Compound microbial agent and application thereof

Abstract

The invention belongs to the technical field of agriculture, and particularly provides a compound microbial agent and application thereof. The compound microbial agent is prepared from bacillus velezensis RKN1111, bacillus subtilis N-3 and paenibacillus mucilaginosus HX-02, and the compound microbial agent is prepared from bacillus velezensis RKN1111, bacillus subtilis N-3 and paenibacillus mucilaginosus HX-02, and the compound microbial agent is prepared from bacillus velezensis RKN1111, bacillus subtilis N-3 and paenibacillus mucilaginosus HX-02. The compound microbial agent can effectively improve the pH and EC of saline alkali soil, increase the content of alkali-hydrolyzable nitrogen, available phosphorus and quick-acting potassium in the soil, and play a significant role in increasing the plant height, stem diameter, chlorophyll content and aboveground biomass of corn plants in coastal saline-alkali soil.

Description

Technical Field

[0001] This invention belongs to the field of agricultural technology, specifically relating to a compound microbial agent and its application. Background Technology

[0002] Saline-alkali land is a major obstacle to the sustainable development of agriculture worldwide. Coastal saline-alkali land, in particular, has accumulated excessive salt over a long period of time, resulting in high electrical conductivity (EC) and high pH value. In addition, the soil particles are compacted and have poor aeration and permeability. This not only directly damages the growth environment of plant roots and inhibits nutrient absorption efficiency, but also leads to a severe deficiency and low availability of readily available nutrients such as alkaline nitrogen, available phosphorus, and available potassium in the soil. Ultimately, this results in slow crop growth, sharp reduction in yield, and even large-scale abandonment of farmland.

[0003] To address the challenges of saline-alkali soil improvement, various technologies have been developed both domestically and internationally, including physical methods (such as deep tillage and leaching for desalination), chemical methods (such as the application of gypsum and humic acid-based amendments), and biological methods. Physical methods suffer from drawbacks such as large engineering workload, high cost, short-term effectiveness, and potential damage to soil structure. While chemical amendments can quickly lower soil pH or alleviate salinity, long-term application can lead to secondary soil pollution and fails to fundamentally improve soil nutrient supply capacity and microbial community structure. In contrast, microbial improvement technology, with its environmentally friendly, efficient, and sustainable advantages, has become a research hotspot and development direction for saline-alkali land improvement.

[0004] In existing technologies, some single microbial strains or simple compound microbial agents have been attempted to improve saline-alkali land, but their functions are often limited: most microbial agents only focus on improving a single indicator (such as only reducing soil pH or only increasing the content of a certain type of nutrient), making it difficult to simultaneously solve the multiple core problems of "high pH, ​​high EC, and low nutrients" in saline-alkali soil; some compound microbial agents have unstable improvement effects due to a lack of targeted strain screening and weak synergistic effects between strains, and their effect on promoting plant growth is limited, failing to meet the actual needs of agricultural production in saline-alkali land for improving the comprehensive physical and chemical properties of soil and synergistically enhancing crop growth.

[0005] Chinese patent CN 118344995A discloses the application of Bacillus belye RKN1111 in saline-alkali land management and plant growth promotion, but it only affects pH and has little effect on EC. Chinese patent CN120647479A provides a bio-organic fertilizer with soil improvement functions, which includes a compound microbial agent. However, this compound microbial agent needs to be used in conjunction with adsorbent materials such as vermiculite and bamboo charcoal powder, and auxiliary substances such as alkaline cores to achieve the desired effect. Whether it can achieve key soil improvement indicators (such as reducing pH and EC, and increasing the content of alkaline nitrogen, available phosphorus, and readily available potassium) when used alone is not clearly stated. Summary of the Invention

[0006] The purpose of this invention is to provide a compound microbial agent and its application. The compound microbial agent is particularly suitable for coastal saline-alkali soils with high EC values. It has multiple improvement effects, strong adaptability, and is eco-friendly. It is of great practical significance for promoting the growth of plants in saline-alkali land and promoting the green development of agriculture in saline-alkali land.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention first provides a compound microbial agent, which is composed of Bacillus belye ( Bacillus from Velez RKN1111, Bacillus subtilis ( Bacillus subtilis N-3 and gelatinous Bacillus ( Paenibacillus mucilaginosus Composition of HX-02; The Bacillus belyssus RKN1111 was deposited on December 12, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.29300. The Bacillus subtilis N-3 was deposited on May 22, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34633. The gelatinous Bacillus HX-02 was deposited on June 14, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30949.

