Method for improving saline-alkali soil by using microbial agent

Abstract

The invention belongs to the technical field of modern agriculture, and relates to a soil improvement method, in particular to a method for improving saline-alkali soil by using a microbial agent, which comprises the following steps: applying a Jinghua alkaline halogen bacillus BH-8 microbial agent to saline-alkali soil, and the concentration of the Jinghua alkaline halogen bacillus BH-8 in the microbial agent is 109 CFU / mL; compared with a blank control group, the saline-alkali soil fertility and various enzyme activities are obviously improved by applying the alkaline halobacter micranthum BH-8 serving as a microbial agent to the saline-alkali soil, and the alkaline halobacter micranthum BH-8 serving as the microbial agent is applied to the saline-alkali soil. The method is scientific, reliable and good in repeatability, has important scientific value, provides important reference for saline-alkali soil improvement, is beneficial to play an important role in saline-alkali soil improvement, and improves the saline-alkali soil improvement efficiency.

Description

Technical field:

[0001] The invention belongs to the field of modern agricultural technology and relates to a method for improving soil, and specifically relates to a method for improving saline-alkali land by using a microbial agent. The saline-alkali land is improved by using the microbial Huajinpu alkaline halobacter agent, which can significantly increase the content of organic matter, alkaline nitrogen, available phosphorus, and available potassium in the saline-alkali land and various soil enzyme activities, thereby achieving the purpose of improving the saline-alkali land. Background technology:

[0002] Soil salinization destroys the physical and chemical structure of the soil and has a significant impact on the growth and yield of crops. In areas with soil salinization, the soil exhibits the characteristics of high salt content and strong alkalinity. Soil salinization also reduces soil permeability, affects the water absorption activity of plants, and leads to problems such as physiological dehydration. Salt-alkali stress in salt-affected soils can lead to excessive accumulation of reactive oxygen species in plants, causing cell tissue damage and hindering normal plant growth. It also has a negative impact on soil microbial communities, deteriorates soil biological properties, and hinders the growth and development of plants and crops. In recent years, soil salinization has intensified due to the influence of natural and human factors. At present, the global area of ​​saline-alkali land exceeds 833 million hectares, accounting for 8.7% of the earth's surface, and the area of ​​soil salinization is increasing at a rate of more than 2 million hectares per year. China is a country with widespread saline-alkali land, and its saline-alkali land area is equivalent to 40% of the total cultivated land area in the country, which has a serious impact on food and environmental security. Therefore, the problem of saline-alkali land management urgently needs research and solutions.

[0003] In recent decades, previous researchers have conducted a lot of research on the management and improvement of saline-alkali land, mainly focusing on physical improvement, chemical improvement and biological improvement. Physical improvement mainly improves soil structure, enhances soil permeability, reduces evaporation and improves soil salt leaching efficiency through traditional methods such as deep plowing and straw mulching. Chemical improvement refers to the addition of chemical amendments such as biochar, marl and desulfurized gypsum to saline-alkali soil. These amendments can replace sodium and chloride ions in the soil, improve soil porosity, inhibit the accumulation of salt in the topsoil layer, and improve the physical and chemical properties of the soil and the structure of the microbial community. Biological improvement refers to the management and improvement of saline-alkali soil by applying organic fertilizers, microbial fertilizers and planting salt-tolerant plants. The gradual maturity of biological improvement technology has provided a new way for the restoration of saline-alkali land. Therefore, how to choose appropriate management methods, effectively manage saline-alkali land, reduce the degree of soil salinization, and restore and improve soil productivity has become an important issue to be solved. Summary of the invention:

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for improving saline-alkali land using microbial agents. By adding microbial agents to improve saline-alkali land, the organic matter, alkaline nitrogen, available phosphorus, available potassium content and various soil enzyme activities of the saline-alkali land can be significantly increased, thereby achieving the purpose of improving the saline-alkali land.

[0005] To achieve the above object, the present invention provides a method for improving saline-alkali land using a microbial agent, wherein the Hwajinpu alkaline halobacter BH-8 agent is applied to the saline-alkali soil, wherein the concentration of the Hwajinpu alkaline halobacter BH-8 in the agent is 1 to 3×10 9 CFU / mL; the solvent in the bacterial agent is distilled water; the application amount is 100mL of bacterial agent per 1kg of soil.

