Method for synergistically improving nitrate type secondary salinized soil by earthworms and microorganisms

Abstract

The invention relates to a method for synergistically improving nitrate type secondary salinized soil by earthworms and microorganisms. The method comprises the following steps: (1) carrying out field preparation and fertilization pretreatment on facility degraded soil; (2) preparing and applying a bacillus licheniformis liquid fungicide; and (3) putting the Willilow cavity earthworms. Organic materials are applied to provide nutrition for growth of salt-reducing bacteria and earthworms, then bacillus licheniformis is applied to preliminarily reduce soil salt content, and a proper growth environment is provided for cavity earthworms. Then the put cavity earthworms grow and propagate in the soil, the activity of the cavity earthworms improves the salt-containing plough layer structure, increases the soil porosity and the soil stability aggregate proportion, promotes the propagation of bacillus licheniformis and soil microorganisms, improves the soil micro-ecology, and promotes the soil nutrient substance circulation and the absorption and utilization of crops. The salt content of the nitrate type secondary salinization soil can be effectively reduced, the soil fertility of the soil is improved, the growth of crops is accelerated, and the sustainable development of agriculture is promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of degraded soil improvement, and particularly relates to a method for synergistically improving nitrate-type secondary salinized soil by earthworms and microorganisms. The earthworms used are Eisenia wiliami, and the microorganisms are Bacillus licheniformis domesticated by salt tolerance. Background Art

[0002] With the increasingly prominent global resource and environmental problems, soil salinization has received wide attention. China is a country with relatively serious soil salinization problems, and the area of saline soil accounts for 1 / 10 of the global saline soil area. Moreover, in recent years, under the influence of natural and human factors, the soil stressed by salinization is still developing continuously, seriously affecting arable land resources and crop yields, and being unfavorable to the sustainable development of agriculture. Among them, nitrate-type secondary salinization is a soil salinization problem caused by the excessive accumulation of nitrates in the soil, mainly seen in long-term production greenhouse facilities, and is an important cause of greenhouse continuous cropping obstacles. Different from coastal saline soil and inland saline-alkali soil, the secondary salinization problem of continuous cropping obstacle soil in protected agriculture mostly shows excessive accumulation of nitrates, which is the dominant factor of crop physiological obstacles. In the salt ion composition of such secondary salinized soil, the main cations are Ca 2+ and Na + , and the main anion is NO 3- , and SO 4 2- takes the second place. Fertilization methods, planting years, crop types and management levels will all affect its salinization degree, resulting in large changes in soil salt content and salt ions. With the increase in the service life of greenhouse facilities, the problem of soil salt accumulation becomes more and more prominent, posing a long-term threat to soil health, ecological environment and food security, and effective monitoring and management measures need to be taken for comprehensive treatment. The planting area of greenhouse facilities in China is huge, and the nitrate-type secondary salinization of soil has become an important problem faced by China's agricultural development.

[0003] At present, some studies have dealt with soil salinization problems through biological measures and found that soil microorganisms not only participate in soil nutrient transformation, but also can alleviate the osmotic stress caused by high salt content in the soil to a certain extent, and have good effects on saline soil improvement. Some nitrate-degrading bacteria play an important role in the nitrate transformation process. They promote the soil nitrogen cycle through denitrification and assimilatory nitrate reduction, reducing the NO 3 - content in the soil. Nitrate-reducing bacteria have strong ecological competitiveness in special nutritional habitats, indicating their important role in soil improvement. However, the effect of only using microbial agents is still limited, and in reality, it is necessary to continuously improve the level of soil improvement, so it is necessary to cooperate with other biological resources to further enhance the improvement effect.

[0004] In existing soil treatment research, CN109156108A discloses a comprehensive treatment method for saline-alkali soil. This method is mainly used for saline-alkali soil and is different from secondary salinized soil. This method is cumbersome to operate, requires the cooperation of planting alkali-tolerant crops, has a long operation time and slow effect, and cannot meet the rapid and efficient tillage requirements of greenhouse facilities. In addition, although CN109197010A discloses a method for jointly repairing compacted soil with a powdery microbial combination and earthworms, which combines earthworms and microorganisms for soil repair, it uses a composite strain and mainly repairs compacted soil, and does not conduct special research on nitrate-type secondary salinized soil.