[0008] Furthermore, the total viable count of the compound microbial agent is not less than 8.4 × 10⁻⁶. 8 CFU / gram.

[0009] Furthermore, the viable count of the Bacillus subtilis N-3 is not less than 2.8 × 10⁻⁶. 8 CFU / gram.

[0010] Furthermore, the preparation method of the above-mentioned compound microbial agent includes the following steps: (1) Take the refrigerated Bacillus belyss RKN1111, Bacillus subtilis N-3 and Bacillus spp. HX-02 and inoculate them separately into sterilized LB liquid medium. After seed culture and scale-up fermentation, the fermentation products of each strain are centrifuged under preset conditions to remove the cells and collect the supernatant to obtain Bacillus belyss RKN1111 fermentation broth, Bacillus subtilis N-3 fermentation broth and Bacillus spp. HX-02 fermentation broth respectively. (2) The fermentation broth of Bacillus subtilis RKN1111, the fermentation broth of Bacillus jelly-like N-3, and the fermentation broth of Bacillus belyss HX-02 are mixed in a volume ratio of 1:1:1 to obtain the compound microbial agent.

[0011] The present invention also provides the application of the above-mentioned composite microbial agent in the following aspects: (1) Improve the physical and chemical properties of coastal saline-alkali soil, including reducing soil pH and EC value, and increasing soil alkaline nitrogen, available phosphorus and available potassium content; (2) Promote the growth of plants in coastal saline-alkali land; (3) Prepare bio-organic fertilizer or plant growth promoter.

[0012] Compared with the prior art, the present invention has at least the following advantages: (1) Multiple soil improvement effects: The compound microbial agent described in this invention can effectively reduce the pH and EC values ​​of coastal saline-alkali soil, significantly reduce the soluble salt content and alkalinity, and at the same time, through nitrogen fixation, phosphorus solubilization, potassium solubilization and metabolic activities, greatly increase the content of available nutrients and organic matter in the soil, fundamentally improving the micro-ecological environment of saline-alkali soil.

[0013] (2) Functional complementarity and synergistic effect: The Bacillus berleis RKN1111, Bacillus subtilis N-3 and Bacillus spp. HX-02 selected in this invention have significant functional complementarity. After the three are combined, they show a significant synergistic growth-promoting effect in the early stage of maize growth, and are superior to single-strain treatment in multiple indicators such as plant height and stem diameter.

[0014] (3) Specificity and practicality: The compound microbial agent described in this invention is specifically designed for the soil characteristics (high pH, ​​high salinity) and crop growth needs of coastal saline-alkali land. It has been verified by pot and field trials (taking corn as an example) that the effect is significant and has good application prospects and promotion value. Detailed Implementation

[0015] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0016] Example 1: Isolation, screening and identification of Bacillus subtilis N-3 1. Test Sample The Bacillus subtilis N-3 used in this embodiment was obtained from the Biotechnology Engineering Innovation Center of Hebei University. It was deposited at the China General Microbiological Culture Collection Center on May 22, 2025, with accession number CGMCC No. 34631.

[0017] Bacillus isolation medium: yeast extract 0.5, peptone 2.0, glucose 2.0, K2HPO4 1.5, (NH4)2SO4 0.5, MgSO4·7H2O 0.5, tricalcium phosphate 5.0, agar 18-20, distilled water 1000mL.

