Complex microbial inoculant as well as preparation method and application thereof

Abstract

The invention relates to a complex microbial inoculant as well as a preparation method and application thereof, and belongs to the technical field of microorganisms. In the composite microbial inoculant, the ratio of the microbial inoculant of the Peribacillusfrigitorirans NMI04 to the microbial inoculant of the bacillus amyloliquefaciens QZB03 to the microbial inoculant of the bacillus subtilis NMC07 is (1 to 5) to (1 to 5) to (1 to 5); the number of effective viable bacteria in the complex microbial inoculant is 1 * 10 < 8 >-1 * 10 < 11 > CFU / g. The complex microbial inoculant disclosed by the invention has a remarkable capability of inhibiting pathogenic bacteria of crops, and has a good biological control effect. The growth vigor of crops can be obviously improved, and the growth-promoting capability is excellent. The salt resistance and the cold resistance of crops can be induced, so that the efficient growth of the crops is promoted, and the yield and the quality of the crops are greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a composite bacterial agent, a preparation method and an application thereof. Background Art

[0002] Bacillus is widely distributed in nature, boasting abundant populations, strong reproductive capacity, stable physical and chemical properties, and a broad antimicrobial spectrum, making it one of the most highly regarded and extensively studied microbial biocontrol agents. As the dominant microbial community in microecological environments, Bacillus possesses strong disease prevention, growth promotion, and stress resistance capabilities. Therefore, Bacillus and its biocontrol agents can be used to prevent and treat root, leaf, fruit, and flower diseases of various crops, promoting normal plant growth and development while also enhancing crop nutrient utilization and significantly improving plant resistance. As a safe and effective microbial resource, Bacillus plays an increasingly important role in modern agricultural production. With technological advancements and growing market demand, its application areas will continue to expand, making a greater contribution to achieving sustainable agricultural development goals.

[0003] Microbial agents utilize the life activities of microorganisms to increase nitrogen, available phosphorus, and potassium in the soil, convert soil substances that cannot be directly utilized by crops into nutrients that can be absorbed and utilized, stimulate crop production, or inhibit the activity of plant pathogens, thereby increasing soil fertility, improving crop nutritional conditions, and increasing crop yields. Microbial agents are typically used in a rational combination based on their functions and characteristics, encompassing multiple improvement functions and ensuring significant results. They can increase crop yields and significantly improve soil properties. In recent years, the compaction and decreased fertility of soil caused by the excessive use of chemical fertilizers have led to reduced crop returns, prompting a trend to discover new microbial agents to partially or gradually replace chemical products.

[0004] Currently, the research and application of microbial agents has evolved from single-strain agents to multi-strain complexes. The shift from a single bacterial strain with a single function to a multifunctional complex microbial consortium is an inevitable trend in the long-term development of microbial agents. Complex microbial agents, comprising one or more beneficial microorganisms and a microbial carrier, contain a certain number of live bacteria. They can rapidly replenish beneficial bacteria in the soil, inhibit harmful bacteria, regulate soil pH, improve soil fertility, enhance plant resistance to pests and diseases, and promote plant growth, effectively overcoming the shortcomings of single-strain agents. In the long term, compared to single strains, complex agents can ensure long-term and significant effects, independent of changes in the external and soil environments. With growing environmental awareness, people are increasingly concerned about the environmental pollution caused by the excessive use of chemicals in agricultural production. Complex microbial agents, as low-carbon, natural, pollution-free, and non-toxic products, can improve the soil ecological environment for crop growth and enhance soil fertility through the metabolic activities of beneficial microorganisms. They can also stimulate and induce crop resistance by secreting trace amounts of active substances, thereby promoting efficient crop growth and significantly increasing crop yield and quality. Composite microbial inoculants are a key tool in modern agriculture and ecological development. They not only help improve crop productivity but also provide new ideas and technical means for addressing global environmental issues. In the current context of developing green agriculture, ecological agriculture, and sustainable agriculture, composite microbial inoculants have broad application prospects. Summary of the Invention

[0005] In view of the problem that the existing single bacterial agent is not effective in resisting plant diseases and low temperature, the present invention provides a composite bacterial agent and its preparation method and application to solve the above problems. Peribacillus frigoritolerans , Bacillus amyloliquefaciens and Bacillus subtilis are compounded to obtain a composite bacterial agent, which improves the plant's disease resistance and low temperature resistance while also improving the plant's salt tolerance.