[0005] When the applicant studied the synergistic effect of earthworms and microorganisms, it was found that in nitrate-type secondary salinized soil, not all earthworms have good survival rates. Although earthworms can loosen the soil structure, in nitrate-type secondary salinized soil, earthworms first need to survive in order to further improve the soil structure. Moreover, the present invention found that in nitrate-type secondary salinized soil or after applying Bacillus licheniformis liquid bacterial agent, not all species of earthworms can survive. The present invention only found that Metaphire guillelmi has excellent ability to synergistically improve nitrate-type secondary salinized soil with Bacillus licheniformis.

[0006] The present invention provides a method for synergistically improving nitrate-type secondary salinized soil by using earthworms and functional microorganisms. Bacillus licheniformis, as a functional microorganism widely present in soil, has been proven to have significant effects in reducing soil nitrate content and improving soil physical and chemical properties. Metaphire guillelmi, a surface species earthworm widely distributed in the southeastern part of China, has a good effect on improving soil structure through its life activities, can increase soil porosity and the proportion of stable aggregates, promote the transformation of soil organic matter and nutrient cycling, and can also improve the soil microecological environment by regulating the microbial community structure. These characteristics make Metaphire guillelmi have important application value in soil ecological restoration and agricultural sustainable development. Summary of the Invention

[0007] The present invention aims at the problem of nitrate-type secondary salinization in continuously cropped facility soil, and provides a method for synergistic improvement using Metaphire guillelmi and Bacillus licheniformis. The present invention provides nutrients for the growth of salt-reducing bacteria and earthworms by applying organic materials, and then applies Bacillus licheniformis to initially reduce the soil salinity, providing a suitable growth environment for Metaphire guillelmi; then Metaphire guillelmi is put in so that it can grow and reproduce in the soil, and its activities will further improve the soil structure of the saline tillage layer, increase the soil porosity and the proportion of soil stable aggregates. Therefore, the activities of earthworms can, in turn, promote the reproduction of Bacillus licheniformis and soil microorganisms, improve the soil microecology, and promote the cycling of soil nutrients and the absorption and utilization by crops. The remarkable feature of this method is to promote the colonization of earthworms in the secondary saline tillage soil, play the role of its "soil ecological engineer", improve the improvement level of nitrate-type secondary saline soil, and create a better environment for crop growth. The present invention can also show good improvement effects in severely nitrate-type secondary saline soil.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for synergistically improving nitrate-type secondary saline soil by earthworms and microorganisms, comprising the following steps:

[0010] Step (1), field preparation and pre-fertilization treatment of nitrate-type secondary saline soil: weeding the plot, excavating drainage ditches, applying base fertilizer, surface-applying decomposed organic materials, and drip-irrigating with water, wherein: the base fertilizer is agricultural chemical fertilizer, and the decomposed organic materials are debris with a particle size less than 2 cm obtained by decomposing and crushing a mixture of livestock manure or agricultural fiber waste and livestock manure;

[0011] Step (2), preparation and application of liquid bacterium agent of Bacillus licheniformis: preparing a liquid bacterium agent from Bacillus licheniformis BL-7 after salt tolerance domestication, and uniformly spraying it on the soil surface;

[0012] Step (3), putting earthworms: putting Metaphire guillelmi into the soil 20 - 40 days after applying the liquid bacterium agent;

[0013] The Bacillus licheniformis is Bacillus licheniformis BL-7, which is a microbial strain domesticated by high-concentration nitrate tolerance, preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCC NO.29417, the preservation date being December 27, 2023, and the preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The BL-7 strain can grow on a high-concentration nitrate medium of 51.2 g / L and has good nitrate degradation ability.

[0014] The described decomposed organic material can provide rich nutrients for the growth of crops and microorganisms, and can also serve as a food source required for the stable colonization of earthworms.

[0015] The described nitrate-type secondary salinized soil is significantly different from coastal saline soil and inland saline-alkali soil, and the nitrate concentration in the soil is greater than 100 mg / kg. The soil showing salt stress during the cultivation process in greenhouse facilities usually exhibits nitrate-type secondary salinized soil.

