A method of improving coastal saline soils

Abstract

This invention discloses a method for improving coastal saline soil, belonging to the agricultural field. To address the technical problems of excessive salinity and low soil biological activity in saline-alkali soils, this invention mainly employs the screening, modification, and cultivation of salt-tolerant cellulose-decomposing bacteria, combined with genetic engineering technology to enhance their cellulose decomposition and salt-alkali tolerance. By constructing a salt-tolerant, highly efficient cellulose-decomposing bacterial fertilizer, soil biological activity is activated, soil structure is improved, and soil fertility is enhanced. This invention can effectively improve the soil quality of saline-alkali land, promote crop growth, and provide technical support for the sustainable utilization of coastal saline soils.

Description

Technical Field

[0001] This invention belongs to the field of agriculture, specifically relating to a method for improving coastal saline soil. Background Technology

[0002] As important land reserve resources, saline-alkali land and dry mountain land have attracted widespread attention for their management, development, and utilization in the context of increasingly scarce global land resources. Salt damage is one of the significant abiotic stresses in agricultural production. According to data from UNESCO and FAO, the global area of ​​saline-alkali land is approximately 954 million hectares. China has a wide area of ​​saline-alkali soil with diverse types. Currently, the area of ​​saline-alkali soil is approximately 36.933 million hectares, residual saline-alkali soil is approximately 44.867 million hectares, and potentially saline-alkali soil is 17.333 million hectares, totaling 99.133 million hectares of saline-alkali land. With the increasing severity of salinization and secondary salinization each year, sustainable agricultural development faces challenges, and soil salinization has become one of the key factors restricting global rice production. On the one hand, large areas of saline-alkali land cannot be used for rice cultivation; on the other hand, secondary soil salinization caused by unreasonable water resource management is becoming increasingly serious in rice-producing areas. Therefore, improving the salt and alkali tolerance of rice through genetic modification is one of the effective means to expand the rice planting area and increase yield.

[0003] Salt-tolerant rice, bred through hybridization, molecular breeding, and other techniques, is a rice variety that can grow under irrigation conditions of 3‰ or higher saline water or in saline-alkali land, and can yield more than 300 kg per mu (approximately 0.067 hectares). It is of great significance for agricultural development, food security, and ecological environmental protection. Although successfully promoted salt-tolerant rice varieties are currently scarce both domestically and internationally, in recent years, Chinese scientists have made significant progress in hybridization technology and molecular marker-assisted breeding, cultivating a series of high-yielding, highly resistant salt-tolerant rice varieties, opening up new pathways for the development and utilization of saline-alkali land.

[0004] China has approximately 1.5 billion mu (100 million hectares) of saline-alkali land, of which about 200 million mu (13.3 million hectares) are suitable for growing salt-tolerant rice. By breeding salt-tolerant rice varieties and promoting corresponding paddy field improvement techniques, not only can grain yields be increased, but the land conflict arising from population growth and industrialization / urbanization can also be alleviated. Salt tolerance in rice is a quantitative trait controlled by multiple genes influenced by the environment, and significant differences in salt tolerance exist among different varieties; these differences are heritable. For a long time, scientists have identified many gene loci related to salt tolerance using methods such as genome-wide association studies (GWAS) and quantitative trait locus (QTL) analysis, laying the foundation for cloning salt tolerance genes. Studies have shown that rice responds differently to salt stress at different growth stages, with the seedling stage and reproductive growth stage being the most sensitive, while the seed germination stage and vegetative growth stage show stronger salt tolerance. Various indicators are used to evaluate rice salt tolerance, including morphological, growth, and physiological indicators such as seedling height, fresh and dry weight of aboveground and root parts, and ion content. Globally, the breeding of salt-tolerant rice varieties has a history of over 70 years. Traditional breeding methods, such as selection of local varieties, pedigree methods, improved mixed pedigree methods, mutation breeding, and shuttle breeding, have been widely used, resulting in salt-tolerant rice varieties such as CSR1 and CSR10. In recent years, molecular marker-assisted selection (MAS) technology has also been used in the breeding of salt-tolerant rice.

[0005] Coastal saline-alkali land refers to saline soils located in coastal areas, where salt primarily originates from seawater intrusion, rising groundwater levels, and evaporative concentration. Common salts include sodium chloride (NaCl), sodium sulfate (Na₂SO₄), and sodium carbonate (Na₂CO₃). The salt concentration in these soils is typically high, sometimes exceeding 0.5%, severely impacting plant growth and metabolism. Poor soil structure leads to salt accumulation in the surface layer, resulting in soil compaction and poor aeration and permeability. The limitations imposed by salinity and alkalinity also affect the accumulation and decomposition of organic matter, resulting in low soil fertility. The soil texture is usually fine and heavy, easily forming a hardened layer.