[0006] The technical solutions of the present invention are as follows: In a first aspect, the present invention provides a composite bacterial agent comprising Peribacillus frigoritolerans NMI04, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )QZB03 and Bacillus subtilis ( Bacillus subtilis ) NMC07. Peribacillus frigoritoleransNMI04 is deposited in the General Microorganism Center of China Culture Collection of Microorganisms with the deposit number CGMCC No 31153, the deposit date is July 3, 2024, and the address of the deposit institution is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Bacillus amyloliquefaciens QZB03 is deposited in the General Microorganism Center of China Culture Collection of Microorganisms with the deposit number CGMCC No 31155, the deposit date is July 3, 2024, and the address of the deposit institution is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Bacillus subtilis NMC07 is deposited in the General Microorganism Center of China Culture Collection of Microorganisms with the deposit number CGMCC No31151, the deposit date is July 3, 2024, and the address of the deposit institution is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] Furthermore, in the composite bacterial agent, by weight, Peribacillus frigoritolerans Solid inoculum of NMI04: solid inoculum of Bacillus amyloliquefaciens QZB03: solid inoculum of Bacillus subtilis NMC07 = (1-5): (1-5): (1-5); the effective viable bacteria count in the composite inoculum is 1×10 8 ~1×10 11 CFU / g.

[0008] In a second aspect, the present invention provides a method for preparing the composite bacterial agent, which is as follows: will contain Peribacillus frigoritolerans The solid inoculum containing NMI04, the solid inoculum containing Bacillus amyloliquefaciens QZB03 and the solid inoculum containing Bacillus subtilis NMC07 were mixed evenly to obtain a composite inoculum.

[0009] Furthermore, it contains Peribacillus frigoritolerans The preparation method of solid inoculant of NMI04 is as follows: Will Peribacillus frigoritolerans NMI04 was activated on NA plate culture medium, and a ring of activated Peribacillus frigoritolerans NMI04 was inoculated into LB medium and cultured at 35°C and 180 rpm for 8-24 h. Peribacillus frigoritolerans NMI04 seed liquid; inoculate the seed liquid into the fermentation medium, continue to ferment for 24 to 48 hours under the conditions of inoculation amount 6%, 35℃, 300rpm, and ventilation volume 1:1.2, until the spore rate is above 90% and the fermentation is completed to obtain a fermentation liquid; then add 5% to 10% diatomaceous earth by volume to the fermentation liquid, spray dry it at 120℃, and obtain a fermentation liquid containing Peribacillus frigoritolerans NMI04 solid inoculant.

[0010] Furthermore, the preparation method of the solid bacterial agent containing Bacillus amyloliquefaciens QZB03 is as follows: Bacillus amyloliquefaciens QZB03 was activated on NA plate culture medium, and a loop of the activated Bacillus amyloliquefaciens QZB03 was inoculated into LB culture medium and cultured at 35°C and 180 rpm for 8-24 hours to obtain a seed liquid of Bacillus amyloliquefaciens QZB03; the seed liquid was inoculated into a fermentation medium, and fermentation was continued for 24-48 hours under the conditions of inoculum amount of 6%, 35°C, 300 rpm, and ventilation volume of 1:1.2, until the spore rate exceeded 90%, thereby completing the fermentation and obtaining a fermentation liquid; then, 5%-10% diatomaceous earth was added to the fermentation liquid by volume, and the liquid was spray-dried at 120°C to obtain a solid inoculum containing Bacillus amyloliquefaciens QZB03.