[0018] 2. Isolation and screening of Bacillus subtilis strains Weigh 10.0 g of soil sample (collected in 2023 from a greenhouse in Shouguang, Shandong), add 90.0 mL of sterile water containing glass beads, let stand for 20 min, shake thoroughly on a rotary shaker for 30 min, then heat in an 80℃ water bath for 20 min, and then cool rapidly. Use a sterile pipette to take 1.0 mL of the above soil suspension and dilute it 1 to 10 times to obtain 1×10⁻⁶ ppm. 1 1×10 2 1×10 3 1×10 4 Soil suspension diluted 10 times... Using a sterile pipette, aspirate 10... times... -2 10 -3 and 10 -4 0.1 mL of the gradient soil suspension was evenly spread on the isolation culture medium plate and incubated at 30℃ for 36-72 h.

[0019] 3. Purification Single colonies with a clear zone were picked and transferred to NA medium slant, incubated at 30°C for 24 h, and stored at 4°C for later use. The colony characteristics of the isolated strains were recorded.

[0020] 4. Identification 4.1 Morphological identification Colony morphology observation: The purified strain was inoculated onto LB plates and incubated at 37°C for 24 hours. The colony shape, color, edge, surface texture, elevation, and whether pigment was produced were recorded.

[0021] Individual morphological observation: Single colonies were picked and Gram staining was performed (crystal violet primary staining → iodine mordant staining → 95% ethanol decolorization → safranin counterstaining). Observation under an optical microscope (1000×): Bacillus subtilis is a Gram-positive bacillus with rod-shaped cells (0.7~0.8μm×2.0~3.0μm), arranged singly or in chains; using the spore staining method (malachite green heating staining → safranin counterstaining), oval spores were observed, located in the center or near the end of the cell, and the sporangia did not swell (typical characteristic).

[0022] 4.2 Molecular biological identification DNA extraction: Total DNA of the bacterial strain was extracted and purified using a bacterial genomic DNA extraction kit, and DNA integrity was verified by 1% agarose gel electrophoresis.

[0023] PCR amplification: Universal primers for Bacillus 16S rRNA were used. Sequencing and alignment: The PCR product was purified and sent to a sequencing company for sequencing to obtain the 16S rRNA gene sequence; the sequence was submitted to the GenBank database and compared with known sequences using the BLAST tool. If it is similar to Bacillus subtilis (…), then… Bacillus subtle The homology of the type strain is ≥99%. Based on the morphological and physiological-biochemical characteristics, the strain is identified as Bacillus subtilis.

[0024] 4.3 Identification Results 4.3.1 Morphological identification results: Based on colony morphology observation and individual morphological staining identification, the morphological characteristics of this isolated and purified strain are similar to those of Bacillus subtilis (B. subtilis). Bacillus subtilis The typical characteristics are completely identical, as follows: Colony morphology: After aerobic incubation at 37°C for 24-48 hours on LB solid medium, round colonies are formed. Colonies grow and reproduce rapidly; the surface is rough and opaque, off-white or slightly yellow, without transparency or translucency, and the edges are neat, without serrations or spreading. When growing in liquid medium, it often forms wrinkled mold, indicating it is an aerobic bacterium.

[0025] Individual morphology: Gram staining result was positive (G). + Under a 1000× oil immersion optical microscope, the bacteria appear purple, rod-shaped, and 0.6–0.9 × 1.0–1.5 μm in size, arranged singly or in short chains. After staining with a modified Schaeffer-Fulton spore staining method, oval spores are visible, located in the center or near the end of the bacterial cell. After spore formation, the bacterial cell does not swell, lacks a capsule, has peritrichous flagella, and is motile. This closely matches the specific spore characteristics of Bacillus subtilis.

[0026] 4.3.2 Molecular biological identification (16S rRNA gene sequencing) results: The sequence was submitted to the GenBank database, and homology analysis was performed between it and known sequences in the NCBI nucleic acid database using the BLAST tool.

[0027] The comparison results showed that the 16S rRNA gene sequence of this strain (gene sequence database accession number NR074540.1, as shown in SEQ ID NO.1) was similar to that of Bacillus subtilis ( Bacillus subtilis The homology with the type strain (such as ATCC6051) was over 99%. Combined with the aforementioned morphological identification results, the isolated and purified strain was determined to be Bacillus subtilis. Bacillus subtilis ).