[0006] The high groundwater level in coastal areas easily leads to an increase in soil salinity, further exacerbating salinization. Biodiversity is low, with limited plant species: the high-salt and high-alkali environment restricts the growth of many plants, with only a few salt-tolerant species able to adapt. Microbial activity is restricted; the high-salt and high-alkali environment inhibits the activity of soil microorganisms, affecting soil biological activity and nutrient cycling. The biggest production problem is soil infertility, resulting in low rice yields. The poor economic benefits of coastal saline-alkali land have prevented it from being widely accepted by farmers, necessitating a significant increase in organic matter and soil fertility improvement. Extensive planting of vegetation and application of straw are important methods to address the soil infertility of coastal saline-alkali land. How to effectively decompose cellulose in saline-alkali soil, cultivate soil organisms, and activate soil biota is a problem that needs to be solved. Cultivating salt-tolerant and highly efficient cellulose-decomposing bacteria to improve the living environment of salt-tolerant organisms in the soil is an effective method for improving coastal saline soil.

[0007] Cellulose-decomposing bacteria can be used for the pretreatment and fermentation of biomass, converting plant cellulose into fermentable sugars to produce renewable energy sources such as bioethanol and biobutanol. Utilizing these bacteria can accelerate the decomposition of agricultural waste such as crop straw and livestock manure, reducing environmental pollution while producing organic fertilizer. In the food, pharmaceutical, and paper industries, the cellulase produced by cellulose-decomposing bacteria can improve product quality and production efficiency. Furthermore, cellulose-decomposing bacteria can degrade organic pollutants in soil and water, promoting environmental restoration and purification.

[0008] In recent years, with the development of genomics and metabolic engineering, researchers have been able to modify cellulose-decomposing bacteria using gene editing technology to increase the yield and activity of their cellulases. For example, genetically engineering Clostridium thermocellum can significantly improve its cellulose decomposition efficiency, thereby reducing biomass conversion costs.

[0009] The coastal saline-alkali areas of South China are characterized by humid air, moderate temperatures, and a high incidence of seasonal salinization and soil salinity. The cultivation technology for salt-tolerant rice in these areas involves a comprehensive application of multiple aspects, including soil improvement, variety selection, water and fertilizer management, salinity control, and pest and disease control. This technology has significant potential for widespread application in saline-alkali regions across China. For severely saline-alkali coastal lands with a salt content exceeding 0.8%, through rational fertilization, physiological and ecological regulation, plant protection, effective water management, and organic and green technologies, the goal is to achieve salt-tolerant rice with excellent plant and leaf morphology, good grain filling, full and plump grains, good coloring, and no premature senescence in the later stages of growth, with a yield exceeding 300 kg per mu (approximately 0.067 hectares).

[0010] The salinization and infertility of coastal saline-alkali soils in South China are a key reason for the lack of economic benefits in rice cultivation. In saline-alkali soils, microbial activity is restricted, affecting soil biological activity and nutrient cycling. By using transgenic and gene-editing technologies, highly efficient salt- and alkali-tolerant cellulose-decomposing bacteria can be screened, modified, and cultivated to improve their growth capacity, salt tolerance, cellulose decomposition ability, and other stress resistance. This can be used to produce highly efficient salt- and alkali-tolerant cellulose-decomposing bacterial fertilizers, which can revitalize soil biology and improve soil fertility.

[0011] Cellulose-decomposing bacteria are a class of microorganisms capable of breaking down cellulose, mainly distributed in environments such as soil, compost, plant residues, and water bodies. These bacteria and fungi degrade complex cellulose molecules into simple sugars by secreting cellulases (including endoglucanases, exoglucanases, and β-glucosidases), which can then be further metabolized and utilized by the microorganisms. Screening for cellulose-decomposing bacteria with specific properties found in nature and further improving them using advanced biotechnology can create new types of microorganisms better suited to specific application scenarios. Currently, it is possible to modify cellulose-decomposing bacteria using gene editing technology to increase the yield and activity of their cellulases. For example, genetic engineering of Clostridium thermocellum can significantly improve its cellulose decomposition efficiency, thereby reducing biomass conversion costs. The main salt- and alkali-tolerant cellulose-decomposing bacteria include: ① Bacteria: *Halomonas*, *Halobacterium*, *Bacillus halodurans*, and *Pseudomonas halophila*. ② Fungi: *Aspergillus terreus* and *Penicillium chrysogenum*

[0012] Salt- and alkali-tolerant cellulose-decomposing bacteria maintain intracellular osmotic balance by accumulating compatible solutes (such as betaine, glycine betaine, etc.); they can grow and metabolize in high pH environments (such as pH 9-11), adapting to high-alkaline environments by regulating cell membrane permeability and intracellular pH buffering systems; they secrete a variety of cellulases, including endoglucanase, exoglucanase, and β-glucosidase, which effectively degrade cellulose into glucose.

