Bio-fertilizer for promoting pea growth under salt stress, its preparation method and application method

Abstract

This invention discloses a bio-fertilizer for promoting pea growth under salt stress, along with its preparation and application methods. The bio-fertilizer, by weight, comprises the following components: 15-25 parts of bacterial solution, 50-70 parts of pea straw, 5-15 parts of urea, and 1-10 parts of calcium phosphate. The bacterial solution is a mixture of one or more of *Bacillus velezensis*, *Bacillus thuringiensis*, and *Brevibacterium frigoritolerans*. This bio-fertilizer plays a crucial role in alleviating salt stress and promoting pea growth. Its growth-promoting properties under salt stress alleviate the inhibitory effect of salt stress on pea growth, and to a certain extent, improve pea plant height, root length, and root quality, resulting in overall healthy plant growth.

Description

Technical Field

[0001] This invention belongs to the field of bio-fertilizer technology, specifically relating to a bio-fertilizer that promotes pea growth under salt stress, as well as its preparation and application methods. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Soil salinization is a major cause of the reduction in arable land, and mitigating the inhibitory effect of salt stress on plant growth is crucial for improving land utilization and crop yields. Currently, methods for mitigating soil salt stress mainly include water management improvements, agricultural technology improvements, chemical reagent improvements, and plant improvement. However, these methods suffer from drawbacks such as high cost, long timeframes, and unsustainability.

[0004] Peas thrive in cool, humid climates, are cold-hardy but not heat-tolerant. Seedlings can withstand temperatures as low as 5°C, with an optimal growing temperature of 12-16°C and an optimal pod-setting temperature of 15-20°C. Temperatures above 25°C result in low fertilization rates, fewer pods, and lower yields. Soil acidity below pH 5.5 increases susceptibility to disease and reduces pod setting. Peas have deep roots, are somewhat drought-tolerant but not waterlogged-tolerant, and poor drainage during sowing or seedling stages can easily lead to root rot. Drought during flowering can also hinder fertilization, resulting in empty or shriveled pods. Currently, there are few bio-fertilizers specifically formulated for peas on the market, and existing bio-fertilizers are inconsistent in their effectiveness, often causing ineffectiveness, seedling burn, and empty or shriveled pods. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a bio-fertilizer that promotes pea growth under salt stress, along with its preparation and application methods. By using this bio-fertilizer for crop growth, salt stress can be alleviated, and pea growth can be promoted.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] In a first aspect, the present invention provides a bio-fertilizer for promoting pea growth under salt stress, comprising the following components by weight: 15-25 parts of bacterial solution, 50-70 parts of pea straw, 5-15 parts of urea, and 1-10 parts of calcium phosphate, wherein the bacterial solution is a mixture of one or more of Bacillus velezensis, Bacillus thuringiensis, and Brevibacterium frigoritolerans.

[0008] In some embodiments, the bio-fertilizer, by weight, comprises the following components: 15-25 parts of bacterial solution, 50-70 parts of pea straw, 5-15 parts of urea, and 3-7 parts of calcium phosphate.

[0009] In some embodiments, the OD of the bacterial culture 600nm It ranges from 0.3 to 2.

[0010] Preferably, the CFU ratio of Bacillus velezensis, Bacillus thuringiensis and Brevibacterium frigoritolerans in the bacterial culture is 0.8-1.2:0.8-1.2:0.8-1.2.

[0011] More preferably, the CFU ratio of Bacillus velezensis, Bacillus thuringiensis and Brevibacterium frigoritolerans in the bacterial solution is 1:1:1.

[0012] Secondly, the present invention provides a method for preparing the bio-fertilizer that promotes pea growth under salt stress, comprising the following steps:

[0013] One or more of Bacillus velezensis, Bacillus thuringiensis and Brevibacterium frigoritolerans are inoculated into a culture medium and cultured to obtain a seed culture medium. The seed culture medium is then cultured at a constant temperature with shaking to obtain a bacterial culture.

[0014] The bio-fertilizer is obtained by mixing the bacterial solution with pea straw, urea and calcium phosphate in a certain proportion.

[0015] Preferably, the temperature for isothermal shaking culture of the seed culture medium is 26-30℃, and the culture time is 48-72h.

[0016] Further preferred, after culturing the seed culture medium, the OD of the bacterial culture medium is increased. 600nm 0.3-2 is available for standby.