[0016] Further, the base fertilizer in step (1) is a common commercial compound fertilizer, and the dosage is determined according to the needs of the subsequent crops to be planted. Generally, about 45 g / m 2 can meet the requirements of most crops.

[0017] Further, the preparation steps of the decomposed organic material in step (1) are as follows: Mix the livestock and poultry manure or the mixture of agricultural fiber waste and livestock and poultry manure, and carry out aerobic preliminary fermentation at a fermentation temperature of 45 - 60 °C for 40 days, then turn the pile every 7 - 10 days, and turn the pile 2 - 3 times to obtain the decomposed organic material. The livestock and poultry manure preferably includes livestock and poultry manure such as cow dung produced by surrounding farms.

[0018] Further, the method of surface-applying the decomposed organic material in step (1) is to evenly sprinkle the decomposed organic material on the soil surface and mix it evenly by shallow tillage of 8 - 15 cm.

[0019] Further, the application rate of the decomposed organic material is 10 - 20 kg / m 2 .

[0020] Further, the liquid bacterial agent in step (2) contains spores formed by the described Bacillus licheniformis strain ≥ 2×10 8 cfu / mL. Preferably, the liquid bacterial agent also contains a protective agent. The protective agent is a commonly used type for preparing liquid bacterial agents, including but not limited to at least one of trehalose, sucrose, glycerol, sorbitol, propylene glycol, benzoate, and sorbate.

[0021] As an example, the preparation method of the liquid bacterial agent is as follows: Expand and culture Bacillus licheniformis BL-7 until the number of spores produced in the culture solution ≥ 1×10 9 cfu / mL, add a protective agent to the culture solution and stir to mix, then the liquid bacterial agent is obtained. According to the specific implementation method, after Bacillus licheniformis BL-7 is cultured in a seed medium for 24 h, it is inoculated into a nitrate medium at an inoculation amount of 1% (v / v) and cultured for 24 h, and finally inoculated into a spore production medium at an inoculation amount of 2% (v / v) and cultured for 36 h to obtain a culture solution. Add 1 g of trehalose to every 100 mL of the culture solution and stir to mix, then the liquid bacterial agent is obtained.

[0022] Furthermore, the application rate of the liquid bacterial agent is 45 - 55 mL / m 2 , which is evenly sprayed on the soil surface. The liquid bacterial agent is sprayed once every 15 days after applying the decomposed organic materials, 22.5 - 27.5 mL / m each time 2 , and it is applied twice in total.

[0023] Furthermore, in step (3), the cocoons of Eisenia fetida are clear, have good activity, and their body weight is between 1.5 - 3 g. Preferably, the Eisenia fetida in step (3) is pre-cultured for 5 - 10 days before being put in.

[0024] According to the specific implementation manner, the method for pre-culturing Eisenia fetida before being put in is: using pollution-free garden soil as the breeding substrate, putting Eisenia fetida into the breeding substrate for 5 - 10 days, and maintaining the soil water content at 30 - 40%.

[0025] Furthermore, the application rate of the Eisenia fetida is 45 - 55 g / m 2 , and it is put in the soil near the irrigation water belt.

[0026] By using the method of synergistically improving nitrate-type secondary salinized soil with earthworms and microorganisms of the present invention, about one month after putting in Eisenia fetida, the soil salinity decreases and the soil ecological environment improves, and at this time, crops can be planted. After planting crops such as tomatoes, conventional field management including measures such as irrigation, drainage, and heat preservation is adopted. Preferably, the field management is that when the soil moisture is lower than 10% m / m, irrigation is required to make the soil moisture reach 60 - 70% of the field water holding capacity.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] 1) Multi-stage synergistic improvement mode: The improvement processes of earthworms and microorganisms are divided into multiple stages, and each stage is targeted at different problems of the soil for targeted improvement. For example, in the initial stage, microorganisms are mainly used to degrade nitrates. In the middle stage, earthworms are used to improve the soil structure, promote the circulation of soil nutrients and reduce salinity. In the later stage, during the growth process of crops, the soil is further improved through the combined action of the bacterial agent and earthworms, realizing the overall restoration of the soil ecosystem.