[0013] In conclusion, how to utilize cellulose-decomposing bacteria to improve coastal saline soil is an urgent problem to be solved. Summary of the Invention

[0014] The purpose of this invention is to provide a method for improving coastal saline soil. This method involves screening out Trichoderma reesei, which is capable of efficiently decomposing cellulose under high salinity and alkalinity conditions, and utilizing the strong cellulase protein secretion ability of Trichoderma reesei as a filamentous fungus to improve coastal saline soil.

[0015] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0016] A method for improving coastal saline soil includes the following steps:

[0017] 1) Collect Trichoderma reesei strains;

[0018] 2) Using a cellulose-containing culture medium, the strains were cultured under different concentrations of NaCl to screen out strains with strong salt stress tolerance.

[0019] 3) Cellulase activity was determined by the filter paper activity method, and strains with the highest cellulase production under high-salt conditions were screened.

[0020] 4) Genetically modify the strains using the CRISPR / Cas9 system to increase the yield and activity of cellulase and hemicellulase;

[0021] 5) Propagate the strain under laboratory conditions to ensure that it is the strain with the desired performance, and then inoculate the strain into the field for propagation;

[0022] 6) The obtained Trichoderma strain was inoculated into the coastal saline soil to modify it.

[0023] Furthermore, the Trichoderma reesei strains in step 1) are purchased or collected from salt lakes, marine sediments, and other high-salinity areas.

[0024] Furthermore, in step 2), the culture system used is the high-throughput microbioreactor BioLector, and the NaCl mass concentration gradient in the culture medium is 0.8%, 1.2%, 1.6%, and 2.0%.

[0025] Furthermore, in step 2), cellulose is the sole carbon source of the culture medium, and the components of the culture medium also include (NH4)2SO4, KH2PO4, MgSO4·7H2O, CaCl2·2H2O, NaCl, 1,4-piperazine dipropanesulfonic acid buffer, urea, casein peptone, and trace elements.

[0026] Further, the screening step in step 3) includes: staining the fungal cell wall with the fluorescent dye Calcofluor white using a disc fluorescence microscope, detecting the growth of Trichoderma reesei and the cellulase production in each well, and selecting strains with good growth and the highest cellulase production.

[0027] Furthermore, step 4) involves genetically engineering the strain, including:

[0028] An expression element composed of a shortened chb1 promoter, red fluorescent protein, and cbh2 terminator was used to transform Trichoderma reesei to construct an RFP signal tag.

[0029] Select the target gene and construct an expression vector;

[0030] The constructed expression vector was introduced into Trichoderma reesei, and the vector carried an antibiotic resistance gene as a selection marker. Cells that integrated the target gene were selected from the transformants.

[0031] Furthermore, step 4) involves selecting the target gene to construct the expression vector, which includes:

[0032] Select the Trichoderma reesei xyn1 constitutive promoter and the pyruvate decarboxylase 1 constitutive promoter;

[0033] Select the target genes: β-glucosidase encoding gene ELA3A, mutant xyr1 gene and Aspergillus niger invertase encoding gene suc1;

[0034] Optimize signal peptides to ensure that the encoded proteins are correctly secreted into the extracellular environment;

[0035] Multiple copies of the ELA3A and xyr1 genes were inserted into the host genome to increase gene expression levels.

[0036] Remove the extracellular protease genes slp1 and pep1.

[0037] Furthermore, step 4) involves validating the expression of the genetically engineered strain, including:

[0038] Gene expression detection: The relative expression levels of ELA3A, xyr1, suc1, slp1, and ace1 genes were detected by quantitative real-time PCR to confirm whether the genes were successfully expressed.

[0039] Protein detection: The presence and expression level of the target protein are detected by Western blotting or ELISA; and

[0040] Enzyme activity assay: The cellulase activity in the culture medium was measured to evaluate the expression effect.

[0041] Further, step 4) involves testing the cellulose degradation performance of the genetically engineered strain, including the following steps:

[0042] Using different types of cellulose sources as substrates, the degradation efficiency of the modified strains on the substrates was tested.

[0043] The degradation rates of cellulose by wild-type and modified strains under the same conditions were compared to quantify the improvement effect.

[0044] Furthermore, in step 5), before the strain is inoculated into the field, the soil is pretreated, including improving soil texture, increasing aeration and water retention capacity, and adding organic fertilizer to improve soil fertility.

