Complex microbial inoculant and application thereof in improvement of saline-alkali soil and promotion of alfalfa growth

Abstract

The invention relates to the technical field of microorganisms, in particular to a complex microbial inoculant and application thereof in improvement of saline-alkali soil and promotion of alfalfa growth. And the complex microbial inoculants comprise pickle bacillus amyloliquefaciens HM-1 and salt-tolerant nitrogen-fixing cyanobacteria HH0518. According to the method, the pickle bacillus oceanae HM-1 and the salt-tolerant nitrogen-fixing cyanobacteria HH0518 are jointly applied, crop growth and soil health are improved through the synergistic effect of a biological symbiotic system, the method has the advantages of being environmentally friendly, low in consumption, sustainable and the like, and the economic, feasible and efficient biological remediation method for improving the saline-alkali soil is provided; a new biotechnology path is provided for agricultural ecological restoration, and the method has important application value for realizing efficient utilization of saline-alkali soil resources and agricultural green development.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a compound microbial agent and its application in improving saline-alkali soil and promoting alfalfa growth. Background Technology

[0002] Soil salinization is one of the major ecological problems hindering sustainable agricultural development globally. Salinization leads to soil structure deterioration, nutrient loss, reduced aeration and permeability, and weakened microbial activity, severely impacting crop growth and yield. High salt concentrations and alkaline pH in saline-alkali land cause osmotic pressure imbalances in plant roots, hindering nutrient absorption and disrupting physiological processes, resulting in low crop survival rates, low biomass, and reduced quality.

[0003] Alfalfa, a high-quality perennial leguminous forage, possesses strong nitrogen-fixing ability through its roots and high nutritional value, making it widely used in feed production and soil improvement. However, in saline-alkali environments, alfalfa growth is significantly inhibited, manifested as low germination rate, reduced biomass, and decreased protein content. This not only affects alfalfa yield and quality but also limits its application potential in saline-alkali land improvement and livestock development.

[0004] To improve saline-alkali soil environments and enhance plant growth performance, scholars both domestically and internationally have conducted research on various improvement technologies, including chemical improvement (such as applying gypsum and acidic substances), physical improvement (such as salt removal and flushing), and biological improvement (such as using microorganisms and plant symbiotic systems to improve soil). Among these, microbial improvement methods have received widespread attention due to their green, sustainable, and eco-friendly advantages. Bacteria can improve soil physicochemical properties and promote plant growth by secreting organic acids, extracellular polysaccharides, and growth-promoting substances; microalgae can fix carbon dioxide, release oxygen, and enhance soil microecological activity through photosynthesis. However, current research on the combined application of bacteria and microalgae in saline-alkali soil improvement and alfalfa growth promotion is still in the exploratory stage. Most of the bacteria and algae currently involved are isolated from neutral soil environments. When applied to saline-alkali soil improvement, they often exhibit weak colonization ability and low survival rates, which significantly affects their effectiveness in improving saline-alkali soil.

[0005] Therefore, developing a biological improvement technology for saline-alkali land based on the synergistic effect of bacteria and microalgae is of great theoretical significance and practical value for improving the soil environment, promoting the healthy growth of alfalfa, and realizing the efficient utilization of saline-alkali land resources. Summary of the Invention

[0006] To address the aforementioned technical challenges, this invention, for the first time, screened and constructed a composite symbiotic system of salt-tolerant bacteria and salt-tolerant microalgae specifically for saline-alkali soil environments. Unlike traditional neutral bacterial-algae systems, this invention selects bacterial-algae strains with high salt stress tolerance and growth-promoting potential, enabling them to grow and colonize stably in high-salt, high-pH environments, effectively overcoming the poor survival ability of existing bacterial-algae systems in saline-alkali soils. The combination of these two systems is mutually beneficial and interdependent, thus more effectively helping alfalfa absorb the soil nutrients needed for growth, improving the physicochemical properties of saline-alkali soil, and promoting alfalfa growth, providing a feasible approach for the development of plant growth promoters.

[0007] Furthermore, this invention optimizes the ratio and application method of fungi and algae based on the physiological characteristics of alfalfa's limited growth in saline-alkali soils. Through rhizosphere symbiosis, it promotes root development and enhances nutrient absorption, thereby significantly increasing alfalfa germination rate, biomass, and protein content. This design not only improves the soil environment but also enhances the quality of forage crops, demonstrating significant agricultural application value.

[0008] Based on this, the following technical solution is proposed.