[0028] Example 2: Preparation of functional strain inoculants and compound microbial inoculants 1. Preparation of Bacillus belyssus RKN1111 fermentation broth The Bacillus belye RKN1111 used in this embodiment was isolated from soil samples around Baoding City, Hebei Province by the Agricultural Environment and Microecology Laboratory of the Innovation Center for Biotechnology of Hebei University. It is preserved at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 29300.

[0029] First, *Bacillus belye* RKN1111 was inoculated from a test tube slant into 50 mL of LB broth and cultured at 38°C and 200 rpm for 18 h to obtain the seed culture (pH 7). Then, 1 mL of the seed culture was added to 250 mL of LB broth and cultured at 38°C and 200 rpm for 36 h to obtain the fermentation broth (pH 7). After fermentation, the count was 2.8 × 10⁻⁶. 8 cfu / mL.

[0030] 2. Preparation of Bacillus subtilis N-3 fermentation broth The Bacillus subtilis N-3 used in this embodiment was isolated from soil samples around Baoding City, Hebei Province by the Agricultural Environment and Microecology Laboratory of the Innovation Center for Biotechnology of Hebei University. It is preserved at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 34633.

[0031] First, Bacillus subtilis N-3 was inoculated onto a test tube slant into 50 mL of LB liquid medium and cultured at 37°C and 190 rpm for 18 h to obtain the seed culture (pH 7). Then, 1 mL of the seed culture was added to 250 mL of LB liquid medium and cultured at 37°C and 190 rpm for 36 h to obtain the fermentation broth (pH 7). After fermentation, the count was 2.8 × 10⁻⁶. 8 cfu / mL.

[0032] 3. Preparation of fermentation broth of Bacillus jellyii HX-02 The gelatinous Bacillus HX-02 used in this embodiment was isolated from soil samples around Baoding City, Hebei Province by the Agricultural Environment and Microecology Laboratory of the Innovation Center for Biotechnology of Hebei University. It is preserved at the China General Microbiological Culture Collection Center with accession number CGMCC No. 29300.

[0033] First, *Bacillus spp.* HX-02 was inoculated from a test tube slant into 50 mL of LB liquid medium and cultured at 38°C and 220 rpm for 22 h to obtain the seed culture (pH 7). Then, 1 mL of the seed culture was added to 250 mL of LB liquid medium and cultured at 38°C and 220 rpm for 48 h to obtain the fermentation broth (pH 7). After fermentation, the count was 2.8 × 10⁻⁶. 8 cfu / mL.

[0034] 4. Preparation of compound microbial agents The above-obtained Bacillus vesiculosus RKN1111 fermentation broth, Bacillus subtilis N-3 fermentation broth, and Bacillus lentigines HX-02 fermentation broth were mixed in an equal volume ratio of 1:1:1.

[0035] Example 3: Determination of the effects of functional strains and their combinations on pH and EC in saline-alkali soil. Experimental treatment The pot experiment used sieved saline-alkali soil (collected from Huanghua, Cangzhou, Hebei Province, with basic physicochemical properties of pH 9.12 and EC 5.46 ds / m), with each pot containing a uniform 450 g of soil.

[0036] Four treatments and one control were set up: treatment 1, treatment 2, treatment 3, and treatment 4. Treatment 1: 20 ml of the gelatinous Bacillus thuringiensis HX-02 fermentation broth prepared in Example 1 was diluted 100 times and then used to irrigate the soil; Treatment 2: 20 ml of the Bacillus berberis RKN1111 fermentation broth prepared in Example 1 was diluted 100 times and then used to irrigate the soil; Treatment 3: 20 ml of Bacillus subtilis N-3 fermentation broth prepared in Example 1 was diluted 100 times and then used to irrigate the soil; Treatment 4: 20 ml of the compound microbial agent prepared in Example 1 was diluted 100 times and then used to irrigate the soil; Control group (CK): This treatment did not use any microbial agents, but instead used clean water to irrigate the soil.