[0045] Furthermore, the methods for inoculating the strain into the field in step 5) include:

[0046] Direct sowing method: The prepared fungal spores or mycelium are directly mixed into the soil;

[0047] Mixed sowing method: Mix the fungal inoculum with the crop seeds and sow them together; or

[0048] Spraying or irrigation: Use an irrigation system or sprayer to evenly apply a suspension containing fungi to the surface of the farmland.

[0049] The beneficial effects achieved by this invention are as follows:

[0050] 1. This invention improves soil salinity tolerance, activates soil biological activity, and promotes sustainable agricultural production by modifying the soil.

[0051] 2. This invention improves the salt resistance and yield of rice by selecting highly adaptable salt-tolerant rice varieties, thereby reducing the use of pesticides and fertilizers and lowering agricultural pollution;

[0052] 3. This invention reduces the accumulation of salt on the surface through scientific and reasonable water management, promotes salt leaching, effectively inhibits the upward movement of salt, and improves the rice growing environment;

[0053] 4. This invention improves overall yield and effectively alleviates salinity barriers by regulating salinity and implementing zoned planting in saline-alkali areas, allowing for the planting of suitable varieties for plots with different salinity gradients.

[0054] 5. This invention promotes rice cultivation on saline-alkali land, reduces the occurrence of pests and diseases, lowers production risks, and produces green, high-quality products rich in minerals, meeting the market demand for healthy food.

[0055] 6. This invention combines precision agriculture technology to achieve real-time monitoring of soil salinity, moisture status, and crop growth status, optimizing irrigation, fertilization, and pest and disease control, thereby improving agricultural production efficiency. Attached Figure Description

[0056] Figure 1 These are photos showing the growth of regular rice before and after the improvement of saline-alkali soil.

[0057] Figure 2 These are photos showing the growth of salt-tolerant rice planted before and after the improvement of saline-alkali soil. Detailed Implementation

[0058] The present invention will be further described in detail below with reference to the embodiments, but these embodiments should not be used to interpret the limitation of the scope of protection of the present invention.

[0059] This invention proposes a method for improving coastal saline soil by cultivating salt-tolerant and highly efficient cellulose-decomposing bacteria to improve the living environment of salt-tolerant organisms in the soil. Microbial activity in saline soils is restricted, affecting soil biological activity and nutrient cycling. This invention uses transgenic and gene-editing technologies to screen, modify, and cultivate highly efficient salt-tolerant and cellulose-decomposing bacteria, improving their growth capacity, salt tolerance, cellulose decomposition ability, and other stress resistances, to produce a highly efficient salt-tolerant and cellulose-decomposing bacterial fertilizer, thereby activating soil biological activity and improving soil fertility.

[0060] (1) Collecting strains

[0061] Source: Trichoderma reesei strains can be purchased directly (e.g., ATTC_56765) or collected from salt lakes, marine sediments and other high-salinity areas.

[0062] Differences in characteristics: Strains from different sources vary considerably in salt tolerance and cellulase production.

[0063] (2) Preliminary screening

[0064] Culture medium: A culture medium containing cellulose as the sole carbon source was used, with different concentration gradients of NaCl (0.8%, 1.2%, 1.6%, 2.0%) added to simulate a high-salt environment.

[0065] Cultivation system: The BioLector system, a high-throughput microbioreactor, is suitable for high-throughput fermentation of Trichoderma reesei, and can obtain morphological, cellulase yield, and growth kinetic data similar to those of traditional shake-flask fermentation.

[0066] The culture conditions were: temperature 30℃, shaking speed 1200rpm, round plate model M2P-MTP-R48-B, and culture volume of 1000μL per well.

[0067] The culture medium also included: (NH4)2SO4 7.6 g / L, KH2PO4 2.6 g / L, MgSO4·7H2O 0.5 g / L, CaCl2·2H2O 0.23 g / L, NaCl 0.05 g / L, 1,4-piperazine dipropanesulfonic acid buffer 33 g / L (0.1 M), urea 0.3 g / L, casein peptone 2 g / L, and trace elements 2.5 mL / L. All cultures were inoculated from spore suspensions, with a final concentration of 106 spores / mL.

[0068] Screening criteria: to screen for strains with strong salt stress tolerance.

[0069] (3) Cellulase experiment

[0070] Experimental methods: The standard filter paper activity (FPA) assay was performed in a 96-well conical bottom PCR plate, and the cellulase activity in the culture supernatant was measured in a 60 μL reaction volume.

[0071] Morphological analysis: The fungal cell walls were stained with Calcofluor white (100 mg / L) using a disc fluorescence microscope to detect the growth of Trichoderma reesei and the production of cellulase in each well.

[0072] Screening criteria: Select strains that grow well under high-salt conditions and produce the highest cellulase yield.

