of 15 mL LB liquid culture medium at an inoculation amount of 1%, and shake and culture at 30 °C and 200 rpm for 24 h to prepare a strain fermentation broth. Then take 15 mL of the fermentation broth of each strain and compound the strain fermentation broths in a ratio of 1:1 between each other to prepare a compound bactericide; Step 2: Compound the fermentation broth into a microbial compound bactericide according to a biomass ratio of 1:1, dilute the compound bactericide to OD600nm = 0.6~0.8, and use a pot experiment to screen for a highly efficient microbial compound bactericide; Step 3: Put the rice seeds into distilled water and incubate them at room temperature for 24 h. Then transfer the rice to a glass petri dish lined with filter paper and incubate it at room temperature for 60 h until all the rice shows white; dry the test saline-alkali soil in a cool and dark place, sieve it to remove impurities, take 240 g of the test soil and put it into a flower pot with a diameter of 8 cm, sow 12 white rice seeds in each flower pot, and transfer them to a light incubator for day and night treatment. The culture conditions are 30 °C during the day, 24 °C at night, the humidity is set at 75%, and the light is 16 h; Step 4: 7 days after sowing, mix the compound bactericide into the soil, and the effective viable count is 5×10 7 CFU / mL, and apply the bactericide to meet 3×10 7 CFU / g soil, 30 mL of the bacterial liquid is used as a positive control, and 30 mL of sterile water is used as a blank control. Immediately observe and record the growth of the rice, and record the root length, seedling height, dry weight, fresh weight and crop growth rate of the rice after 10 days; Crop growth rate (g pot-1 d-1) = (W2 - W1) / t; W1 and W2 are the dry weights of the above-ground plants (g pot-1) measured for the first and second times; t is the time difference between the two measurements.

[0010] Preferably, the microbial inoculant is composed of Bacillus subtilis with an effective viable bacteria concentration of 1×10 5 ~9×10 7 CFU / mL, Bacillus licheniformis with an effective viable bacteria concentration, and Bacillus cereus with an effective viable bacteria concentration, mixed in a volume ratio of 1:1:1.

[0011] Preferably, the saline-alkali land is deeply plowed in the depth range of 0 - 30 cm, and then a microbial compound inoculant is added to the soil, mixed evenly with the soil in the depth range of 10 - 20 cm of the surface layer of the saline-alkali land, and the soil is balanced for 2 days.

[0012] Preferably, the seeding rate of the pre-treated rice seeds is 1.5 - 2 kg / mu, and the seeding depth is 3 - 5 cm. Preferably, in the preservation tube strains: Bacillus subtilis is Bacillus subtilis CGMCC No. 8734, Bacillus licheniformis is Bacillus licheniformis CGMCC NO. 6155, and Bacillus cereus is Bacillus cereus CGMCC No. 4348.

[0013] Preferably, the microbial inoculant is composed of Bacillus subtilis with an effective viable bacteria concentration of 1×10 5 ~9×10 7 CFU / mL, Bacillus licheniformis with an effective viable bacteria concentration, and Bacillus cereus with an effective viable bacteria concentration, mixed in a volume ratio of 1:1:1. Compared with the related technology, a method for jointly repairing saline-alkali land soil by microorganisms and plants provided by the present invention has the following beneficial effects: The microbial strains used in the present invention not only have the ability to tolerate saline-alkali, but also have the ability to promote the stress resistance and growth promotion of rice. Since it is very difficult for rice to survive in saline-alkali land and the yield is very low, the saline-alkali-tolerant rice used in this experiment is used. Applying this strain can improve the saline-alkali degree of the soil and increase the rice yield. The rice roots and stems will loosen the soil and improve the soil air permeability, further improving the living environment for the strain. The EC value of the soil jointly repaired by rice and saline-alkali-tolerant microorganisms can be reduced by 78.9%. Description of the Drawings

[0014] Figure 1 It is a schematic diagram of the test data of Example 1 of a method for jointly repairing saline-alkali land soil by microorganisms and plants provided by the present invention; Figure 2Schematic diagram of the EC value data of Example 1 of a method for jointly repairing saline-alkali soil by microorganisms and plants provided by the present invention; Figure 3 Schematic diagram of the comparison of the combined plant height of Example 2 of a method for jointly repairing saline-alkali soil by microorganisms and plants provided by the present invention; Figure 4 Schematic diagram of the comparison of the combined fresh weight, dry weight, and chlorophyll of Example 2 of a method for jointly repairing saline-alkali soil by microorganisms and plants provided by the present invention. Detailed implementation manners

[0015] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Embodiment

[0016] A method for jointly repairing saline-alkali soil by microorganisms and plants: Step 1: Pick the strains in the preservation tube and inoculate them into a test tube of LB liquid medium (5 mL / vial), and shake and culture overnight at 30 °C and 200 rpm; inoculate the seed liquid into a test tube of 15 mL LB liquid culture medium at an inoculation amount of 1%, and shake and culture at 30 °C and 200 rpm for 24 h to prepare a strain fermentation broth. Take 15 mL of the fermentation broth of each strain, and compound the strain fermentation broths pairwise in a ratio of 1:1 to prepare a compound microbial agent.