[0037] All of the above irrigations were applied once every 5 days, for a total of 4 applications.

[0038] 2. Sampling Soil samples were collected before the first irrigation (0 days), and at 12 days and 20 days. Using a sampling shovel, topsoil and 5-8cm deep soil samples were taken from three different locations in each group of potted soil samples. The samples were mixed together as one sample, air-dried, ground, and sieved. The pH, EC and available nutrients of the soil samples were then measured.

[0039] 3. Experimental Results As shown in Tables 1 and 2, the soil pH and EC values ​​decreased to varying degrees under all treatment groups, and the pH values ​​of all single-strain treatments were lower than those of the control group. Furthermore, the pH value of the compound microbial agent group was the lowest at all time points, demonstrating superior performance compared to all single-strain treatments. This indicates that the synergistic effect of the three microorganisms was the most effective in reducing soil pH and EC values.

[0040] Table 1. Changes in soil pH under different bacterial strain treatments.

[0041] Table 2. Changes in soil EC values ​​(ds / m) under different strain treatments

[0042] Example 4: Determination of the effect of functional strains and their combinations on available nutrients in saline-alkali soil The available nitrogen (AN), available phosphorus (AP), and available potassium (AK) of soil samples under different treatments in Example 2 were determined using a soil nutrient analyzer.

[0043] The experimental results show that by 20 days, the levels of available nitrogen (Table 3), available phosphorus (Table 4), and available potassium (Table 5) in the soil were significantly increased in all inoculant treatments. However, with the extension of treatment time, the soil effect under the compound microbial agent treatment was the most significantly improved.

[0044] Table 3. Changes in soil available nitrogen content (mg / kg) under different strain treatments

[0045] Table 4. Changes in available phosphorus content in soil under different strain treatments (mg / kg)

[0046] Table 5. Changes in available potassium content in soil under different bacterial strain treatments (mg / kg) Example 5: Determination of the effects of functional microbial agents and their combination on growth indicators of potted maize 1. Experimental Methods Corn (Dicai "635") at the germination to two-leaf stage was transplanted into pots. (The soil samples were taken from the Nandagang Experimental Area of ​​Huanghua City, Cangzhou. The pH of the soil was 9.12 and the EC value was 5.46 ds / m. The corn was treated with different inoculants by root irrigation. The first root irrigation was performed when the corn was transplanted into the pots, and then the root irrigation was performed every four days for a total of five times.)

[0047] (1) Experimental group treatment: Treatment 1: Irrigation water (200ml) + Bacillus vesiculosus RKN1111 inoculum prepared in Example 1 (20ml); Treatment 2: Irrigation water (200ml) + Bacillus subtilis N-3 inoculum prepared in Example 1 (20ml); Treatment 3: Irrigation water (200ml) + Bacillus thuringiensis HX-02 bacterial agent prepared in Example 1 (20ml); Treatment 4: Irrigation water (200ml) + compound microbial agent prepared in Example 1 (20ml).

[0048] (2) Control group treatment: irrigation water (220ml).

[0049] During the third and fifth root irrigation treatments, the height of the corn plants (distance from the soil surface to the new leaves) was measured with a measuring tape; stem diameter was measured (selecting a typical part of the stem with good stem diameter, measuring the aboveground biomass (after drying at 100℃ for 5 hours), measuring the stem diameter at the same location with a measuring tape, and then calculating the stem diameter based on the stem shape); and chlorophyll content was measured using a leaf area meter (the standard for test leaves: mature leaves at the top of the plant, leaves that have turned dark green, and the same selected leaf location was used for measurement in different treatments).

[0050] 2. Experimental Results The corn in the blank treatment group only grew until day 20 and died completely by day 30. The results of plant height, stem diameter, chlorophyll content, and aboveground biomass measurements are shown in Tables 6, 7, 8, and 9, respectively. It can be seen that the compound microbial agent group showed the best growth.

[0051] Table 6. Changes in maize plant height (cm) under different bacterial strain treatments.