[0006] The Alkalihalobacillus hwajinpoensis BH-8 is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing), and the deposit number is CGMCC No. 30829.

[0007] The formula of the improved LB medium is: 5 g / L yeast powder, 10 g / L peptone, 5 g / L magnesium sulfate heptahydrate, 2.5 g / L potassium chloride, 0.05 g / L iron sulfate heptahydrate, 2-12 g / L sodium chloride; pH is 7.2.

[0008] After the application of the Huajinpu alkaline halobacter BH-8 bacterial agent, the soil organic matter, quick-acting potassium, alkaline nitrogen and other environmental factors all have positive responses after being treated, and the Huajinpu alkaline halobacter BH-8 plays a significant role in these positive responses.

[0009] Compared with the prior art, the present invention uses a biological improvement method to apply the Huajinpu alkaline Halobacter bacteria agent to improve the soil fertility of saline-alkali land. By comparing with the blank control group, it is found that the use of the Huajinpu alkaline Halobacter BH-8 bacteria agent significantly improves the soil fertility and various enzyme activities of saline-alkali land. The method of the present invention is scientific and reliable, has good repeatability, has important scientific value, provides an important reference for saline-alkali land improvement, helps to play an important role in saline-alkali land improvement, and improves the efficiency of saline-alkali land improvement. Description of the drawings:

[0010] Figure 1 The constructed Hwajinpo Alkalihalobacillus developmental tree involved in the present invention is identified as Alkalihalobacillus hwajinpoensis through 16S rDNA sequencing. The strain belongs to the class Bacilli, the order Bacillales, the family Bacillaceae, and is a Hwajinpo Alkalihalobacillus.

[0011] Figure 2 This is a morphological characteristic diagram of the BH-8 strain involved in the present invention.

[0012] Figure 3 The present invention relates to a BH-8 strain.

[0013] Figure 4 This is a diagram showing the spore staining results of the BH-8 strain involved in the present invention.

[0014] Figure 5 The figure is a growth curve diagram of the BH-8 strain involved in the present invention under different NaCl concentrations.

[0015] Figure 6 The figure is a schematic diagram of the change results of the soil alkaline nitrogen (AN) content involved in the present invention, wherein CK is the control group; B is the group where the BH-8 bacterial agent was applied.

[0016] Figure 7 The figure is a schematic diagram of the change results of the soil available phosphorus (AP) content involved in the present invention, wherein CK is the control group; B is the group where the BH-8 bacterial agent was applied.

[0017] Figure 8 It is a schematic diagram of the change results of soil organic matter (OM) content involved in the present invention, wherein CK is the control group; B is the group where BH-8 bacterial agent was applied.

[0018] Fig. 9 It is a schematic diagram of the change results of soil pH involved in the present invention, wherein CK is the control group; B is the group where BH-8 bacterial agent was applied.

[0019] Fig.10 The figure is a schematic diagram of the change results of the soil available potassium (AK) content involved in the present invention, wherein CK is the control group; B is the group where the BH-8 bacterial agent was applied.

[0020] Fig.11 The figure is a schematic diagram of the change results of soil catalase activity (S-CAT) involved in the present invention, wherein CK is the control group; B is the group where BH-8 bacterial agent was applied.

[0021] Fig.12 The figure is a schematic diagram of the change results of soil nitrate reductase activity (S-NR) involved in the present invention, wherein CK is the control group; B is the group where BH-8 bacterial agent was applied.

[0022] Fig.13 The figure is a schematic diagram of the change results of soil alkaline phosphatase activity (S-ALP) in different treatment groups involved in the present invention, wherein CK is the control group; B is the group treated with BH-8 bacterial agent.

[0023] Fig.14The figure is a schematic diagram of the change results of soil acid phosphatase activity (S-ACP) in different treatment groups involved in the present invention, wherein CK is the control group; B is the group treated with BH-8 bacterial agent.