[0017] Step 2: Compound the fermentation broth into a microbial compound microbial agent according to a biomass ratio of 1:1, dilute the compound microbial agent to OD600nm = 0.6 - 0.8, and use a pot experiment to screen for an efficient microbial compound microbial agent.

[0018] Step 3: Put the rice seeds into distilled water, incubate at room temperature for 24 h, then transfer the rice to a glass petri dish lined with filter paper and incubate at room temperature for 60 h until all the rice shows white tips. Air-dry the test saline-alkali soil in a cool and dark place, sieve it to remove impurities, take 240 g of the test soil and put it into a flower pot with a diameter of 8 cm, sow 12 rice seeds with white tips in each flower pot, and transfer them to a light incubator for day and night treatment. The culture conditions are 30 °C during the day, 24 °C at night, the humidity is set at 75%, and the light is 16 h.

[0019] Step 4: 7 days after sowing, mix the compound microbial agent into the soil, with the effective viable count being 5×10 7 CFU / mL, and apply the microbial agent to meet 3×10 7CFU / g soil. 30 mL of the bacterial solution was used as the positive control, and 30 mL of sterile water was used as the blank control. Observe and record the growth of rice immediately. After 10 days, record the root length, seedling height, dry weight, fresh weight of rice, and the crop growth rate.

[0020] Crop growth rate (g pot-1 d-1) = (W2 - W1) / t; W1 and W2 are the dry weights of the above-ground plants in the first and second measurements (g pot-1); t is the time difference between the two measurements Example

[0021] In another example, inoculate a test tube of LB liquid medium (5 mL / bottle), and shake and culture overnight at 30 °C and 200 rpm; inoculate the seed liquid into a test tube of 15 mL LB liquid culture medium at an inoculation amount of 1%, and shake and culture at 30 °C and 200 rpm for 24 h to prepare a strain fermentation broth. Take 30 mL of the fermentation broth of each strain and mix it with 240 g of soil. 30 mL of the bacterial solution was used as the positive control, and 30 mL of sterile water was used as the blank control. Observe and record the growth of rice immediately. After 10 days, record the root length, seedling height, dry weight, fresh weight of rice, and the crop growth rate. After the test, measure the EC value in the soil respectively. CK is the soil without any treatment, CK1 is the soil with only the bacterial solution and no plants, and CK2 is the soil without the bacterial solution and only plants. Among them, the data of Bacillus subtilis are better Bacillus subtilis , Bacillus cereus Bacillus cereus , Bacillus licheniformis Bacillus licheniformis Three strains of bacteria.

[0022] Among them, the microbial inoculant is composed of Bacillus subtilis with an effective viable bacteria concentration of 1×10 5 ~9×10 7 CFU / mL, Bacillus licheniformis with an effective viable bacteria number concentration, and Bacillus cereus with an effective viable bacteria number concentration, mixed in a volume ratio of 1:1:1.

[0023] Among them, the depth range of 0 - 30 cm of the saline-alkali land is deeply plowed, and then a microbial compound inoculant is added to the soil and mixed evenly with the soil at a depth of 10 - 20 cm on the surface of the saline-alkali land, and the soil is balanced for 2 days.

[0024] Among them, the seeding rate of the pre-treated rice seeds is 1.5 - 2 kg / mu, and the seeding depth is 3 - 5 cm. Among them, in the preservation tube strains: Bacillus subtilis is Bacillus subtilis CGMCC No.8734, Bacillus licheniformis is Bacillus licheniformis CGMCC NO.6155, and Bacillus cereus is Bacillus cereus CGMCC No.4348.

[0025] Among them, the microbial inoculant is composed of Bacillus subtilis with an effective viable bacteria concentration of 1×10 5 ~9×10 7 CFU / mL, Bacillus licheniformis with an effective viable bacteria number concentration, and Bacillus cereus with an effective viable bacteria number concentration, mixed in a volume ratio of 1:1:1.