[0011] Furthermore, the preparation method of the solid inoculum containing Bacillus subtilis NMC07 is as follows: Bacillus subtilis NMC07 was activated on a NA plate culture medium, and a loop of the activated Bacillus subtilis NMC07 was inoculated into an LB culture medium and cultured at 35°C and 180 rpm for 8 to 24 hours to obtain a Bacillus subtilis NMC07 seed liquid; the seed liquid was inoculated into a fermentation medium, and fermentation was continued for 24 to 48 hours under the conditions of an inoculum amount of 6%, 35°C, 300 rpm, and an aeration volume of 1:1.2, until the spore rate exceeded 90%, thereby completing the fermentation and obtaining a fermentation liquid; then, 5% to 10% diatomaceous earth was added to the fermentation liquid by volume, and the liquid was spray-dried at 120°C to obtain a solid inoculum containing Bacillus subtilis NMC07.

[0012] Furthermore, the LB medium components are as follows: peptone 10 g / L, NaCl 5 g / L, yeast extract 10 g / L, sterile water, pH=7.2.

[0013] In a third aspect, the present invention provides a use of the above-mentioned composite bacterial agent in preventing and treating cucumber wilt.

[0014] In a fourth aspect, the present invention provides an application of the above-mentioned composite bacterial agent in promoting the growth of Chinese cabbage.

[0015] In a fifth aspect, the present invention provides an application of the above-mentioned composite bacterial agent in improving the tolerance of crops to soil salinity and alkali.

[0016] In a sixth aspect, the present invention provides an application of the above-mentioned composite bacterial agent in improving the low temperature resistance of crops.

[0017] The beneficial effects of the present invention are: The composite microbial agent of the present invention is a composite agent composed of three beneficial microorganisms, which can effectively overcome the shortcomings of the use of a single agent. Compared with a single strain, the composite agent can ensure long-term and significant effects, and is not easily affected by changes in the external environment and soil environment. The composite agent of the present invention has a significant ability to inhibit crop pathogens and has a good biological control effect. It can significantly improve crop growth and has excellent growth-promoting ability. It can also induce salt tolerance and cold tolerance in crops, thereby promoting efficient crop growth and significantly improving crop yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0019] Figure 1 In Example 1 of the present invention Peribacillus frigoritolerans Growth diagram of NMI04 in NA medium.

[0020] Figure 2 In Example 1 of the present invention Peribacillus frigoritolerans Gram staining and spore staining of NMI04; A is the Gram staining image, and B is the spore staining image.

[0021] Figure 3 This is a graph showing the growth of Bacillus amyloliquefaciens QZB03 in NA medium in Example 1 of the present invention.

[0022] Figure 4 These are the Gram staining and spore staining images of Bacillus amyloliquefaciens QZB03 in Example 1 of the present invention; wherein A is the Gram staining image and B is the spore staining image.

[0023] Figure 5 This is a graph showing the growth of Bacillus subtilis NMC07 in NA medium in Example 1 of the present invention.

[0024] Figure 6 These are the Gram staining and spore staining images of Bacillus subtilis NMC07 in Example 1 of the present invention; wherein A is the Gram staining image and B is the spore staining image.

[0025] Figure 7 This is a diagram showing the biocontrol capability verification results in Example 3.

[0026] Figure 8 This is a graph showing the growth-promoting ability verification results in Example 4.

[0027] Figure 9This is a graph showing the results of verifying the salt tolerance of crops in Example 5 (0.2% salinized soil).

[0028] Figure 10 This is a diagram showing the verification results of improving the salt tolerance of crops in Example 5 (0.4% salinized soil).

[0029] Figure 11 This is a diagram showing the verification results of improving the low temperature tolerance of crops in Example 6. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0031] Example 1 1. Peribacillus frigoritolerans Isolation and identification of NMI04 (1) Sampling: On April 10, 2024, soil was collected from a tomato greenhouse in Hohhot, Inner Mongolia Autonomous Region (111.8548°E, 40.8297°N) and placed in a ziplock bag for future use.