[0009] This invention first provides a compound microbial agent, comprising: Bacillus subtilis (Kimchi Bacillus) Oceanobacillus kimchii HM-1 and salt-tolerant nitrogen-fixing cyanobacteria ( Leptolyngbya angustata HH0518; the preservation number of the Bacillus oryzae HM-1 for kimchi is CGMCC No. 30790; the preservation number of the salt-tolerant nitrogen-fixing cyanobacterium HH0518 is CGMCC No. 41197.

[0010] This invention discovers that the combined application of Bacillus oryzae HM-1 (for kimchi) and the salt-tolerant nitrogen-fixing cyanobacterium HH0518 can regulate the saline-alkali soil environment through their synergistic effect, achieving the dual benefits of improving saline-alkali soil and promoting alfalfa growth.

[0011] Among them, *Bacillus oryzae* HM-1 was isolated from the rhizosphere soil of alfalfa in saline-alkali land. Its preservation information is as follows: Preservation number: CGMCC No. 30790; Classification and nomenclature: *Bacillus oryzae*. Oceanobacillus kimchii Depository Institution: China General Microbiological Culture Collection Center, China; Depository Address: Institute of Microbiology, No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, 100101, China; Depository Date: May 28, 2024.

[0012] Salt-tolerant nitrogen-fixing cyanobacteria ( Leptolyngbya angustata HH0518 was isolated from Huanghua soil, and its preservation information is as follows: preservation number: CGMCC No. 41197; classification and naming: Leptolyngbya angustataDepository Institution: China General Microbiological Culture Collection Center, China; Depository Address: Institute of Microbiology, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China; Depository Date: May 23, 2024.

[0013] This invention utilizes a synergistic mechanism between bacteria and algae to form a mutually beneficial symbiotic ecosystem in a saline-alkali environment. *Bacillus oryzae* HM-1, through the secretion of organic acids and phosphatases, converts insoluble phosphorus into readily absorbable forms for plants; it also possesses nitrogen-fixing capabilities, increasing the available nitrogen content in the soil. The nitrogen-fixing cyanobacterium HH0518 further fixes atmospheric nitrogen and converts it into ammonia nitrogen, enhancing soil nutrient supply and significantly improving soil fertility. The bacteria-algae system improves soil structure and physicochemical properties through various metabolites: organic acids neutralize alkaline ions, lowering soil pH; extracellular polysaccharides promote soil particle aggregation, forming stable aggregate structures and improving water retention and aeration; and the accumulation of photosynthetic products increases soil organic matter and cation exchange capacity, thereby enhancing buffering capacity and fertility. The oxygen and organic carbon produced by microalgae photosynthesis provide growth conditions for bacteria, while bacterial metabolites (such as IAA and GA growth hormones) stimulate plant root development. The symbiotic metabolic system formed by these two organisms promotes carbon and nitrogen cycling in the rhizosphere and enhances nutrient absorption efficiency, thus accelerating alfalfa growth. The symbiotic system of bacteria and algae can promote the accumulation of osmotic regulators (such as proline and soluble sugars) in plants and enhance their salt and alkali resistance; at the same time, it can improve the redox state of the rhizosphere, reduce salt ion stress, and enhance the overall stress resistance and physiological vitality of plants.

[0014] Preferably, the mass ratio of the Bacillus oryzae HM-1 to the salt-tolerant nitrogen-fixing cyanobacterium HH0518 is 1:(1~2).

[0015] Preferably, the mass ratio of the Bacillus oryzae HM-1 to the salt-tolerant nitrogen-fixing cyanobacterium HH0518 is 1:(1.8~2).

[0016] Furthermore, the present invention provides a fertilizer additive or fertilizer containing the aforementioned compound microbial agent.

[0017] Furthermore, the present invention provides a soil conditioner containing the aforementioned compound microbial agent.

[0018] Furthermore, the present invention provides the application of the aforementioned compound microbial agent, the aforementioned fertilizer additive or fertilizer, or the aforementioned soil conditioner in improving saline-alkali soil and / or promoting alfalfa growth.

[0019] Preferably, promoting alfalfa growth manifests in at least one of the following aspects: increasing alfalfa plant height, increasing alfalfa fresh weight, increasing alfalfa dry weight, increasing alfalfa proline content, decreasing alfalfa malondialdehyde content, decreasing alfalfa catalase activity, and increasing alfalfa chlorophyll content.

[0020] Preferably, the improvement of saline-alkali soil is manifested in at least one of the following aspects: reducing soil electrical conductivity and increasing the content of available nitrogen in the soil.

[0021] Preferably, the promotion of alfalfa growth refers to promoting the growth of alfalfa under saline-alkali stress in saline-alkali land.