[0026] Example 2 Pick the strains of Bacillus subtilis, Bacillus cereus, and Bacillus licheniformis in the preservation tube and inoculate them into test tubes of LB liquid medium (5 mL / vial), and shake culture overnight at 30°C and 200 rpm; inoculate the seed liquid into 15 mL of LB liquid culture medium test tubes according to an inoculation amount of 1%, and shake culture at 30°C and 200 rpm for 24 h to prepare the strain fermentation broth. Take 15 mL of the fermentation broth of each strain, and Bacillus subtilis and Bacillus cereus , Bacillus licheniformis Compound them in a ratio of 1:1:1 to prepare 30 mL of compound microbial inoculant, and use it immediately for subsequent experiments.

[0027] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. All equivalent structural or equivalent process transformations made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, are similarly included in the patent protection scope of the present invention.

Claims

1. A method for repairing saline-alkali soil by combining microorganisms and plants, characterized in that: Step 1: Pick the strains from the preservation tube, inoculate them into LB liquid culture medium test tubes, 5 mL / bottle, and shake culture them at 30°C, 200 rpm overnight; inoculate the seed liquid into 15 mL LB liquid culture medium test tube base according to the inoculation amount of 1%, shake culture at 30°C, 200 rpm for 24 h to make strain fermentation liquid, then take 15 mL of the fermentation liquid of each strain, and compound the fermentation liquids of the strains in a ratio of 1:1 to make a composite bacterial agent; Step 2: The fermentation broth is compounded into a microbial composite agent according to a ratio of 1:1 to biomass, the composite agent is diluted to OD600nm=0.6~0.8, and a pot experiment is used to screen a high-efficiency microbial composite agent; Step 3: Put the rice seeds in distilled water and incubate at room temperature for 24 hours. Then transfer the rice to a glass culture dish covered with filter paper and incubate at room temperature for 60 hours until the rice turns white. Dry the saline-alkali soil in a cool and dark place, sieve it to remove impurities, take 240 g of the test soil and put it into a flower pot with a diameter of 8 cm, sow 12 white rice seeds in each flower pot, and move it into a light incubator for day and night treatment. The culture conditions are 30℃ during the day and 24℃ at night, the humidity is set to 75%, and the light is 16 hours. Step 4: 7 days after sowing, mix the compound bacterial agent into the soil, the effective viable count is 5107 CFU / mL, and the bacterial agent is applied to meet 1´108 CFU / g soil. 30 mL of bacterial solution is used as a positive control, and 30 mL of sterile water is used as a blank control. The growth of rice is observed and recorded immediately. After 10 days, the root length, seedling height, dry weight, fresh weight and crop growth rate of rice are recorded; Crop growth rate (g pot-1 d-1) = (W2-W1) / t; W1 and W2 are the aboveground plant dry weights of the first and second measurements (g pot-1); t is the time difference between the two measurements.

2. The method for repairing saline-alkali soil by combining microorganisms and plants according to claim 2, characterized in that: The microbial agent has an effective live bacteria concentration of 1×10 5 ~9×10 7 Bacillus subtilis with an effective viable count of 100 CFU / mL, Bacillus licheniformis with an effective viable count of 100 CFU / mL, and Bacillus cereus with an effective viable count of 100 CFU / mL are mixed in a volume ratio of 1:1:

1.

3. The method for repairing saline-alkali soil by combining microorganisms and plants according to claim 1, characterized in that: The saline-alkali land was deep plowed at a depth of 0 to 30 cm, and then a microbial compound agent was added to the soil and mixed evenly with the soil 10 to 20 cm deep in the surface layer of the saline-alkali land, and the soil was balanced for 2 days.

4. The method for repairing saline-alkali soil by combining microorganisms and plants according to claim 1, characterized in that: The sowing rate of pretreated rice seeds is 1.5-2 kg / mu, and the sowing depth is 3-5 cm.

5. The method for repairing saline-alkali soil by combining microorganisms and plants according to claim 1, characterized in that: Among the strains in the deposited tube: the Bacillus subtilis is Bacillus subtilis CGMCC No.8734, the Bacillus licheniformis is Bacillus licheniformis CGMCC No.6155, and the Bacillus cereus is Bacillus cereus CGMCC No.4348.

6. The method for repairing saline-alkali soil by combining microorganisms and plants according to claim 5, characterized in that: The effective live bacteria concentration of microbial agents is 1×10 5 ~9×10 7 Bacillus subtilis with an effective viable count of 100 CFU / mL, Bacillus licheniformis with an effective viable count of 100 CFU / mL, and Bacillus cereus with an effective viable count of 100 CFU / mL are mixed in a volume ratio of 1:1:1.

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