[0029] 2) Application of Pheretima guillemi: Pheretima guillemi used in the present invention is a widely distributed earthworm species, which is widely distributed in the southeastern region of China. Compared with the toxicological model species Eisenia foetida, Pheretima guillemi has stronger field adaptability. It has a large body size, strong anti-interference ability, and low demand for organic matter, and is more suitable for the farmland soil environment. By supplementing the nutrients of organic materials and preliminarily reducing salt by functional strains, a suitable growth environment is provided for earthworms. Their activities such as feeding, burrowing, and excretion significantly improve the soil structure, increase the porosity and the proportion of stable aggregates, and further promote the decline of soil salinity and the improvement of soil ecological environment.

[0030] 3) Application of highly salt-tolerant strains: Bacillus licheniformis is a common Bacillus species, which is widely present in environments such as soil, water bodies, and the surfaces of plants, and is an important microbial resource. The Bacillus licheniformis BL-7 strain after high-salt tolerance domestication has simple culture conditions and convenient operation, and can effectively repair secondary salinized soil, reduce the use of chemical fertilizers, and reduce nitrogen loss. Description of the Drawings

[0031] Figure 1 Changes in earthworm density, biomass, and the abundance of Bacillus licheniformis before and after different treatments;

[0032] Figure 2 Changes in the gene abundance of nitrogen cycle functional microorganisms under different treatments;

[0033] Figure 3 Analysis of tomato plant indicators and yields under different treatments;

[0034] Figure 4 Changes in the experimental plots during the improvement process;

[0035] Figure 5 Colonization of earthworms in the BE treatment and BM treatment. Specific Implementation Methods

[0037] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and should not be used to limit the protection scope of the present invention.

[0038] Example 1 Domestication of Bacillus licheniformis and Preparation of Liquid Bacterial Agent

[0039] Seed medium: Peptone 10 g / L, beef extract 10 g / L, yeast extract 3 g / L, glucose 10 g / L, dipotassium hydrogen phosphate 2 g / L, sodium chloride 1 g / L, adjust the pH to 6.5 with NaOH solution, and use after autoclaving;

[0040] Nitrate medium (51.2 g / L potassium nitrate): Mix the seed medium and the high-concentration nitrate solution (weigh 102.4 g of potassium nitrate, dissolve it in pure water and make up to 1 L, use after autoclaving) in a volume ratio of 1:1;

[0041] Sporulation medium: 10 g / L corn flour, 5 g / L soybean cake powder, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.1 g / L manganese sulfate, adjust the pH to 7.0 with NaOH solution, use after autoclaving.

[0042] Culture Bacillus licheniformis BL-7 (CGMCC NO.29417) in the seed medium at 30 °C and 200 r / min on a shaker for 24 h. Inoculate the seed liquid into the nitrate medium at a seed amount of 1% (v / v) and culture it at 30 °C and 200 r / min on a shaker for 24 h. Finally, inoculate it into a 5-L conical flask pre-filled with 2 L of sterilized sporulation medium at a seed amount of 2% (v / v) for culture. The culture conditions are: 37 °C, 300 r / min, ferment for 36 h, and the number of spores produced reaches 1×10 9 cfu / mL. Add 1% (W / V) trehalose to the culture solution and stir to mix, then the liquid bacterial agent containing Bacillus licheniformis BL-7 (CGMCC NO.29417) is obtained. After detection, the liquid bacterial agent contains spores formed by the strain ≥2×10 8 cfu / mL.

[0043] Pre-culture of earthworms for the experiment in Example 2

[0044] The pre-culture in this example is to improve the later repair effect. When implementing the present invention, healthy and uncontaminated earthworms can also be directly selected for application.