[0017] Thirdly, the present invention provides a method for using the bio-fertilizer that promotes pea growth under salt stress, comprising the following steps:

[0018] The pea seeds are sterilized, then soaked in the bacterial solution for a set time, dried, and then germinated.

[0019] Transplant the germinating seedlings into the soil, apply the bio-fertilizer around the roots of the seedlings, and they will grow.

[0020] In some embodiments, pea seeds are soaked in the bacterial solution for 2-6 hours.

[0021] In some embodiments, pea seeds are dried at room temperature (20-35°C) for 1-2 hours.

[0022] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:

[0023] (1) The bio-fertilizer of this invention plays an important role in alleviating salt stress and promoting pea growth. Its life-promoting properties under salt stress conditions alleviate the inhibition of pea growth by salt stress, and to a certain extent improve the plant height, root length and quality of peas, resulting in good growth of the whole plant.

[0024] The bio-fertilizer provided by this invention has a significant effect on improving saline-alkali soil, greatly improving the salt tolerance of peas, and promoting pea growth. The fresh weight of seedlings, fresh weight of roots, plant height, and root length are all significantly increased compared with the control. It also significantly reduces soil pH and significantly increases the content of available phosphorus, available potassium, organic matter, and alkaline nitrogen.

[0025] (2) This bio-fertilizer has a synergistic effect on promoting pea growth under salt stress. Under salt stress, the dominant bacteria in the fertilizer work together to complement each other's strengths. Brevibacterium frigoritolerans enhances the ACC deaminase activity, phosphorus solubilization, and siderophore capacity of the growth-promoting system, significantly reducing the ethylene level in the plant. Bacillus thuringiensis compensates for the insufficient nitrogen fixation capacity of Brevibacterium frigoritolerans under high salt stress. Bacillus Velezensis enhances the ACC deaminase activity in the growth-promoting system. Each strain exerts all its abilities to provide supplementary nutrition to the plant, significantly improving the overall growth level of the plant.

[0026] (3) In view of the growth habits of peas, this invention introduces beneficial bacteria such as Bacillus velezensis, Bacillus thuringiensis and Brevibacterium frigoritolerans to make a bio-fertilizer for pea growth. This bio-fertilizer can promote the rapid growth of peas, which is beneficial to seedling emergence and the development of rhizobia, and reduces root rot, empty pods or shriveled pods. Attached Figure Description

[0027] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0028] Figure 1This is a pea potted plant experiment. (A) Blank (B) This biological fertilizer (C) Commercial fertilizer 1 (D) Commercial fertilizer 2 (E) Commercial fertilizer 3;

[0029] Figure 2 The effects of different bio-fertilizers on pea growth parameters: (A) plant height (B) root length (C) fresh weight and (D) dry weight.

[0030] Figure 3 This is a comparison diagram of the growth of three strains at different salt concentrations in an embodiment of the present invention;

[0031] Figure 4 This is a comparison chart of the germination rates of pea seeds treated with different strains and different combinations of strains at different salt concentrations in an embodiment of the present invention. The germination rate order corresponding to each salt concentration is the same as the order corresponding to the legend.

[0032] Figure 5 This is a comparison chart of the germination rates of pea seeds treated with different mixed strains at different salt concentrations in an embodiment of the present invention. The germination rate order corresponding to each salt concentration is the same as the order corresponding to the legend. Detailed Implementation

[0033] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] The present invention will be further described below with reference to the embodiments.

[0035] Example 1

[0036] A bio-fertilizer for promoting pea growth under salt stress was developed. The experimental bio-fertilizer was based on an inoculum of 10% of the soil mass. The weight ratio of bacterial solution: pea straw: urea: calcium phosphate was 20:65:10:5. The bacterial solution was a mixture of one or more of *B. velezensis*, *B. thuringiensis*, and *B. frigoritolerans*. The OD of the bacterial solution was... 600nm The CFU ratio of Bacillus velezensis, Bacillus thuringiensis, and Brevibacterium frigoritolerans in the bacterial culture was 1:1:1.

[0037] The three strains, Bacillus velezensis, Bacillus thuringiensis, and Brevibacterium frigoritolerans, are all commercially available.

[0038] Bacillus velezensis, strain number CICC 20025, was purchased from the China Industrial Microbial Culture Collection Center.

[0039] Bacillus thuringiensis, strain number CICC 23706, was purchased from the China Industrial Microbial Culture Collection Center.

[0040] The cold-resistant short bacillus *Brevibacterium frigoritolerans*, with accession number CGMCC 1.10488, was purchased from the China General Microbiological Culture Collection Center.