[0073] (4) Genetic engineering modification

[0074] By knocking out genes that inhibit cellulase production using the CRISPR / Cas9 system and overexpressing key cellulase genes, the yield of cellulase can be synergistically increased, and the activity levels of cellulase and hemicellulase, especially β-glucosidase and xylanase, can be enhanced, thereby achieving a comprehensive performance improvement.

[0075] The specific operating procedure is as follows:

[0076] ① Construction of RFP signal tag: Trichoderma reesei was transformed with an expression element consisting of a shortened chb1 promoter, red fluorescent protein (RFP), and cbh2 terminator.

[0077] ② Select the target gene and construct an expression vector, including:

[0078] Promoters: The constitutive promoters Trichoderma reesei xyn1 and pyruvate decarboxylase 1 (pdc1) were selected.

[0079] Target genes: including the β-glucosidase encoding gene ELA3A, the mutant xyr1 gene, and the Aspergillus niger invertase encoding gene suc1.

[0080] Signal peptide optimization: ensuring that the encoded protein is correctly secreted into the extracellular environment.

[0081] Multiple copy insertion: Multiple copies of the ELA3A and xyr1 genes can be inserted into the host genome to increase gene expression levels.

[0082] Gene knockout: Removal of extracellular protease genes slp1 and pep1.

[0083] The above genes are explained as follows:

[0084] ELA3A gene: encodes β-glucosidase, which can effectively degrade cellulose in plant materials.

[0085] xyr1 transcription factor: a major regulator of cellulase-encoding genes. Constitutive expression of xyr1 can significantly increase overall enzyme yield, and point mutations in amino acid AF at position 824 can alleviate the inhibition of carbon catabolites.

[0086] SUC1 gene: The invertase-encoding gene of Aspergillus niger, which can confer the ability of Trichoderma reesei to utilize sucrose.

[0087] SLP1 and PEP1 gene deletion: increases the protein secretion rate of Trichoderma reesei, further enhancing the secretion efficiency of cellulase.

[0088] ③ Transformation and screening: The constructed vector was introduced into Trichoderma reesei using protoplast transformation. The antibiotic resistance gene carried by the vector was used as a selection marker to select cells that successfully integrated the target gene from a large number of transformants.

[0089] ④ Expression verification:

[0090] Gene expression detection: The relative expression levels of ELA3A, xyr1, suc1, slp1, and ace1 genes were detected by quantitative real-time PCR (qRT-PCR) to confirm whether the genes were successfully expressed.

[0091] Protein detection: The presence and expression level of the target protein are detected by Western blotting or ELISA.

[0092] Enzyme activity assay: The cellulase activity in the culture medium was measured to directly assess the expression effect.

[0093] ⑤ Cellulose degradation performance test:

[0094] Substrate selection: Different types of cellulose sources, such as microcrystalline cellulose and filter paper, were used as substrates to test the degradation efficiency of the modified strains on the substrates.

[0095] Degradation rate analysis: The degradation rates of cellulose by wild-type and modified strains under the same conditions were compared to quantify the improvement effect.

[0096] For the construction of a fluorescent Trichoderma reesei strain (RFP1), the shortened chb1 promoter controls the expression of weak fluorescence in the RFP1 strain, with the fluorescence intensity increasing with the production of cellulase. This characteristic is suitable for observing the dynamics of cellulase production and can assist in strain screening and direct testing of the degradation efficiency of modified strains for different carbon source substrates.

[0097] (5) Exploration of large-scale application

[0098] Strains that have been successfully developed under small-scale laboratory conditions undergo further cultivation on a larger scale, including pilot-scale fermenter operation and commissioning of industrial-grade production equipment, involving the following processes:

[0099] ① Preparation of inoculum:

[0100] Propagation process: Start with laboratory scale and gradually increase the culture volume until sufficient biomass is reached for field application.

[0101] Quality control: Ensure that the inoculum is free from contamination by other microorganisms and has high reproductive capacity and target functional characteristics.

[0102] ②Prepare the field environment:

[0103] Soil pretreatment: Improve soil texture, increase aeration and water retention capacity, and add appropriate amounts of organic fertilizer if necessary to improve soil fertility.

[0104] Environmental monitoring: Understand local climate conditions and select the most suitable time for fungal survival for release.

[0105] ③ For field applications, the following three methods can be selected:

[0106] Direct sowing method: The prepared fungal spores or mycelium are directly mixed into the soil, which is suitable for small experimental areas or local treatment.

[0107] Mixed sowing method: The fungal inoculum is mixed with the crop seeds and sown together, so that the fungi can establish a symbiotic relationship in the early stage of seed germination.