[0045] (1) Preliminary screening of earthworms: Drawida willsi and Eisenia fetida are from farms. First, conduct a preliminary screening, and select earthworms with clear clitellum and good activity for pre-culture. The weight of Drawida willsi is between 1.5 - 3 g, and the weight of Eisenia fetida is between 0.2 - 0.7 g. During the screening process, eliminate individuals with damaged body surfaces, slow movement or too light weight to ensure that the earthworms used for pre-culture are healthy and have good growth potential;

[0046] (2) Preparation of breeding substrate: During pre-culture, select uncontaminated garden soil with medium fertility as the breeding substrate, and the collection depth is 0 - 30 cm. Air-dry the collected garden soil naturally and remove impurities such as weeds, stones and large insects;

[0047] (3) Breeding environment control: Use standard breeding boxes with dimensions of 60 cm in length, 45 cm in width, and 40 cm in height. Put 30 kg of treated garden soil into each breeding box, ensure the uniform thickness of the soil, and maintain the soil moisture content between 30 - 40%. Keep the soil moisture by spraying water regularly and covering the surface with straw. Control the breeding temperature at 18 - 22 °C and pre-culture for 5 - 10 days. During the pre-culture period, observe the activities of earthworms and the soil moisture every day, and regularly record the growth of earthworms, including weight changes and reproduction.

[0048] Through the above pre-culture process, ensure that earthworms have a good growth state and adaptability before being put into the field experiment, laying a foundation for the subsequent field experiment.

[0049] Example 3 Field experiment on the synergistic improvement of secondary saline-alkali soil by Eisenia foetida and Bacillus licheniformis

[0050] The field experiment was carried out at Youran Ecological Farm, Maqiao Town, Minhang District, Shanghai, with a longitude of 121.3384°E and a latitude of 30.9963°N. The planting mode of this base is mainly facility cultivation, and crops such as Chinese cabbage, chili peppers, and tomatoes are planted in the greenhouse all year round. The long-term facility cultivation has led to an increasingly serious problem of soil salinization, affecting the growth and yield of crops, and effective improvement methods are urgently needed. The specific implementation steps are as follows:

[0051] I. Steps of the field experiment

[0052] (1) Dehydrate the cow dung generated from livestock and poultry breeding around the farm initially and then put it into the fermentation tank, mix it fully with rice husks to form a fermentation pile, carry out aerobic primary fermentation at a fermentation temperature of 45 - 60 °C for 40 days, and then turn the pile every 7 - 10 days, turning the pile 2 - 3 times to obtain mature cow dung. After the mature cow dung is broken into debris with a particle size less than 2 cm, it is reserved for use;

[0053] (2) After weeding the plot, dig drainage ditches and conduct the experiment in the form of plots. Take Eisenia fetida as the control, adopt a 3×2 two-factor interaction design, that is, 3 levels of earthworms (no earthworms inoculated, inoculated with Eisenia fetida (E), inoculated with Eisenia andrei (M)) and 2 levels of microbial agents (no microbial agent applied, applied with the liquid microbial agent of Bacillus licheniformis prepared in Example 1 (B)). The plot size is 6 m × 2.5 m, with an area of 15 m 2 , a total of 18 plots are set up. To prevent earthworms from escaping and reduce the influence between plots, separate the plots with a waterproof membrane, with a depth of about 40 cm and 20 cm higher than the soil surface. Each plot is set with two ridges, the ridge width is about 1.0 m, and the interval is 0.5 m;

[0054] (3) Earthworms feed on decomposed cow dung and other substances. According to the principle of equal nitrogen fertilization, chemical fertilizers and decomposed cow dung are used for fertilization. The base fertilizer used is 15-15-15 Lion Horse brand compound fertilizer, with a dosage of 45g / m 2 . The cow dung was applied on the surface, and shallow tillage was used to mix evenly. The treatment group with cow dung alone was used as the control. Therefore, there were 6 treatments in this experiment: CK (cow dung alone), B (cow dung + bacterial agent), E (cow dung + earthworm), M (cow dung + earthworm), BE (cow dung + bacterial agent + earthworm), BM (cow dung + bacterial agent + earthworm, that is, earthworm-Bacillus licheniformis combined treatment), and each treatment was repeated 3 times. After the plot was set up, drip irrigation tape was laid and 15kg / m of decomposed cow dung was applied. 2 , watering is stable for one week, and the situation of the plot after watering is stable for one week is as follows Figure 4 (C)

[0055] (4) After the soil is stable, evenly spray the cultured Bacillus licheniformis liquid inoculant on the soil surface, once every 15 days, for a total of two applications. One month after the application of the inoculant, add William earthworms and Eisenia fetida to the soil. When adding, evenly spread the earthworms at a certain distance near the irrigation water belt of the plot, observe the earthworms' entry into the soil, and replace the earthworms with poor activity if necessary. Plant crops after the earthworms have stabilized in the soil for one month. The application rate of liquid inoculant each time is 25mL / m 2 , the earthworm dosage is 50g / m 2 ; The situation of the plot after earthworm-Bacillus licheniformis combined treatment is as follows Figure 4 (D)