[0022] Preferably, the saline-alkali land is Huanghua saline-alkali land.

[0023] Preferably, the alfalfa is purple alfalfa.

[0024] Furthermore, the present invention provides the application of the aforementioned compound microbial agent in the preparation of saline-alkali soil conditioners, salt-alkali stress-resistant plant growth promoters, or salt-alkali stress-resistant fertilizers.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention abandons chemical amendments and improves crop growth and soil health through the combined application of Bacillus oryzae HM-1 and salt-tolerant nitrogen-fixing cyanobacterium HH0518. It has the advantages of being green, low-cost, and sustainable, and provides an economical, feasible, and efficient bioremediation method for improving saline-alkali soil. It offers a new biotechnological path for agricultural ecological restoration and has important application value for achieving efficient utilization of saline-alkali land resources and green agricultural development. Attached Figure Description

[0026] Figure 1 This is a diagram showing the growth results of alfalfa.

[0027] Figure 2 The effect of combined bacterial and algal treatment on plant height of alfalfa in saline-alkali environments.

[0028] Figure 3 The effect of combined bacterial and algal treatment on the fresh weight of alfalfa plants under saline-alkali conditions.

[0029] Figure 4 The effect of combined bacterial and algal treatment on the dry weight of alfalfa plants under saline-alkali conditions.

[0030] Figure 5 The effect of combined bacterial and algal treatment on malondialdehyde (MDA) in alfalfa plants under saline-alkali conditions.

[0031] Figure 6 The effect of combined bacterial and algal treatment on proline content in alfalfa plants under saline-alkali conditions.

[0032] Figure 7 The effect of combined bacterial and algal treatment on catalase (CAT) in alfalfa plants under saline-alkali conditions.

[0033] Figure 8The effect of combined bacterial and algal treatment on chlorophyll content in alfalfa plants under saline-alkali conditions.

[0034] Figure 9 The effect of combined bacterial and algal treatment on the electrical conductivity (EC) of saline-alkali soil.

[0035] Figure 10 The effect of combined bacterial and algal treatment on the available nitrogen (AN) content in saline-alkali soil.

[0036] In the figure, different lowercase letters indicate that there are significant differences between different treatments at this level (p<0.05). Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the embodiments provided in this specification, where specific techniques or conditions are not specified, they are performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0038] Example 1: Preparation of a mixture of strain HM-1 and algal strain HH0518 Strain HM-1 was cultured in liquid at 37℃ and 200 rpm for 3 days. After culture, the bacterial cells were collected by centrifugation and washed three times with sterile water. The collected bacterial cells were resuspended in sterile water to prepare a bacterial suspension. The culture medium for HM-1 liquid culture consisted of: 10 g / L tryptone, 5 g / L yeast extract, 100 g / L NaCl, and pH=8.5.

[0039] Algal strain HH0518 was cultured in liquid at 25°C and 200 rpm for 5 days. After culture, the algae were collected by centrifugation and washed three times with sterile water. The collected algae were then resuspended in sterile water to prepare an algal suspension. The culture medium for HH0518 liquid culture consisted of: Na₂NO₃ 0.15 g / L, K₂HPO₄ 0.04 g / L, MgSO₄·7H₂O 0.075 g / L, CaCl₂·2H₂O 0.036 g / L, Citric acid 0.006 g / L, Ferric ammonium citrate 0.006 g / L, EDTANa₂ 0.001 g / L, NaCO₃ 0.02 g / L, H₃BO₃ 2.86 mg / L, MnCl₂·4H₂O 1.86 mg / L, ZnSO₄·7H₂O 0.22 mg / L, Na₂MoO₄·H₂O 0.39 mg / L, CuSO₄·5H₂O 0.08 mg / L, Co(NO₃)₂·6H₂O 0.05 mg / L, with a pH of 7.1.

[0040] The bacterial suspension and algal suspension were mixed to obtain a bacterial-algal composite microbial preparation.

[0041] Example 2: Effects of combined use of strain HM-1 and algal strain HH0518 on alfalfa growth in saline-alkali soil. 1. The experimental soil was collected from saline-alkali land in Huanghua City, Cangzhou City, Hebei Province. After the soil sample was naturally air-dried and impurities were removed, it was sieved through a 20-mesh sieve for later use. The plant base fertilizer was thoroughly mixed with the sieved soil, and then evenly filled into plastic flower pots with dimensions of 7×7×8 cm, with each pot containing 210 g of soil.

[0042] 2. Preparation of bacterial strains and algal strains.