[0024] Fig.15 The figure is a schematic diagram of the change results of soil urease activity (S-URE) in different treatment groups involved in the present invention, wherein CK is the control group; B is the group treated with BH-8 bacterial agent. Specific implementation method:

[0025] The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0026] Embodiment 1:

[0027] This embodiment involves the screening and identification of Halobacterium alkalicum BH-8, and the specific steps are as follows:

[0028] (I) Screening and isolation of Hwajinpo alkaline halobacter: Take 1 g of soil sample and add it into a 250 mL conical flask containing 100 mL of enrichment medium, shake and mix, and place it in a constant temperature shaker at 30°C for shaking culture for 2 to 3 days; use physiological saline to gradient dilute the cultured bacterial solution and spread it on a plate, place the plate in a constant temperature and humidity chamber at 30°C and culture until colonies grow on the plate; use an inoculation loop to pick up colonies with good growth and repeatedly streak them on a solid enrichment medium to obtain purified single colonies.

[0029] The formula of the enrichment medium is: 5 g / L yeast powder, 10 g / L peptone, 5 g / L magnesium sulfate heptahydrate, 2.5 g / L potassium chloride, 0.05 g / L iron sulfate heptahydrate and 3 g / L NaCl, with a pH of 7.2.

[0030] The preparation method of the enrichment medium is as follows: 5g yeast powder; 10g peptone; 5g magnesium sulfate heptahydrate; 2.5g potassium chloride; 0.05g ferric sulfate heptahydrate; 3g NaCl. Adjust the pH to 7.2, add distilled water to 1L, sterilize with high temperature and wet heat for 30min, and place at room temperature for use.

[0031] The sodium chloride concentration in the acclimation medium was set to 3 g / L, 5 g / L, 8 g / L, 10 g / L, 12 g / L, 15 g / L, 18 g / L, and 20 g / L. The purified single colony was streaked on the solid acclimation medium, and the sodium chloride concentration was gradually increased until a single colony that could tolerate a sodium chloride concentration of 20 g / L was obtained.

[0032] The formula of the acclimation medium is 5 g / L yeast powder, 10 g / L peptone, 5 g / L magnesium sulfate heptahydrate, 2.5 g / L potassium chloride, 0.05 g / L iron sulfate heptahydrate, and the NaCl content is adjusted according to experimental requirements; the pH is 7.2.

[0033] The culture medium composition is: 5g yeast powder; 10g peptone; 5g magnesium sulfate heptahydrate; 2.5g potassium chloride; 0.05g ferrous sulfate heptahydrate; the NaCl content is adjusted according to the experimental requirements. Adjust the pH to 7.2, add distilled water to 1L. After high temperature and wet heat sterilization for 30 minutes, place it at room temperature for use.

[0034] After enrichment culture, 8 different strains were isolated. After domestication, a moderately halophilic bacterium was finally screened out. This strain can grow on a medium with a NaCl concentration of 20 g / L. It was named BH-8. 16S rDNA sequencing was performed on the BH-8 strain to determine its species, and the colony morphology of the BH-8 strain was recorded, and its physiological, biochemical and enzyme activity characteristics were analyzed.

[0035] (II) 16S rDNA sequencing results:

[0036] The total DNA of the BH-8 strain was extracted using a bacterial genomic DNA kit, and the extracted DNA product was subjected to agarose gel electrophoresis, where clear bands were visible, proving that the extracted bacterial DNA group was intact and not broken.

[0037] The 16S rDNA sequencing results were compared with the known species sequences in Genbank, and the 16S rDNA analysis phylogenetic tree was constructed using Mega 11 software ( Figure 1 ), it was found that the BH-8 strain belonged to the same branch as Alkalihalobacillus hwajinpoensis JSM076093 in the phylogenetic tree, had high similarity with Salibacterium nitratireducens SMB4, Alkalihalobacillus algicola AB423f, and Pseudalkalibacillus caeni HB172195, and had the highest sequence homology with Alkalihalobacillus hwajinpoensis SW72 (Pseudalkalibacillus hwajinpoensisSW-72), which was 99.30%. It was known that the BH-8 strain was an Alkalihalobacillus hwajinpoensis.