[0052] Table 7. Changes in maize stalk diameter (mm) under different bacterial strain treatments.

[0053] Table 8. Changes in chlorophyll content (SPAD) under different bacterial strain treatments.

[0054] Table 9. Changes in corn dry weight (g) under different bacterial strain treatments.

[0055] Example 6: Determination of the effects of functional strains on maize plant growth and yield 1. Experimental Materials Crops and varieties: Corn, Dicai "635"; The tested soil was the same as that used in Example 2 (pH 9.12 EC 5.46 ds / m).

[0056] 2. Experimental Methods This experiment included two treatments, using a large-area comparison without replication. Each large area was 1 mu (approximately 0.16 acres), with 5500 plants per mu. Irrigation and fertilization were carried out separately in each large area. Except for fertilization, all other agricultural operations were the same for each treatment. 5500 plants were planted per mu.

[0057] Treatment 1: Control area [(NH4)2HPO4] 20 kg / mu; Treatment 2: Demonstration area [(NH4)2HPO4] 10kg / mu, compound microbial inoculant 10kg / mu (effective viable count 8.4×10⁻⁶) 8 CFU / gram).

[0058] Both the control and demonstration areas were fertilized at the jointing and tasseling stages, respectively. The control area was fertilized twice with 20 kg of (NH4)2HPO4 each time; the demonstration area was fertilized twice, each time with 10 kg / mu of (NH4)2HPO4 + 10 kg / mu of compound microbial inoculant from Example 1. Irrigation was carried out three times during the growing season, at the jointing, heading, and grain-filling stages.

[0059] During the corn harvest season, the two treatment areas were harvested separately, and a five-point sampling method was used, with each point covering an area of ​​50m². 2 Plant height and biomass were investigated, and the results are shown in Table 10.

[0060] Table 10 Changes in maize plant size and yield under different bacterial strain treatments.

[0061] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A compound microbial agent, characterized in that, It is caused by Bacillus belyssus ( Bacillus velezensis RKN1111, Bacillus subtilis ( Bacillus subtilis N-3 and gelatinous Bacillus ( Paenibacillus mucilaginosus The composition is HX-02; the preservation number of Bacillus belyssus RKN1111 is CGMCC No.29300; the preservation number of Bacillus subtilis N-3 is CGMCC No.34633; and the preservation number of gelatinous Bacillus HX-02 is CGMCC No.30949.

2. The compound microbial agent according to claim 1, characterized in that, The total viable count of the compound microbial agent is not less than 8.4 × 10⁻⁶. 8 CFU / gram.

3. The compound microbial agent according to claim 2, characterized in that, The viable count of the Bacillus subtilis N-3 is not less than 2.8 × 10⁻⁶. 8 CFU / gram.

4. The method for preparing the composite microbial agent according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Take the refrigerated Bacillus belyss RKN1111, Bacillus subtilis N-3 and Bacillus spp. HX-02 and inoculate them separately into sterilized LB liquid medium. After seed culture and scale-up fermentation, the fermentation products of each strain are centrifuged under preset conditions to remove the cells and collect the supernatant to obtain Bacillus belyss RKN1111 fermentation broth, Bacillus subtilis N-3 fermentation broth and Bacillus spp. HX-02 fermentation broth respectively. (2) The fermentation broth of Bacillus subtilis RKN1111, the fermentation broth of Bacillus jelly-like N-3, and the fermentation broth of Bacillus belyss HX-02 are mixed in a volume ratio of 1:1:1 to obtain the compound microbial agent.

5. The application of the composite microbial agent according to claim 1 in improving the physical and chemical properties of coastal saline-alkali soil.

6. The application according to claim 5, characterized in that, The improvement of the physical and chemical properties of coastal saline-alkali soil includes reducing soil pH and EC values, and increasing the content of available nitrogen, available phosphorus, and available potassium in the soil.

7. The application of the compound microbial agent according to claim 1 in promoting the growth of plants in coastal saline-alkali land.

8. The application of the compound microbial agent according to claim 1 in the preparation of bio-organic fertilizer or plant growth promoter.

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