[0032] (2) Separation: 5 g of rhizosphere soil collected in step (1) was added to a conical flask containing 45 mL of sterilized pure water, and placed in a water bath at 100°C for 10 minutes. After standing, the upper layer of solution was diluted to samples of different concentration gradients, and spread on nutrient agar (NA) plate culture medium using a spreader, and inverted cultured at 35°C for 24 hours.

[0033] (3) Purification: Select single colonies with different morphologies, and purify the strains of each single colony by streaking on NA plates three times. Screen the strains according to the different colony morphologies and determine the numbers. Store them in test tube slant and store them in a refrigerator at 4°C. Use them as test bacteria for later tests. At the same time, store them in a 50% sterilized glycerol tube at -20°C for future use.

[0034] (4) Molecular identification A single colony of strain NMI04 was streaked onto a NA plate and incubated at 35°C for 12 hours. The incubated NA plate was then sent to Sangon Biotech (Shanghai) Co., Ltd. for strain identification. The 16S rDNA sequence is shown in SEQ ID NO. 1.

[0035] The sequence obtained was submitted to Gen-Bank for homology comparison, and the strain was preliminarily determined to be Peribacillus frigoritolerans . Peribacillus frigoritolerans NMI04 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No.31153 and the deposit date July 3, 2024. The address of the deposit institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0036] 2. Isolation and identification of Bacillus amyloliquefaciens QZB03 (1) Sampling: On April 10, 2024, soil was collected from a ginger field in Qingzhou City, Shandong Province (118.5935° east longitude, 36.8055° north latitude) and placed in a ziplock bag for future use.

[0037] (2) Separation 5 g of rhizosphere soil collected in step (1) was added to a conical flask containing 45 mL of sterilized pure water, and placed in a water bath at 100°C for 10 minutes. After standing, the upper layer of solution was diluted to samples of different concentration gradients, and spread on nutrient agar (NA) plate culture medium using a spreader, and inverted cultured at 35°C for 24 hours.

[0038] (3) Purification Single colonies with different morphologies were selected, and the strains of each single colony were streaked and purified three times by NA plate. The strains were screened and numbered according to the different colony morphologies, and stored in test tube slants and stored in a 4°C refrigerator. They were used as test bacteria for later experiments and placed in 50% sterile glycerol tubes at -20°C for storage until use.

[0039] (4) Molecular identification A single colony of strain QZB03 was streaked onto a NA plate and incubated at 35°C for 12 hours. The incubated NA plate was then sent to Sangon Biotech (Shanghai) Co., Ltd. for strain identification. The 16S rDNA sequence is shown in SEQ ID NO. 2.

[0040] The sequence was submitted to GenBank for homology comparison, and the strain was preliminarily identified as Bacillus amyloliquefaciens. Bacillus amyloliquefaciens QZB03 was deposited with the General Microbiology Center of the China Culture Collection Administration under the deposit number CGMCC No. 31155, dated July 3, 2024. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0041] 3. Isolation and identification of Bacillus subtilis NMC07 (1) Sampling: On April 10, 2024, soil was collected from a lettuce field in Hohhot City, Inner Mongolia Autonomous Region (111.7856°E, 40.5891°N) and placed in a ziplock bag for future use.

[0042] (2) Separation: 5 g of rhizosphere soil collected in step (1) was added to a conical flask containing 45 mL of sterilized pure water, and placed in a water bath at 100°C for 10 minutes. After standing, the upper layer of solution was diluted to samples of different concentration gradients, and spread on nutrient agar (NA) plate culture medium using a spreader, and inverted cultured at 35°C for 24 hours.

[0043] (3) Purification: Select single colonies with different morphologies, and purify the strains of each single colony by streaking on NA plates three times. Screen the strains according to the different colony morphologies and determine the numbers. Store them in test tube slant and store them in a refrigerator at 4°C. Use them as test bacteria for later tests. At the same time, store them in a 50% sterilized glycerol tube at -20°C for future use.