[0038] The Huajinpu alkaline halobacter BH-8 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​May 31, 2024, with the deposit number CGMCC No. 30829.

[0039] (III) Physiological, biochemical and enzymatic characteristics of the Huajinpu alkaline Halobacterium strain BH-8:

[0040] like Figure 2 As shown, by observing the colony morphology, it can be seen that the BH-8 colonies are yellow, the surface is moist and smooth, and the Gram staining result is positive.

[0041] like Figure 3 As shown, the microscopic morphology of the BH-8 strain was observed by scanning electron microscopy, and it was found that the BH-8 strain was overall long and strip-shaped without flagella.

[0042] like Figure 4 As shown, the spore production of Halobacterium spp. Hwajinpo was observed by using the Schaeffer-Fulton staining method, in which the spores were stained green and the vegetative cells were stained red.

[0043] The results of physiological and biochemical and enzyme activity characteristics are shown in Table 1. The VP test results of BH-8 strain were negative. It could not use glucose to produce pyruvate; it could not produce indole; it could produce H2S; it could not reduce nitrate; it could not use citrate; it had catalase and oxidase activities, but no urease activity. It could not use inositol and D(+)-galactose, but could use glucose, sucrose, lactose, D(-)-fructose, mannitol, and starch as the only carbon source.

[0044] Table 1 Biochemical and enzymatic characteristics of BH-8 strain

[0045] Experiment Name result VP - <![CDATA[ Indole Generate]]> - <![CDATA[H2S generation]]> + Nitrate reduction - Citrate Utilization - Starch hydrolysis + Catalase + <![CDATA[ Urease ]]> - Oxidase +

[0046] Embodiment 2:

[0047] This embodiment relates to a method for preparing the Huajinpu alkaline halobacterium BH-8 bacterial agent, and the specific steps are as follows:

[0048] Determination of the optimal salt concentration of BH-8 strain: 100 mL of liquid culture medium was placed in each 250 mL conical flask, and the NaCl concentrations were adjusted to 2 g / L, 5 g / L, 8 g / L, 10 g / L, 12 g / L, and 15 g / L, respectively. Three parallels were set for each group. After high-temperature sterilization, the culture was cooled to room temperature and inoculated with 1 mL of thawed preserved BH-8 bacterial solution. The culture was placed in a 30°C constant temperature shaker for shaking culture. The absorbance of the bacterial solution at 600 nm was measured every 2 hours until the absorbance value no longer increased. Graphpad prim9 software was used to fit the growth curve, and the growth curve of the BH-8 strain under different NaCl conditions was plotted to determine the NaCl concentration tolerance range of the BH-8 strain.

[0049] The formula of the liquid culture medium is 5 g / L yeast powder, 10 g / L peptone, 5 g / L magnesium sulfate heptahydrate, 2.5 g / L potassium chloride, 0.05 g / L iron sulfate heptahydrate, and the NaCl content is adjusted according to experimental requirements; the pH is 7.2.

[0050] The growth curve of BH-8 strain under different NaCl concentration environments was drawn ( Figure 5 ), it can be clearly observed that the BH-8 strain has the ability to grow and reproduce in the range of NaCl concentration between 2g / L and 12g / L. However, as the NaCl concentration gradually increases, the growth rate of the absorbance value of the bacterial solution gradually slows down, and its maximum absorbance value also decreases, indicating that the growth of the strain is significantly inhibited. Despite this, the BH-8 strain still exhibits a high salt tolerance and can withstand a maximum NaCl concentration of 12g / L. When the NaCl concentration reaches 15g / L, the absorbance value of the bacterial solution remains almost unchanged, indicating that the strain cannot grow effectively in this environment. It is worth mentioning that when the NaCl concentration is in the range of 2g / L to 5g / L, the growth condition of the BH-8 strain is most ideal, so the following selection of a NaCl culture medium with a concentration of 3g / L and the BH-8 strain is carried out for an in-depth exploration of the culture conditions.