[0108] Spraying or irrigation: Use an irrigation system or sprayer to evenly apply a suspension containing fungi to the surface of the farmland.

[0109] (6) Coastal saline soil improvement

[0110] The obtained Trichoderma strain was inoculated into coastal saline soil to modify the coastal saline soil.

[0111] The application of this method is as follows:

[0112] (1) Soil improvement and conditioning:

[0113] By constructing a complete irrigation and drainage system and regularly rinsing or changing the water, the salt concentration in the topsoil can be reduced; multiple irrigations and drainage should be carried out before rice planting; organic materials such as rice straw, green manure, and compost should be added; and inorganic amendments such as gypsum, phosphogypsum, and furfural residue should be used to neutralize sodium ions in the soil.

[0114] (2) Freshwater seedling raising:

[0115] Rice seedlings have weak salt tolerance, hybrid seeds are expensive, and freshwater resources are relatively abundant in the coastal areas of South China. Therefore, salt-tolerant rice should be cultivated by seedling raising and transplanting, avoiding seedling raising on saline-alkali land and the direct transplanting technique. The seedling raising method can be flexibly selected according to specific circumstances.

[0116] (3) Select rice varieties with strong adaptability:

[0117] Salt-tolerant rice variety selection: Select salt-tolerant rice varieties that have been bred or genetically engineered to ensure normal growth and development under certain salt concentrations, such as the "seawater rice" series. These varieties possess strong salt tolerance, salt uptake capacity, salt excretion mechanisms, and the ability to maintain ion balance. Hybrid seawater rice exhibits vigorous physiological metabolism, demonstrating stronger resistance to adverse conditions, and its yield is less affected by environmental factors. New salt-tolerant rice varieties can not only reduce the use of pesticides and fertilizers, lowering agricultural pollution, but also promote the improvement of saline soils.

[0118] Regional adaptability testing: Variety adaptability tests are conducted based on the climate characteristics (such as high temperature, high humidity, typhoons, etc.) and soil types of the coastal areas of South China to ensure that the selected varieties can not only tolerate salt but also cope with other environmental pressures.

[0119] (4) Scientific and rational water management:

[0120] Flooding management: Freshwater irrigation creates a water layer covering the soil surface, reducing salt accumulation on the surface and promoting salt leaching downwards. Combining shallow irrigation with timely field drying effectively inhibits salt migration upwards while preventing the accumulation of reducing substances caused by prolonged flooding.

[0121] Saltwater irrigation: When water resources are scarce and soil salinity is evenly distributed, low-salinity seawater can be used for irrigation in appropriate amounts. However, the salt concentration must be strictly monitored to avoid excessive salt content from damaging the rice, and fresh water should be used in conjunction to leach out the salt.

[0122] (5) Salt regulation and saline-alkali zone planting:

[0123] Salinity monitoring: Regularly test soil salinity and adjust agronomic measures such as irrigation and fertilization based on the monitoring results. For severely saline plots, local improvement or zoned planting can be carried out, planting rice varieties with different salt tolerance in suitable salinity gradient plots to improve overall yield.

[0124] Salinity and alkalinity barrier mitigation: Set up physical or chemical barriers in the field, such as salt-proof films and isolation ditches, to prevent salt from migrating into the planting area and protect rice roots from the effects of high-salt environments.

[0125] (6) Pest and disease control and technological support:

[0126] Rice cultivation in saline-alkali lands is less susceptible to pests and diseases, requires less pesticide and fertilizer use, and produces green, high-quality products rich in minerals, meeting people's demand for healthy food and effectively reducing the production risks caused by salt stress.

[0127] Targeted prevention and control: Focus on pests and diseases specific to saline-alkali environments, such as diseases caused by salt-tolerant pathogens and insect pests unique to saline-alkali land. Effective control can be achieved through a combination of biological control, chemical control, or resistant varieties.

[0128] Precision agriculture technology application: Utilizing modern information technologies such as remote sensing monitoring, drone field patrols, and the Internet of Things, soil salinity, moisture status, and crop growth status are monitored in real time to guide precision irrigation, fertilization, and pest and disease control.

[0129] The following is a specific embodiment:

[0130] 1. Collect strains

[0131] Trichoderma reesei strains (IHEM_5652 / ATCC_56765) were cultured in a medium containing cellulose as the sole carbon source, with four different NaCl concentrations (0.8%, 1.2%, 1.6%, and 2.0%) added. Strains were inoculated and cultured stepwise from low to high NaCl concentrations. Strains that could survive at 1.6% or 2% NaCl concentrations were selected and further cultured in a high-throughput microbioreactor (BioLector) to screen for strains with high cellulase production.