[0056] (5) The experimental plot was planted with tomatoes at a planting density of 1,800 plants / mu. During the growth period of tomatoes, the same farmland management measures were adopted in each field treatment, such as irrigation, manual weeding, and real-time monitoring of soil and air temperature and humidity. In order to ensure the normal activity of earthworms, no pesticides were used during the entire experimental process.

[0057] 2. Sample Collection and Measurement

[0058] Soil samples and earthworm samples were collected before and after each treatment to observe the growth of tomatoes and count the yield. Multiple soil samples were randomly collected using an "S" shape. The sampling tool used was a military shovel, and the sampling depth was 0 to 20 cm in the tillage layer. After sufficient mixing, 1 kg was retained by quartering to form a soil sample. Gravel, plant roots and other impurities visible to the naked eye were removed by hand picking, and fresh soil samples were stored in self-sealing bags and brought back to the laboratory. Some soil samples were frozen in a refrigerator at -80°C for DNA extraction. The remaining soil samples were naturally air-dried and sieved for the determination of the physical and chemical properties of the soil.

[0059] In each plot, 1 sample plot of 1.0 m × 1.0 m was randomly selected, and all earthworms in the soil layer of 0 - 40 cm were collected by handpicking method. The number of earthworms and biomass in the collected earthworm samples were recorded. Three earthworms from each treatment were quickly frozen in liquid nitrogen and stored in a refrigerator at -80 °C. The remaining earthworms were anesthetized and fixed, then stored in specimen tubes filled with absolute ethanol. The internal and external characteristics of the earthworm specimens were observed under a dissection microscope. Based on the morphological characteristics, anatomical characteristics and COI gene sequences of the earthworms, the species were identified.

[0060] During the full fruit stage of tomatoes, 18 plants were randomly selected from each treatment to measure various growth indicators, and the yield data were recorded each time. After the tomatoes were harvested, the single fruit weight was measured.

[0061] Determination of soil physical and chemical and biological indicators: Take air-dried soil and measure the following indicators with reference to soil agrochemical analysis methods: pH value (potentiometric method, NY / T 1121.2 - 2006); total water-soluble salts (EC value, conductivity method); organic matter content (potassium dichromate volumetric method - external heating method, NY / T 1121.6 - 2006); total nitrogen content (Kjeldahl method, NY / T 1121.24 - 2012); available phosphorus content (NaHCO 3 extraction - molybdenum antimony anti-colorimetric method, NY / T 1121.7 - 2014); available potassium content (NH 4 OAc extraction - flame photometry method); soil nitrate nitrogen content (ultraviolet spectrophotometry, GB / T 32737 - 2016); soil ammonium nitrogen content (indophenol blue colorimetry); soil bulk density and porosity (weighing method, NY-T 1121.4 - 2006).

[0062] Analysis of soil nitrogen cycle functional gene abundance and earthworm colonization: Weigh 0.25 g of fresh soil and extract DNA using Shengong column soil DNA extraction kit. Use NanoDrop spectrophotometer to detect the quality and concentration of the extracted DNA. Use ABI7500 fluorescence quantitative PCR instrument to perform quantitative PCR (qPCR) to analyze the abundance of functional microbial genes related to nitrogen cycle processes such as denitrification (nosZ, nirK), nitrate dissimilatory reduction (napA), nitrogen fixation (nifH), etc. Select specific primers for Bacillus licheniformis BL18AF: 5'-GTCAACGACACAATTTCCCCGT-3', BL18AR: 5'-AGCTCCCTCAGGCGGCAATT-3' to analyze the abundance of Bacillus licheniformis in the soil. Select standard primers to analyze the abundance of bacteria, fungi, archaea and protozoa in the soil. The colonization of Eisenia fetida and Eisenia andrei was mainly analyzed by the density and biomass of earthworms in the soil of each treatment, and the number of earthworms per square meter (number / m 2 ) and the weight of earthworms (g / m 2) and identify earthworm species through stereoscopic microscopy examination.