[0044] (4) Molecular identification A single colony of strain NMC07 was streaked onto a NA plate and incubated at 35°C for 12 hours. The incubated NA plate was then sent to Sangon Biotech (Shanghai) Co., Ltd. for strain identification. The 16S rDNA sequence is shown in SEQ ID NO. 3.

[0045] The sequence was submitted to GenBank for homology comparison, and the strain was preliminarily identified as Bacillus subtilis. Bacillus subtilis NMC07 was deposited with the General Microbiology Center of the China Culture Collection of Microorganisms under the deposit number CGMCC No. 31151, dated July 3, 2024. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0046] Example 2 The preparation method of the composite bacterial agent comprises the following steps: (1) Peribacillus frigoritolerans NMI04 was activated on NA plate culture medium, and a ring of activated Peribacillus frigoritolerans NMI04 was inoculated into LB medium, wherein the LB medium composition is as follows: peptone 10g / L, NaCl 5g / L, yeast extract 10g / L, sterile water, pH = 7.2; cultured at 35°C, 180 rpm for 8-24h, and obtained Peribacillus frigoritoleransNMI04 seed liquid; the seed liquid was inoculated into the fermentation medium, and the fermentation was continued for 24 to 48 hours under the conditions of inoculation amount 6%, 35°C, 300rpm, and ventilation volume 1:1.2, until the spore rate was more than 90% and the fermentation was completed to obtain a fermentation liquid; then 5% to 10% diatomaceous earth was added to the fermentation liquid by volume ratio, and spray-dried at 120°C to obtain a fermentation liquid containing Peribacillus frigoritolerans NMI04 solid inoculant.

[0047] (2) Bacillus amyloliquefaciens QZB03 was activated on NA plate culture medium, and a loop of the activated Bacillus amyloliquefaciens QZB03 was inoculated into LB culture medium, wherein the LB culture medium composition is as follows: peptone 10 g / L, NaCl 5 g / L, yeast extract 10 g / L, sterile water, pH = 7.2; cultured at 35 ° C, 180 rpm for 8 to 24 h to obtain Bacillus amyloliquefaciens QZB03 seed liquid; the seed liquid was inoculated into the fermentation medium, and fermented for 24 to 48 h under the conditions of inoculation amount 6%, 35 ° C, 300 rpm, and ventilation volume 1:1.2, until the spore rate exceeded 90% and the fermentation was completed to obtain a fermentation liquid; then, 5% to 10% diatomaceous earth was added to the fermentation liquid by volume, and spray-dried at 120 ° C to obtain a solid bacterial agent containing Bacillus amyloliquefaciens QZB03.

[0048] (3) Activate Bacillus subtilis NMC07 on NA plate medium, take a loop of activated Bacillus subtilis NMC07 and inoculate it into LB medium, where the LB medium composition is as follows: peptone 10 g / L, NaCl 5 g / L, yeast extract 10 g / L, sterile water, pH = 7.2; culture at 35 ° C, 180 rpm for 8-24 h to obtain Bacillus subtilis NMC07 seed liquid; inoculate the seed liquid into the fermentation medium, and continue to ferment for 24-48 h under the conditions of inoculation amount 6%, 35 ° C, 300 rpm, and ventilation volume 1:1.2, until the spore rate is more than 90% and the fermentation is completed to obtain a fermentation liquid; then, add 5%-10% diatomaceous earth to the fermentation liquid by volume, and spray dry it at 120 ° C to obtain a solid inoculum containing Bacillus subtilis NMC07.

[0049] (4) Contain Peribacillus frigoritolerans The solid inoculum of NMI04, the solid inoculum containing Bacillus amyloliquefaciens QZB03, and the solid inoculum containing Bacillus subtilis NMC07 were mixed according to the following mass ratio, and the mixing ratio is shown in Table 1 below.