[0051] Embodiment 3:

[0052] This example involves an experiment on the effect of the Huajinpu alkaline halobacterium BH-8 bacterial agent on saline-alkali soil remediation, and the specific steps are as follows:

[0053] 1. Experimental design

[0054] 200g of air-dried coastal saline-alkali soil was placed in a black plastic pot with filter paper at the bottom to prevent soil loss during irrigation. The basic physical and chemical properties of the soil are as follows: soil conductivity 91.7ms / m, density 2.64g / cm3, total salt content 2.6g / kg, total nitrogen 0.89g / kg.

[0055] A control group (CK) was set up, and 20 mL of sterile water was applied; a group (B) was given Hwajinpu alkaline Halobacterium BH-8, and 20 mL of 10 9 CFU / ml of BH-8 bacterial agent. After adding different treatments, the topsoil and the applied bacterial agent need to be stirred evenly.

[0056] The preparation method of the bacterial agent used in the Huajinpu alkaline halobacter group (B) is as follows: after culturing the BH-8 strain in a modified LB medium to the logarithmic phase, the bacterial solution is centrifuged, and the precipitate is added with sterile water and resuspended to 10 9 CFU / mL of bacterial suspension.

[0057] The formula of the improved LB medium is: 5 g / L yeast powder, 10 g / L peptone, 5 g / L magnesium sulfate heptahydrate, 2.5 g / L potassium chloride, 0.05 g / L iron sulfate heptahydrate, 3 g / L NaCl; pH is 7.2.

[0058] This experiment was conducted indoors, with 3 replicates per group. An equal amount of sterile water was added every 3 days to maintain soil moisture at 60%. After 3 days of incubation, samples were taken four times on the 1st (treatment day), 7th, 14th, and 21st days. Each time, 25 g of soil was sampled using the five-point sampling method. The 15 g soil samples were air-dried under natural conditions, ground and sieved, and the changes in soil physical and chemical properties and enzyme activity were determined. The results are shown in Figure 6- Fig.15 shown.

[0059] (II) Results of the effects of the Huajinpu alkaline halobacterium BH-8 bacterial agent on the physical and chemical properties of saline-alkali soil:

[0060] The basic physical and chemical properties of the soil changed to varying degrees through the application of the Hwajinpu alkaline Halobacterium BH-8 bacterial agent (B), such as Figure 6-Figure 10 shown.

[0061] from Figure 6 It can be seen that compared with the control group (CK), after applying the Huajinpu Alkaline Halobacter BH-8 bacterial agent (B), the alkaline nitrogen (AN) content of the soil increased significantly on the 1st day (used on the same day), 7th day, 14th day and 21st day, and was the highest on the day of application. As time went on, the alkaline nitrogen content of the soil gradually decreased and basically reached stability on the 14th and 21st days.

[0062] from Figure 7 From the changes in soil available phosphorus (AP) content, it can be seen that compared with the control group, the soil available phosphorus content increased significantly on the 1st, 7th and 14th days after the application of BH-8 bacterial agent (B), and the best effect was achieved on the 1st day; the application of Huajinpu alkaline halobacterium BH-8 (B) had a relatively stable effect on the improvement of soil available phosphorus content, and only decreased on the 21st day.

[0063] from Figure 8 From the changes in soil organic matter (OM) content, it can be seen that the treatment group showed a positive trend in the effect on the soil organic matter content. Compared with the control group, Group B significantly increased the organic matter content in the soil. This improvement effect reached its peak on the 14th day after treatment, indicating that this treatment method has a significant effect on increasing soil organic matter. The increase in organic matter content not only plays an important role in improving soil fertility, but also has a positive effect on improving soil water retention and aeration. At the same time, this also helps to promote the activity of soil microorganisms and further enhance the ecological function of the soil.

[0064] from Fig. 9From the changes in soil pH, it can be seen that from the 1st to the 21st day after the application of BH-8 bacterial agent (B), the soil pH decreased significantly, which has positive significance for improving the alkaline environment of coastal saline soil.

[0065] from Fig.10 From the changes in soil available potassium (AK) content, it can be seen that the available potassium content in the soil increased significantly in the first 14 days after the application of BH-8 bacterial agent (B). Available potassium is the main form of potassium absorbed and utilized by plants, and the increase in its content helps to enhance plant resistance and yield.