[0043] Prepare a bacterial-algae mixture according to the method described in Example 1 for later use.

[0044] 3. This experiment included 5 treatments, with 3 replicates for each treatment. These were: a control (CK) treatment (no bacterial strain or algae applied), treatment with only *Bacillus oryzae* HM-1 (A), treatment with only salt-tolerant nitrogen-fixing cyanobacterium HH0518 (B), treatment with a combination of strain HM-1 and algae HH0518 (bacteria-algae mass ratio 1:1) (A1B1), and treatment with a combination of strain HM-1 and algae HH0518 (bacteria-algae mass ratio 1:2) (A1B2). Except for the control group, the total amount of microorganisms applied to each flowerpot in the other treatment groups was 0.06 g to ensure a consistent total amount of microorganisms applied across all treatment groups. This was done twice during the experiment.

[0045] 4. The tested alfalfa variety was Zhongmu No. 3 alfalfa. Uniform and plump alfalfa seeds were disinfected in a 1% sodium hypochlorite solution for 10 minutes, followed by thorough rinsing three times with deionized water to completely remove residual sodium hypochlorite. Moistened filter paper was placed in a petri dish, and the disinfected seeds were evenly spread on top. The dish was then sealed and placed in a dark environment at 25℃ to promote germination. After germination, the seeds were transferred with tweezers to plastic flowerpots filled with saline-alkali soil, with 8 seeds per pot. A thin layer of soil was covered, and the soil was kept moist. Seven days later, thinning was performed, leaving 5 seedlings per pot. Subsequently, bacterial suspension, algae suspension, and a bacterial-algae compound microbial preparation were evenly sprayed onto the soil surface of the corresponding treatment groups. The control group received the same amount of sterile water in the same manner.

[0046] 5. During the cultivation period, the soil moisture content was maintained at approximately 50% of field capacity by regularly irrigating with deionized water. Sixty days after planting, alfalfa plants and rhizosphere soil samples were harvested separately to determine plant growth and physiological indicators, as well as the physicochemical properties of the soil.

[0047] Figure 1 A comparison of alfalfa growth shows that the alfalfa grown after applying microbial-algae fertilizer was significantly better than the group that did not apply microbial-algae fertilizer or the group that applied microbial fertilizer or algae fertilizer alone. Figure 2 As shown, the alfalfa plant height after applying the microbial-algae fertilizer was significantly higher than that of the untreated group, and also significantly higher than that of the group treated with either microbial fertilizer or algae fertilizer alone. Figure 3 The comparison of alfalfa fresh weight shown also demonstrates a significant increase in alfalfa fresh weight after application of the microbial-algae compound fertilizer, and the effect is more pronounced than that of applying microbial fertilizer formulated with HM-1 alone. Figure 4 As shown, the trend of alfalfa dry weight variation is basically consistent with that of fresh weight. Compared with the unfertilized group and the microbial fertilizer group, the application of compound microbial-algae fertilizer can significantly increase the dry weight of alfalfa. Figure 5 To compare the malondialdehyde (MDA) content in alfalfa leaves, the application of microbial-algae fertilizer resulted in a decrease in MDA content, demonstrating better performance than applying microbial or algae fertilizer alone. Figure 6 The proline content in alfalfa leaves was significantly increased after the application of microbial-algae fertilizer compared to other treatment groups. Figure 7 To compare the catalase activity in alfalfa leaves, it was found that the catalase activity in the leaves was significantly reduced after the application of microbial-algae fertilizer. Figure 8 To compare the chlorophyll content in alfalfa leaves, the application of microbial-algae fertilizer significantly increased the chlorophyll content in alfalfa leaves compared to other treatment groups. Figure 9 The figure shows the change in electrical conductivity of saline-alkali soil in flowerpots; the soil electrical conductivity decreased significantly after the treatment was applied. Specific changes in the available nitrogen content in the soil are shown below. Figure 10After applying microbial-algae fertilizer, the content of available nitrogen in the soil increased, with the combined effect of microbial and algae being particularly significant. This indicates that the combined application of strain HM-1 and algae HH0518 reduced oxidative stress on alfalfa, significantly promoted its growth, and the growth-promoting effect was more pronounced than that of applying microbial or algae fertilizer alone.