[0050] Table 1-Mixture of compound microbial agents

[0051] Example 3 Verification of biocontrol capability of composite microbial agents Taking cucumber wilt as the control target, the biocontrol test was carried out using the composite microbial agent provided by the present invention. The experimental period was from September 2024 to November 2024. Five parallel treatments were set up, namely QZB03 group, NMI04 group, NMC07 group, composite microbial agent group and blank group, with 10 replicates in each group. The specific application method was as follows: Peribacillus frigoritolerans The number of viable bacteria in the solid inoculum of NMI04, the solid inoculum containing Bacillus amyloliquefaciens QZB03, the solid inoculum containing Bacillus subtilis NMC07 and the composite inoculum A were all 1×10 8 Take 100 mL of the diluted solution with a CFU / mL and irrigate the roots of 4-week-old cucumber seedlings (the blank group was irrigated with water). After 24 hours of pretreatment, inoculate the cucumber wilt pathogen (Fusarium oxysporum cucumber-specific type) with a turbidity value of 1.0 MCF by root irrigation method, continuously record the disease index and observe the phenotype.

[0052] Disease level: Level 0, no symptoms; Level 1, cotyledons yellowing but not wilting; Level 2, cotyledons wilting; Level 3, cotyledons and true leaves wilting or plants stunted; Level 4, death.

[0053] Disease index = ∑(number of diseased plants at each level × representative value of each level) / (total number of plants surveyed × highest representative value) × 100; Control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100.

[0054] The experimental results are shown in Table 2 and Figure 7 shown.

[0055] Table 2 - Biocontrol capability verification results

[0056] The conditions of the cucumber seedlings treated in each of the above groups are shown in the photos and table. Nearly all plants in the blank group showed symptoms of cucumber wilt, with yellowing and wilting of the lower or entire leaves, and some plants exhibiting stunting symptoms. The single-agent treatment groups (QZB03, NMI04, and NMC07) also exhibited varying degrees of wilt symptoms. In contrast, more than half of the plants in the combined-agent group showed no symptoms and grew well, while the remaining plants showed yellowing of older leaves but did not wilt. This indicates that the combined-agent has a significant control effect on cucumber wilt.

[0057] Example 4 Verification of the growth-promoting ability of composite microbial agents Green stalk cabbage was used as the test object, and a growth promotion experiment was carried out using a composite microbial agent. The experimental time was 2024.09~2024.11. Five parallel treatments were set up, namely QZB03 group, NMI04 group, NMC07 group, composite microbial agent group and blank group, and each group was treated with 10 repetitions. 100mL of the corresponding microbial agent dilution solution (preparation method is the same as Example 3) was taken from the QZB03 group, NMI04 group, NMC07 group and composite microbial agent group for root irrigation treatment. 100mL of clean water was applied to the blank group. Green stalk cabbage was transplanted after the third true leaf unfolded. The first root irrigation treatment was carried out on the second day after transplanting, and the root irrigation treatment was carried out once every 7 days thereafter, for a total of 3 treatments. 7 days after the third root irrigation treatment, the plant height of the green stalk cabbage was measured with a ruler, the stem diameter was measured with a vernier caliper, and the fresh weight of the aboveground part was weighed with a balance. The results are shown in Table 3 and Figure 8 shown.

[0058] Table 3 - Results of growth promotion ability verification

[0059] From Table 3 and Figure 8 The results showed that the application of the composite bacterial agent increased plant height by 9.2%, stem diameter by 19.9%, and fresh weight by 19.1% compared with the blank group, and the growth-promoting effect was significantly better than that of the single bacterial agent treatment group. This shows that the composite bacterial agent has a good growth-promoting effect on green stalk cabbage.

[0060] Example 5 Verification of compound microbial agents to improve crop salt tolerance A growth promotion experiment using a composite microbial agent was conducted on pakchoi (Brachia chinensis) from September 2024 to November 2024. Five parallel treatments were set up: QZB03, NMI04, NMC07, a composite microbial agent, and a blank control, with 10 replicates per treatment.