[0066] In summary, compared with the control group (CK), the addition of BH-8 inoculant (B) significantly increased the content of organic matter and alkaline nitrogen in the soil (p < 0.01), and significantly reduced the pH of coastal saline soil (p < 0.05). For the content of available potassium and available phosphorus in the soil, the application of BH-8 significantly increased their content from 0 to 14 days (p < 0.05), but these effects decreased at 21 days, which may be due to the loss of soil nutrients caused by irrigation.

[0067] (III) Effects of the Huajinpu alkaline Halobacterium BH-8 bacterial agent on enzyme activity in saline-alkali soil:

[0068] The nitrate reductase, alkaline phosphatase, acid phosphatase, urease and catalase activities of the soil after each treatment were detected using existing technology. The results are as follows: Figure 11-15 shown.

[0069] from Fig.11 It can be seen from the changes in catalase activity (S-CAT) in the soil that the catalase activity in the soil presents a fluctuating change, but after the application of BH-8 bacterial agent (B), the catalase activity in the soil can be significantly improved overall.

[0070] from Fig.12 From the changes in soil nitrate reductase activity (S-NR), it can be seen that compared with the control group (CK), the application of Huajinpu Alkaline Halobacterium BH-8 showed an overall upward trend within 21 days, reaching a peak on the 7th to 14th day, indicating that the use of BH-8 bacterial agent can generally increase the nitrate reductase activity in the soil.

[0071] Soil alkaline phosphatase (S-AKP, Fig.13 ), acid phosphatase (S-ACP, Fig.14 ) and urease activity (S-UE, Fig.15) were increased within 21 days after the application of BH-8 bacterial agent (B). The application of BH-8 bacterial agent (B) significantly increased the activities of soil acid phosphatase and alkaline phosphatase within 14 days. The urease activity in the soil showed a significant increase within 21 days after the application of BH-8 bacterial agent, and reached the highest value of enzyme activity on the 7th day.

[0072] In terms of changes in soil enzyme activity, after the application of BH-8 bacterial agent, the activities of soil catalase, soil nitrate reductase, soil acid phosphatase, and soil alkaline phosphatase increased within 14 days after the application of BH-8 bacterial agent (B); the soil urease activity increased significantly within 21 days after the application of BH-8 bacterial agent.

[0073] The above experiments prove that the application of BH-8 bacterial agent can significantly increase the content of alkaline nitrogen, organic matter, available phosphorus and available potassium in coastal saline soil, help lower soil pH, and enhance the activity of soil catalase, nitrate reductase, acid phosphatase, alkaline phosphatase and urease, indicating that the application of BH-8 bacterial agent can effectively improve the nutrient content of coastal saline-alkali soil.

Claims

1. A method for improving saline-alkali land using microbial agents, characterized in that: The Huajinpu alkaline halobacter BH-8 bacterial agent is applied to the saline-alkali soil. The Huajinpu alkaline halobacter BH-8 is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, and the preservation number is CGMCC No.30829.

2. The method for improving saline-alkali land using microbial agents according to claim 1, characterized in that: The concentration of the BH-8 bacterium in the bacterial agent is 1 to 3×10 9 CFU / mL.

3. The method for improving saline-alkali land using microbial agents according to claim 1, characterized in that: The preparation method of Huajinpu alkaline halobacter BH-8 bacterial agent is as follows: after the BH-8 strain is co-cultured in a modified LB medium until the bacterial body is in the logarithmic phase, the bacterial liquid is centrifuged to obtain the precipitate, and sterile water is added to resuspend it to 10 9 CFU / mL, and the bacterial suspension after resuspension was used as the target bacterial agent.

4. The method for improving saline-alkali land using microbial agents according to claim 1, characterized in that: The formula of the improved LB medium is: 5 g / L yeast powder, 10 g / L peptone, 5 g / L magnesium sulfate heptahydrate, 2.5 g / L potassium chloride, 0.05 g / L iron sulfate heptahydrate, 2-12 g / L sodium chloride; pH is 7.

2.

5. The method for improving saline-alkali land using microbial agents according to claim 1, characterized in that: The application amount of the compound bacterial agent is 100 mL per 1 kg of soil.

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