[0048] In summary, this invention combines *Bacillus oryzae* HM-1 (for kimchi) with the salt-tolerant nitrogen-fixing cyanobacterium HH0518, achieving a synergistic effect. Firstly, after treating saline-alkali soil with the combined microbial and algal fertilizer, plant height, fresh weight, and dry weight all significantly increased, with effects superior to applying either the microbial or algal fertilizer alone, indicating that the microbial-algal compound microbial preparation is a more effective growth-promoting bio-fertilizer. After applying the microbial and algal fertilizer, the chlorophyll content of alfalfa leaves significantly increased, promoting photosynthesis and accumulating organic matter for plant growth. The significant increase in proline, an osmotic substance, indicates that the combined application of microbial and algal fertilizer promoted osmotic balance in cells, enhancing leaf resistance. The significantly reduced malondialdehyde content and catalase activity suggest a decrease in oxidative stress on the leaves after applying the microbial and algal fertilizer. After applying the microbial and algal fertilizer, soil electrical conductivity significantly decreased. Electrical conductivity is an important standard for measuring soil salinity; a decrease in soil electrical conductivity may indicate a decrease in sodium content in the soil. + The reduced nitrogen content alleviates soil stress on plant growth. Nitrogen is an essential element for plant growth, but the nitrogen in the air and soil cannot be directly utilized by plants. Only by converting nitrogen into readily available nitrogen can plant growth be effectively promoted. The bacteria and microalgae used in this invention have nitrogen-fixing capabilities, which can convert nitrogen that plants cannot utilize into readily available nitrogen, thereby promoting alfalfa growth. After applying the microbial-algae fertilizer, the content of readily available nitrogen in the soil increases significantly, the amount of nitrogen that plants can absorb increases, and their growth is significantly promoted.

[0049] In summary, the application of microbial and algal fertilizers reduced oxidative stress on alfalfa, enhanced its resistance, and altered the soil environment, increasing soil fertility and promoting alfalfa growth. This resulted in stronger adaptability and growth vigor in saline-alkali environments, improving survival rate and yield, with effects superior to applying microbial or algal fertilizers alone. Furthermore, compared to traditional chemical improvement methods, the combined use of microorganisms and algae is more environmentally friendly and helps establish a healthy soil ecosystem. Through the action of the microbial community, the ecological environment of saline-alkali soil is gradually improved. In conclusion, compared to applying Bacillus or cyanobacteria alone, the combined application of these two methods has a more significant synergistic effect in alleviating plant salt stress, promoting growth, and restoring saline-alkali soil.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A compound microbial agent, characterized in that, include: Bacillus subtilis (Kimchi) Oceanobacillus kimchii HM-1 and salt-tolerant nitrogen-fixing cyanobacteria ( Leptolyngbya angustata HH0518; the preservation number of the Bacillus oryzae HM-1 for kimchi is CGMCC No. 30790; the preservation number of the salt-tolerant nitrogen-fixing cyanobacterium HH0518 is CGMCC No. 41197.

2. The compound microbial agent according to claim 1, characterized in that, The mass ratio of the Bacillus oryzae HM-1 to the salt-tolerant nitrogen-fixing cyanobacterium HH0518 is 1:(1~2).

3. The compound microbial agent according to claim 2, characterized in that, The mass ratio of the Bacillus oryzae HM-1 to the salt-tolerant nitrogen-fixing cyanobacterium HH0518 is 1:(1.8~2).

4. A fertilizer additive or fertilizer, characterized in that, It contains the compound microbial agent as described in any one of claims 1 to 3.

5. A soil conditioner, characterized in that, It contains the compound microbial agent as described in any one of claims 1 to 3.

6. The application of the compound microbial agent according to any one of claims 1 to 3, the fertilizer additive or fertilizer according to claim 4, or the soil conditioner according to claim 5 in improving saline-alkali soil and / or promoting alfalfa growth.

7. The application according to claim 6, characterized in that, Promoting alfalfa growth manifests in at least one of the following aspects: increasing alfalfa plant height, increasing alfalfa fresh weight, increasing alfalfa dry weight, increasing alfalfa proline content, decreasing alfalfa malondialdehyde content, decreasing alfalfa catalase activity, and increasing alfalfa chlorophyll content. And / or, the improvement of saline-alkali soil is manifested in at least one of the following aspects: reducing soil electrical conductivity and increasing the content of available nitrogen in the soil.

8. The application according to claim 6 or 7, characterized in that, The promotion of alfalfa growth refers to promoting the growth of alfalfa under saline-alkali stress in saline-alkali land.

9. The application according to claim 6 or 7, characterized in that, The alfalfa in question is alfalfa.

10. The application of the compound microbial agent according to any one of claims 1 to 3 in the preparation of saline-alkali soil conditioner, salt-alkali stress resistant plant growth promoter, or salt-alkali stress resistant fertilizer.

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