[0061] Soil Pretreatment: Based on moderately salinized soil with a salt content of 0.2% to 0.4%, pretreat the soil. Mix two neutral salts, NaCl and Na₂SO₄, and two alkaline salts, NaHCO₃ and Na₂CO₃, in a ratio of 1:9:9:1. Weigh 0.2% and 0.4% of the soil weight of the mixed salts, respectively. Dissolve the salts and pour them into the soil, mixing thoroughly to create salinized soils with salt contents of 0.2% and 0.4%, respectively.

[0062] Experimental treatment: 100 mL of the corresponding dilution (prepared as in Example 3) was administered to the QZB03, NMI04, NMC07, and composite microbial agent groups for root irrigation. A blank control group was treated with 100 mL of water. The first root irrigation was performed the day after the third true leaf of the pakchoy was transplanted. Thereafter, root irrigation was performed every seven days for a total of three treatments. Seven days after the third root irrigation, the plant height of the pakchoy was measured with a ruler, the stem diameter was measured with a vernier caliper, and the aboveground fresh weight was weighed with a balance. The results are shown in Tables 4 and 5. Figures 9 - 10 shown.

[0063] Table 4 - Growth results of Chinese cabbage

[0064] The experimental results showed that the treatment group treated with the composite microbial agent in 0.2% salinized soil showed a 14.6% increase in plant height, a 20% increase in stem diameter, and a 26% increase in fresh weight compared to the control group. The treatment group treated with the composite microbial agent in 0.4% salinized soil showed a 5% increase in plant height, a 30% increase in stem diameter, and a 38.2% increase in fresh weight compared to the control group. Compared to the 0.2% salinized soil, the control group in 0.4% salinized soil showed an increase in plant height, but a significant decrease in stem diameter and fresh weight, indicating significantly weakened growth. The composite microbial agent treatment group showed no significant changes in plant height, stem diameter, or fresh weight. The single microbial agent treatments, NMI04 and NMC07, also improved the crop's salt stress tolerance to some extent, but the effect was not as significant as the composite microbial agent treatment. These results indicate that the composite microbial agent significantly improved the salt tolerance of Chinese cabbage.

[0065] Example 6 Verification of compound microbial agents to improve crop tolerance to low temperatures Wheat was used as the experimental object, and a growth promotion experiment was conducted using a composite microbial agent. The experimental period was from September 2024 to November 2024. Five parallel treatments were set up: QZB03 group, NMI04 group, NMC07 group, composite microbial agent group, and blank group, with 10 replicates in each treatment group. The treatment groups were 2000-fold dilutions of the four corresponding microbial agents (the viable count was 5×10 7 CFU / mL) as the nutrient solution for wheat hydroponics; the blank group used water as the nutrient solution. After germination, the seed tray was placed in a constant temperature box at 7°C for cultivation. After 15 days, the plant height, root length and single plant weight of the wheat were measured. The results are shown in Table 5 and Figure 11 shown.

[0066] Table 5-7℃ wheat growth results

[0067] The test results showed that at 7°C, the application of the composite bacterial agent did not significantly increase plant height compared to the blank control group, but root length increased by 29.9% and plant weight increased by 77.1%. This shows that the composite bacterial agent can significantly improve wheat's cold tolerance, enabling it to grow robustly in low temperatures.

[0068] Although the present invention has been described in detail with reference to the accompanying drawings and in conjunction with preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be easily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. A composite bacterial agent, characterized in that: include Peribacillus frigoritolerans NMI04, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )QZB03 and Bacillus subtilis ( Bacillus subtilis )NMC07; Peribacillus frigoritolerans NMI04 is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with a deposit number of CGMCC No 31153 and a deposit date of July 3, 2024. The address of the depository institution is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; Bacillus amyloliquefaciens QZB03 is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with a deposit number of CGMCC No 31155 and a deposit date of July 3, 2024. The address of the depository institution is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; Bacillus subtilis NMC07 is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with a deposit number of CGMCC No 31151 and a deposit date of July 3, 2024. The address of the depository institution is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; In the composite bacterial agent, by weight, Peribacillus frigoritolerans Solid bacterial agent of NMI04: solid bacterial agent of Bacillus amyloliquefaciens QZB03: solid bacterial agent of Bacillus subtilis NMC07 = (1-5): (1-5): (1-5); the number of effective live bacteria in the composite bacterial agent is 1×10 8 ~1×10 11 CFU / g.