[0041] like Figure 3 As shown, strains B. velezensis, B. thuringiensis and B. frigoritolerans still exhibit good growth activity under salt stress (up to 10%).

[0042] Pea potted plant experiment

[0043] (1) Seed sterilization

[0044] Pea seeds were used for pot experiments. The seeds were sterilized using the following method: washed with 75% ethanol for 4 min; washed three times with sterile distilled water; soaked in 10% H2O2 for 20 min; and washed with 50 mL of Hoagland nutrient solution.

[0045] (2) Preliminary Experiment

[0046] Sterilized pea seeds were soaked in bacterial solutions with different salt concentration gradients for 4 hours, with 20 seeds in each treatment. Pea seeds soaked in sterile culture medium served as a control. After drying at room temperature for 2 hours, the treated pea seeds were evenly placed in sterile, moist petri dishes for cultivation. Their growth was observed, and their germination rate was calculated.

[0047] In preliminary experiments, there was a significant difference in germination rates between untreated and strain-treated pea seeds under salt stress. For example... Figure 4 As shown, the germination rate of seeds in the control group decreased with increasing salt concentration, while the germination rate of seeds treated with the bacterial strain was higher than that of the control.

[0048] Pea seeds treated with three strains showed the best germination rate compared to those treated with a single strain. Figure 5As shown, the germination rate of pea seeds soaked in the mixed bacterial solution was higher than that of untreated seeds, and the germination rate of seeds treated with all three strains of bacteria was higher than that of seeds treated with only two strains. This sufficiently demonstrates that pea seeds treated with all three strains of bacteria were the most effective in alleviating salt stress and improving germination rate in the mixed bacterial solution. Therefore, in subsequent pot experiments, all three strains of bacteria were used to treat plants to alleviate salt stress, providing a clear contrast between the promoting effects of single strains and mixed bacteria on plant growth under salt stress.

[0049] (3) Seed pre-culture

[0050] Sterilized seeds were soaked in a 1:1:1 bacterial solution of strains B. velezensis, B. thuringiensis, and B. frigoritolerans for 4 hours, then dried at room temperature for 2 hours. The treated plant seeds were then placed in a petri dish (containing moist sterile filter paper) and germinated at 25°C.

[0051] (4) Amplification of bacterial strains

[0052] One tube each of three slant cultures acclimatized to different salinities was taken, and a loopful of bacterial growth was aseptically transferred and inoculated into the corresponding salt concentration culture medium. The culture was then incubated for 12 hours as a "seed" culture medium. A sterile culture medium was used as a control.

[0053] Using sterile pipettes, transfer 100 mL of culture medium with different salinities (0-10% NaCl) into 250 mL Erlenmeyer flasks. Inoculate each flask with 10 mL of "seed" culture medium and incubate at a constant temperature with shaking (28±2℃, 120 rpm) for 48-72 h to allow OD to develop. 600nm It reached 1.5.

[0054] (5) Inoculation of bacterial strains

[0055] Select seedlings of similar size and growth and transplant them into flowerpots containing pre-prepared soil containing bio-fertilizer. Transplant three seedlings into each flowerpot and use seedlings treated with sterile culture solution as a control.

[0056] In the bio-fertilizer, the weight ratio of bacterial solution: pea straw: urea: calcium phosphate is 20:65:10:5;

[0057] Bio-fertilizer accounts for 10% of the soil mass.

[0058] Each flowerpot was inoculated with the same dose of the bacterial strain, as shown in Table 1.

[0059] Table 1. Inoculation of strains

[0060]

[0061] The combination in Table 1 refers to the CFU ratio of Bacillus velezensis, Bacillus thuringiensis, and Brevibacterium frigoritolerans being 1:1:1.

[0062] After 28 days of constant temperature culture at 25℃, the seedlings were removed from the pots, cleaned, and their morphological parameters were measured.

[0063] The results are as follows Figure 1 As shown in the figures, the NaCl stress values ​​for peas in each graph, from left to right, are 0, 50, 100, 200, 300, and 400 mM, respectively. Increased salt stress inhibited pea growth. Adding bio-fertilizers can promote pea growth to varying degrees when exposed to salt stress.