[0132] 2. Cellulase Experiment

[0133] Cellulase activity in culture supernatants was determined using the standard filter paper activity (FPA) assay. The reaction volume was 60 μL in a 96-well conical-bottom PCR plate. Morphological analysis was performed using a disc fluorescence microscope, and the fungal cell walls were stained with Calcofluorwhite (100 mg / L) to detect the growth and cellulase production of *Trichoderma reesei* in each well. The strain with the best growth and highest cellulase production was selected.

[0134] 3. Preservation of strains

[0135] Trichoderma reesei strains were inoculated onto potato dextrose agar (PDA) medium and cultured at 28°C until conidial formation was observed (approximately 5-10 days). The conidia were then suspended in a 20% (v / v) glycerol solution, mycelia were removed by filtration through sterile cotton, quantified using a cell counter, and stored at -80°C.

[0136] 4. Gene editing and modification

[0137] (1) Construction of RFP signal tag: The expression element composed of shortened chb1 promoter, red fluorescent protein (RFP) and cbh2 terminator was transformed into Trichoderma reesei.

[0138] (2) Gene editing: This invention employs a single-plasmid CRISPR / Cas9 system and a label-free donor element. The CRISPR / Cas9 plasmid contains a codon-optimized Streptococcus pyogenes Cas9 gene, a ribozyme-mediated guide RNA (gRNA) sequence, an antibiotic resistance selection marker sequence, and an AMA1 fungal replication sequence.

[0139] First, the donor element carrying the xyn1 constitutive promoter and the β-glucosidase encoding gene ELA3A was transferred into Trichoderma reesei to replace the Bacillus subtilis protease encoding gene slp1, thus obtaining strain T01.

[0140] Then, the donor element of the constitutive promoter sequence of pyruvate decarboxylase 1 (pdc1) and the expression sequence of the mutant xyr1 allele (alanine at position 824 is mutated to valine) was introduced into strain T01 to replace the ace1 gene, thus obtaining strain T02.

[0141] Finally, the invertase-encoding gene suc1 from Aspergillus niger was introduced into the T02 genome to replace the pep1 gene, resulting in the final strain T03.

[0142] These genetic modifications significantly improved the protein secretion rate of the engineered bacteria, with strain T03 achieving an extracellular protein titer of 23.2 g / L. The activities of cellulase and hemicellulase in the resulting enzyme mixture were increased, particularly the specific activities of β-glucosidase and xylanase, which were significantly enhanced.

[0143] ③ Transformation and screening: The constructed vector was introduced into *Trichoderma reesei* using protoplast transformation technology. Cells that successfully integrated the target gene were screened from a large number of transformants using the antibiotic resistance gene carried by the vector as a selection marker.

[0144] ④ Expression Validation: Quantitative real-time PCR (qRT-PCR) was used to detect the relative expression levels of ELA3A, xyr1, suc1, slp1, and ace1 genes to confirm successful gene overexpression. The presence and expression level of the target protein were detected by Western blot or ELISA. Finally, cellulase activity in the culture medium was assessed using an enzyme activity assay to directly verify the functional effect.

[0145] ⑤ Cellulose degradation performance test:

[0146] Substrate selection: Different types of cellulose sources (such as microcrystalline cellulose, filter paper, etc.) were used as substrates to test the degradation efficiency of the modified strains on these substrates.

[0147] Degradation rate analysis: The degradation rates of cellulose by wild-type and modified strains under the same conditions were compared to quantify the improvement effect.

[0148] 5. Exploration of Large-Scale Applications

[0149] The strains successfully cultivated under small-scale laboratory conditions will undergo pilot-scale fermentation and industrial-grade production equipment debugging before being cultivated and applied on a larger scale.

[0150] ① Preparation of inoculum:

[0151] Propagation process: Start at the laboratory scale and gradually increase the culture volume until sufficient biomass is reached for field application.

[0152] Quality control: Ensure that the inoculum is free from contamination by other microorganisms and possesses high reproductive capacity and target functional characteristics.

[0153] ②Prepare the field environment:

[0154] Soil pretreatment: Improve soil texture, increase aeration and water retention capacity, and add appropriate amounts of organic fertilizer to improve soil fertility if necessary.

[0155] Environmental monitoring: Understand the local climate conditions and select the time most favorable for fungal survival for release.

[0156] ③ Field application:

[0157] Direct sowing: The prepared fungal spores or mycelium are directly mixed into the soil, which is suitable for small experimental areas or local treatment.

[0158] Mixing with seeds: Mix the fungal inoculum with crop seeds and sow them together to establish a symbiotic relationship in the early stages of seed germination.

[0159] Spraying or irrigating: Use an irrigation system or sprayer to apply a suspension containing fungi evenly to the surface of the farmland.