2. A method for preparing the composite bacterial agent according to claim 1, characterized in that: The specific method is as follows: will contain Peribacillus frigoritolerans The solid bacterial agent containing NMI04, the solid bacterial agent containing Bacillus amyloliquefaciens QZB03 and the solid bacterial agent containing Bacillus subtilis NMC07 are uniformly mixed to obtain a composite bacterial agent.

3. The method according to claim 2, characterized in that contain Peribacillus frigoritolerans The preparation method of the solid inoculant of NMI04 is as follows: Will Peribacillus frigoritolerans NMI04 was activated on NA plate medium and a ring of activated Peribacillus frigoritolerans NMI04 was inoculated into LB medium and cultured at 35°C and 180 rpm for 8-24 h. Peribacillus frigoritolerans NMI04 seed liquid; inoculate the seed liquid into the fermentation medium, continue to ferment for 24-48 hours under the conditions of inoculation amount 6%, 35°C, 300rpm, and ventilation volume 1:1.2, until the spore rate is more than 90% and the fermentation is completed to obtain a fermentation liquid; then add 5%-10% diatomaceous earth by volume to the fermentation liquid, spray dry it at 120°C, and obtain a fermentation liquid containing Peribacillus frigoritolerans Solid inoculant of NMI04.

4. The method according to claim 2, characterized in that The preparation method of the solid bacterial agent containing Bacillus amyloliquefaciens QZB03 is as follows: Bacillus amyloliquefaciens QZB03 was activated on a NA plate culture medium, and a loop of the activated Bacillus amyloliquefaciens QZB03 was inoculated into an LB culture medium, and cultured at 35°C and 180rpm for 8-24h to obtain a seed solution of Bacillus amyloliquefaciens QZB03; the seed solution was inoculated into a fermentation culture medium, and fermentation was continued for 24-48h under the conditions of an inoculation amount of 6%, 35°C, 300rpm, and an aeration volume of 1:1.2, until the spore rate was more than 90% and the fermentation was completed to obtain a fermentation liquid; then, 5%-10% diatomaceous earth was added to the fermentation liquid by volume, and spray-dried at 120°C to obtain a solid bacterial agent containing Bacillus amyloliquefaciens QZB03.

5. The method according to claim 2, characterized in that The preparation method of the solid bacterial agent containing Bacillus subtilis NMC07 is as follows: Bacillus subtilis NMC07 was activated on a NA plate culture medium, and a loop of the activated Bacillus subtilis NMC07 was inoculated into an LB culture medium, and cultured at 35°C and 180rpm for 8-24h to obtain a seed solution of Bacillus subtilis NMC07; the seed solution was inoculated into a fermentation culture medium, and fermentation was continued for 24-48h under the conditions of an inoculation amount of 6%, 35°C, 300rpm, and an aeration volume of 1:1.2, until the spore rate was more than 90% and the fermentation was completed to obtain a fermentation liquid; then, 5%-10% diatomaceous earth was added to the fermentation liquid by volume, and spray-dried at 120°C to obtain a solid bacterial agent containing Bacillus subtilis NMC07.

6. The method according to any one of claims 3 to 5, characterized in that: The LB medium had the following components: 10 g / L peptone, 5 g / L NaCl, 10 g / L yeast extract, sterile water, pH=7.

2.

7. Use of the composite bacterial agent as claimed in claim 1 in preventing and controlling cucumber wilt.

8. Use of the composite bacterial agent as claimed in claim 1 in promoting the growth of Brassica juncea.

9. Use of the composite bacterial agent as claimed in claim 1 in improving the tolerance of crops to soil salinity and alkali.

10. Use of the composite bacterial agent as claimed in claim 1 in improving the low temperature resistance of crops.

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