[0064] Figure 2 This study investigated the effects of salt-tolerant microorganisms on the growth parameters of pea plants. Pea parameters in the untreated group decreased with increasing salt stress. The effectiveness of the strains was ranked as follows: Bio-fertilizer of this invention > Commercial bio-fertilizer 2 > Commercial bio-fertilizer 1 > Commercial bio-fertilizer 3 > Blank control. Under high salt stress (400 mM), compared to the control group, pea plants treated with this bio-fertilizer showed increases in plant height, root length, fresh weight, and dry weight of 1.78, 2.00, 2.13, and 2.81 times, respectively.

[0065] Commercial microbial fertilizer 1 is Trichoderma harzianum microbial fertilizer, purchased from Hanzheng Yinong Agricultural Technology Co., Ltd.;

[0066] Commercial microbial fertilizer 2 is Bacillus subtilis microbial fertilizer, purchased from Shandong Zhonggu Nongkang Fertilizer Co., Ltd.;

[0067] Commercial microbial fertilizer 3 is Bacillus subtilis microbial fertilizer - Dingheyuan, purchased from Suqian Dinghe Horticulture Co., Ltd.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A bio-fertilizer that promotes pea growth under salt stress, characterized in that: By weight, it includes the following components: The bacterial solution contains 15-25 parts of bacterial culture, 50-70 parts of pea straw, 5-15 parts of urea, and 1-10 parts of calcium phosphate. The bacterial culture is *Bacillus belyssus*. Bacillus velezensis Bacillus thuringiensis Bacillus thuringiensis and cold-resistant short bacilli Brevibacterium frigoritolerans Mixed bacterial solution; In the bacterial solution, Bacillus velezensis , Bacillus thuringiensis and Brevibacterium frigoritolerans The CFU ratio was 0.8-1.2:0.8-1.2:0.8-1.2; The OD of the bacterial solution 600 nm The value is 0.3~2; Bacillus belesiensis Bacillus velezensis The strain number is CICC 20025, and it was purchased from the China Industrial Microbial Culture Collection Center. Bacillus thuringiensis Bacillus thuringiensis The strain number is CICC 23706, and it was purchased from the China Industrial Microbial Culture Collection Center. Brevibacterium thermotolerans Brevibacterium frigoritolerans The accession number is CGMCC 1.10488, and it was purchased from the China General Microbiological Culture Collection Center.

2. The bio-fertilizer for promoting pea growth under salt stress according to claim 1, characterized in that: By weight, it includes the following components: 15-25 parts bacterial solution, 50-70 parts pea straw, 5-15 parts urea, and 3-7 parts calcium phosphate.

3. The bio-fertilizer for promoting pea growth under salt stress according to claim 1, characterized in that: The bacterial solution contained Bacillus belye Bacillus velezensis Bacillus thuringiensis Bacillus thuringiensis and cold-resistant short bacilli Brevibacterium frigoritolerans The CFU ratio is 1:1:

1.

4. The method for preparing the bio-fertilizer for promoting pea growth under salt stress as described in any one of claims 1-3, characterized in that: Includes the following steps: Bacillus berberis Bacillus velezensis Bacillus thuringiensis Bacillus thuringiensis and cold-resistant short bacilli Brevibacterium frigoritolerans One or more bacteria are inoculated into the culture medium and cultured to obtain a seed culture medium. The seed culture medium is then cultured under constant temperature and shaking to obtain a bacterial solution. The bio-fertilizer is obtained by mixing the bacterial solution with pea straw, urea and calcium phosphate in a certain proportion.

5. The method for preparing bio-fertilizer for promoting pea growth under salt stress according to claim 4, characterized in that: The temperature for constant temperature shaking culture of seed culture medium is 26-30℃, and the culture time is 48-72h.

6. The method for preparing bio-fertilizer for promoting pea growth under salt stress according to claim 4, characterized in that: After culturing the seed culture medium, the OD of the bacterial culture was increased. 600 nm 0.3-2 is available for standby.

7. The method of using the bio-fertilizer for promoting pea growth under salt stress as described in any one of claims 1-3, characterized in that: Includes the following steps: The pea seeds are sterilized, then soaked in the bacterial solution for a set time, dried, and then germinated. Transplant the germinating seedlings into the soil, apply the bio-fertilizer around the roots of the seedlings, and they will grow.

8. The method of using the bio-fertilizer for promoting pea growth under salt stress according to claim 7, characterized in that: Pea seeds are soaked in the bacterial solution for 2-6 hours.

9. The method of using the bio-fertilizer for promoting pea growth under salt stress according to claim 7, characterized in that: Pea seeds are dried at room temperature (20-35℃) for 1-2 hours.

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