[0063] Tomato plant measurement indicators and methods: Plant height: Measure the distance from the base of the plant to the growth point with a tape measure. Stem diameter: Measure at 3 cm from the base of the plant with a vernier caliper. Leaf area: Select a complete and normally growing leaf in the middle of the plant, measure its length and width with a tape measure, and then calculate its leaf area.

[0064] III. Analysis of soil improvement results

[0065] Before treatment, the number of earthworms in the experimental plot was scarce and mainly distributed in the positions with less salt content at the edge of the soil clods, such as Figure 1 shown, with an average density of 1.5 earthworms / m 2 , and a biomass of 0.316 g / m 2 . After identification, they were Amynthas hubeiensis and Pontoscolex corethrurus, and the widely distributed native species Metaphire guillelmi was not found. One month after introducing Metaphire guillelmi in the M treatment, the density of earthworms increased to 8.42 earthworms / m 2 , and the biomass increased to 12.6 g / m 2 , much higher than the earthworm population in the experimental plot before treatment, indicating that Metaphire guillelmi has successfully colonized in the secondary saline-alkali soil. However, in the E treatment, i.e., the Eisenia fetida treatment group, there was no significant increase in the density and biomass of earthworms, indicating that Eisenia fetida may not have successfully colonized in the soil, which is highly related to the survival conditions and feeding preferences of Eisenia fetida. In addition, the abundance of Bacillus licheniformis in the treatment group applying the microbial agent was higher than that in the control group (CK) and before treatment, indicating that Bacillus licheniformis has also successfully colonized in the soil. As Figure 5 shown, after applying the Bacillus licheniformis microbial agent and introducing Metaphire guillelmi and Eisenia fetida respectively, the soil texture became loose, a large amount of earthworm feces appeared on the soil surface, and the soil condition began to gradually improve while the two organisms began to colonize in the soil.

[0066] From Figure 2 it can be seen that compared with CK, the combined action of Metaphire guillelmi and Bacillus licheniformis increased the abundances of the nirK, napA, and nosZ functional genes related to the denitrification process, but the individual action of Metaphire guillelmi had little effect on the abundances of the nitrogen cycle functional genes. The soil of the experimental plot before improvement was as Figure 4 (A) shown, and a large amount of green moss appeared on the soil surface after watering the experimental plot, as Figure 4As shown in (B), this is a typical phenomenon of nitrogen excess. The average initial nitrate nitrogen content in the soil is 136.81 mg / kg, which far exceeds the normal range of 5 - 20 mg / kg. According to the statistical results in Table 1, the combined treatment of Eisenia foetida and Bacillus licheniformis (BM) reduced the nitrate nitrogen content to 105.94 mg / kg. At the same time, the problem of soil salt toxicity was significantly improved. The BM treatment reduced the EC from the initial 1250 μS / cm to 592.7 μS / cm, and it basically no longer causes salt toxicity to crops. In the treatment groups with earthworms added, the available phosphorus content increased to varying degrees, probably due to the promotion of phosphorus activation by the phosphatase in the earthworm intestine. In addition, the addition of earthworms optimized the soil physical structure, reduced the bulk density, and increased the total soil porosity, indicating that the burrowing behavior of earthworms and the microbial metabolites can jointly promote the formation of soil aggregates.

[0067] Table 1 Changes in soil physical and chemical properties before and after different treatments

[0068]

[0069] Figure 3 The results showed that there were no significant differences in the plant height and stem diameter of tomato among the treatment groups, and the leaf area of the CK treatment group was higher. Generally speaking, a larger leaf area can improve the photosynthesis efficiency of plants, thereby increasing the growth rate and yield of plants. However, when the leaf area index increases to a certain extent, the field becomes crowded, the light is insufficient, the photosynthetic efficiency will weaken, and the yield will instead decrease. Therefore, the CK treatment has a larger leaf area but a low yield. According to the statistical results of tomato fruit weight, the single treatment of Eisenia fetida (E) and the combined treatment of Eisenia foetida and Bacillus licheniformis (BM) both increased the fruit weight. However, considering the changes in soil physical and chemical properties and the colonization of earthworms, Eisenia foetida and Bacillus licheniformis formed a better cooperative relationship.