[0160] 6. Coastal saline soil remediation

[0161] The obtained Trichoderma strain was inoculated into coastal saline soil to modify the coastal saline soil.

[0162] Figure 1 The image shows a comparison of the growth of regular rice before and after the improvement of saline-alkali soil. Figure 2 The illustration shows a comparison of the growth of salt-tolerant rice before and after the improvement of saline-alkali soil. It can be seen that the improvement of saline-alkali soil by the method of this invention is significantly beneficial to the growth of rice, indicating that the ideal improvement effect has been achieved.

[0163] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Appropriate modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention should be covered within the protection scope of the present invention, which is defined by the claims.

Claims

1. A method for improving a coastal saline soil, characterized by, Includes the following steps: 1) Collect Trichoderma reesei strains; 2) Using a cellulose-containing culture medium, the strains were cultured under different concentrations of NaCl to screen out strains with strong salt stress tolerance. 3) Cellulase activity was determined by the filter paper activity method, and strains with the highest cellulase production under high-salt conditions were screened. 4) Genetically modify the strains using the CRISPR / Cas9 system to increase the yield and activity of cellulase and hemicellulase; 5) Propagate the strain under laboratory conditions to ensure that it is the strain with the desired performance, and then inoculate the strain into the field for propagation; 6) The obtained Trichoderma strain was inoculated into the coastal saline soil to modify it.

2. The method of claim 1, wherein, In step 1), the Trichoderma reesei strain was purchased or collected from salt lakes, marine sediments, and other high-salinity areas.

3. The method of claim 1, wherein, In step 2), the culture system used is the high-throughput microbioreactor BioLector, and the NaCl mass concentration gradient in the culture medium is 0.8%, 1.2%, 1.6%, and 2.0%.

4. The method of claim 1, wherein, In step 2), cellulose is the only carbon source of the culture medium. The culture medium also includes (NH4)2SO4, KH2PO4, MgSO4·7H2O, CaCl2·2H2O, NaCl, 1,4-piperazine dipropanesulfonic acid buffer, urea, casein peptone, and trace elements.

5. The method of claim 1, wherein, The screening steps in step 3) include: using a disc fluorescence microscope, staining the fungal cell walls with the fluorescent dye Calcofluor white, detecting the growth of Trichoderma reesei and the cellulase production in each well, and selecting strains with good growth and the highest cellulase production.

6. The method of claim 1, wherein, Step 4) involves genetically modifying the strain, including: An expression element composed of a shortened chb1 promoter, red fluorescent protein, and cbh2 terminator was used to transform Trichoderma reesei to construct an RFP signal tag. Select the target gene and construct an expression vector; The constructed expression vector was introduced into Trichoderma reesei, and the vector carried an antibiotic resistance gene as a selection marker. Cells that integrated the target gene were selected from the transformants.

7. The method of claim 6, wherein, Step 4) involves selecting the target gene and constructing the expression vector, including: Select the Trichoderma reesei xyn1 constitutive promoter and the pyruvate decarboxylase 1 constitutive promoter; Select the target genes: β-glucosidase encoding gene ELA3A, mutant xyr1 gene and Aspergillus niger invertase encoding gene suc1; Optimize signal peptides to ensure that the encoded proteins are correctly secreted into the extracellular environment; Multiple copies of the ELA3A and xyr1 genes were inserted into the host genome to increase gene expression levels. Remove the extracellular protease genes slp1 and pep1.

8. The method of claim 7, wherein, Step 4) involves validating the expression of the genetically engineered strain, including: Gene expression detection: The relative expression levels of ELA3A, xyr1, suc1, slp1, and ace1 genes were detected by quantitative real-time PCR to confirm whether the genes were successfully expressed. Protein detection: The presence and expression level of the target protein are detected by Western blotting or ELISA; and Enzyme activity assay: The cellulase activity in the culture medium was measured to evaluate the expression effect.

9. The method of claim 1, wherein, Step 4) involves testing the cellulose degradation performance of the genetically engineered strain. The steps include: Using different types of cellulose sources as substrates, the degradation efficiency of the modified strains on the substrates was tested. The degradation rates of cellulose by wild-type and modified strains under the same conditions were compared to quantify the improvement effect.

10. The method of claim 1, wherein, In step 5), before the strain is inoculated into the field, the soil is pretreated, including improving soil texture, increasing aeration and water retention capacity, and adding organic fertilizer to improve soil fertility. Methods of inoculating the strain into the field include: Direct sowing method: The prepared fungal spores or mycelium are directly mixed into the soil; Mixed sowing method: Mix the fungal inoculum with the crop seeds and sow them together; or Spraying or irrigation: Use an irrigation system or sprayer to evenly apply a suspension containing fungi to the surface of the farmland.

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