[0070] In summary, the present invention significantly alleviated the problems of soil salt toxicity and nitrogen excess in secondary salinized soil through microbe - animal interaction. After the successful colonization of Eisenia foetida, the soil structure was effectively improved, promoting the cycling of soil nutrients and the absorption and utilization by crops. It provides a stable and efficient biological combined remediation strategy for the remediation of salinized soil.

Claims

1. A method for improving nitrate-type secondary salinized soil by using earthworms and microorganisms in collaboration, characterized in that: The following steps are involved: Step (1), field preparation and pre-fertilization treatment of nitrate-type secondary salinized soil: weeding the land, digging drainage ditches, applying base fertilizer, surface application of decomposed organic materials, and drip irrigation, wherein: the base fertilizer is agricultural fertilizer, and the decomposed organic materials are livestock and poultry manure or a mixture of agricultural fiber waste and livestock and poultry manure that is decomposed and crushed into fragments with a particle size of less than 2 cm; Step (2), preparation and application of liquid bacterial agent of Bacillus licheniformis: preparing a liquid bacterial agent of Bacillus licheniformis BL-7 after salt tolerance acclimation, and spraying it evenly on the soil surface; Step (3), earthworm placement: 20-40 days after the liquid bacterial agent is applied, Metaphire guillelmi is placed into the soil; The Bacillus licheniformis BL-7 is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCCNO.29417 and a deposit date of December 27, 2023. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The method for improving nitrate-type secondary salinized soil by using earthworms and microorganisms in collaboration according to claim 1, characterized in that: The preparation step of the decomposed organic material in step (1) is: subjecting livestock and poultry manure or a mixture of agricultural fiber waste and livestock and poultry manure to aerobic preliminary fermentation at a fermentation temperature of 45-60° C. for 40 days, and then turning the pile every 7-10 days, for 2-3 times, to obtain decomposed organic material.

3. The method for improving nitrate-type secondary salinized soil by using earthworms and microorganisms in collaboration according to claim 1, characterized in that: The method of applying the decomposed organic material on the surface in step (1) is to evenly sprinkle the decomposed organic material on the soil surface, and shallowly till the soil to a depth of 8-15 cm to mix the material evenly.

4. The method for improving nitrate-type secondary salinized soil by using earthworms and microorganisms in collaboration according to claim 1, characterized in that: The William's earthworms released in step (3) have clear annular bands, good activity, and weigh between 1.5-3g.

5. The method for improving nitrate-type secondary salinized soil by using earthworms and microorganisms in collaboration according to claim 1, characterized in that: The William's earthworms described in step (3) are pre-cultured for 5-10 days before release.

6. The method for improving nitrate-type secondary salinized soil by using earthworms and microorganisms in collaboration according to claim 1, characterized in that: The method for pre-culturing the William earthworms before putting them in is as follows: using uncontaminated garden soil as a culture matrix, putting the William earthworms in the culture matrix for 5-10 days, and maintaining the soil moisture content at 30-40%.

7. The method for improving nitrate-type secondary salinized soil by using earthworms and microorganisms in collaboration according to claim 1, characterized in that: The application amount of the decomposed organic material is 10-20kg / m 2 .

8. The method for improving nitrate-type secondary salinized soil by using earthworms and microorganisms in collaboration according to claim 1, characterized in that: The application amount of the liquid inoculant is 45-55mL / m 2 The liquid inoculant contains ≥2×10 spores formed by the strain 8 cfu / mL.

9. The method for improving nitrate-type secondary salinized soil by using earthworms and microorganisms in collaboration according to claim 1, characterized in that: The preparation method of the liquid bacterial agent is as follows: Bacillus licheniformis BL-7 is cultured until the number of spores produced in the culture solution is ≥ 1×10 9 cfu / mL, add the protective agent to the culture solution and stir to obtain the liquid bacterial agent.

10. The method according to claim 1 for improving nitrate-type secondary growth by synergistic action of earthworms and microorganisms A method for salinizing soil, characterized in that The amount of William's earthworms added is 45-55g / m 2 , Place in the soil near the irrigation water line.

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