Multifunctional microbial agent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a multifunctional microbial agent as well as a preparation method and application thereof, and the technical key point is that the microbial agent comprises bacillus subtilis MZS1 (B.sub MZS1), rhodococcus PG42 (Rhodococcus sp.PG42) and an auxiliary synergist. The auxiliary synergist is an amino acid compound, and the amino acid compound contains 9% of glutamic acid, 3.5% of proline, 6.5% of glycine, 7% of alanine, 7% of valine, 0.5% of methionine, 0.5% of isoleucine, 9% of leucine, 2% of tyrosine, 5% of phenylalanine, 8% of lysine, 6% of histidine and 3% of arginine. According to the preparation method of the multifunctional microbial agent and the using technology of the multifunctional microbial agent in the alpine degraded grassland, the problems that in the prior art, the using cost is high, the using effect is unknown, and the ecological restoration development requirement of the alpine environment cannot be met are solved.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a multifunctional microbial agent and a preparation method and application thereof. Background Art

[0002] Alpine grasslands are not only a vital source of water for rivers, lakes, and oceans, but also a crucial base for the development of plateau animal husbandry. They significantly impact the maintenance of ecosystem functions, including water conservation in the upper reaches of rivers, climate regulation, soil and water conservation, sandstorm control, and biodiversity protection. They play a crucial role in ensuring regional ecological security and responding to global climate change. In recent years, grassland quality has been steadily deteriorating due to factors such as climate warming and human intervention. This degradation has led to a sharp decline in soil fertility, available nutrients, soil aggregates, soil water holding capacity, net nitrification rate, net ammonification rate, and soil nitrogen pools, and an increase in soil compaction and bare areas. This further restricts plant growth, resulting in sparse and short plant populations and a significant shift in the succession of dominant vegetation species. This has led to a significant decline in the structure and function of plant communities, as well as vegetation productivity, diversity, height, cover, and quality. Forage yield and quality have steadily declined, edible forage has decreased, and toxic and noxious weeds have proliferated. This coordinated degradation of soil and vegetation has trapped alpine grasslands in a vicious cycle. Coupled with the characteristics of high altitude, coldness and drought in high-altitude areas, the high-altitude ecosystem is extremely fragile and can easily continue to degrade.

[0003] Currently, research on the ecological restoration of degraded alpine grasslands has yielded a number of research results and theoretical technologies. For example, based on the diagnosis and classification of the current status of degraded alpine grasslands and research on simple and practical technologies for the management of degraded alpine grasslands, a classification and grading management technology for alpine grasslands has been developed. Depending on the degree of degradation, measures such as fencing, sand barrier nets, overseeding, fertilization, artificial grassland establishment, planting and irrigation, and rodent control are used. However, these methods all have significant drawbacks: fencing is time-consuming and has slow and limited effectiveness; while artificial grassland establishment can improve soil conditions to a certain extent, it is limited by the significant soil disturbance caused by plowing; the currently widely promoted grassland improvement technology involves overseeding grass seeds followed by fertilization. However, this method, which transfers fertilizers used in low-altitude farmland to high-altitude, cold climate alpine regions, is poorly targeted and ineffective. Furthermore, the selection of vegetation varies greatly depending on the grassland degradation level, stage of degradation, and soil properties, making it a very labor-intensive and time-consuming task.

[0004] Microbial restoration technology not only has significant advantages such as low investment, good effect, and little impact on the environment, but also because microorganisms, as the link between plants and soil nutrients, have outstanding advantages in the ecological restoration of degraded grasslands. After searching, the national invention patent with application number 202110069822.5 in the prior art discloses a life-promoting Paenibacillus bacterium and its preparation and application. The invention isolates a life-promoting Paenibacillus (Paenibacillus pabuli) B03 from the root of Stipa purpurogena, and applies the metabolites of the strain to forage grass to improve the biochemical fertility of grassland soil, improve the functional stability of grassland soil ecosystem, restore and improve grassland productivity, increase yield, improve quality, and thus promote the development of animal husbandry.

[0005] Furthermore, the national invention patent with application number 202211206946.4 discloses a microbial composite inoculant and preparation method for reseeding natural degraded grassland. The invention mainly uses the resource microorganisms Acinetobacter calcoaceticus HMAJ1 and Pseudarthrobacter sp. NCZ1 isolated from alpine meadows to be inoculated into LB liquid culture medium respectively. After culturing at 15 ° C and 180 rpm for 48 hours, the OD 600 values ​​of the two strains are adjusted to the same value with sterile water. The single strain culture solutions of Acinetobacter calcoaceticus and Pseudarthrobacter sp. NCZ1 are inoculated into new triangular flasks at a volume ratio of 3:7 to obtain a microbial composite inoculant for reseeding natural degraded grassland. The application method of the inoculant is: direct spraying, and the usage amount is 5.0kg-6.0kg / mu.

[0006] The above-mentioned existing patent, a bacterium of the genus Paenibacillus with pro-liferative properties and its preparation and application, mainly applies the metabolites of the strain to animal husbandry, thereby achieving the purpose of improving grassland soil ecology. However, the process of inactivating microorganisms and preparing metabolites increases costs, and the active substance is used once, which is not conducive to the sustainability of the improvement effect.

[0007] The invention describes a microbial composite inoculant specifically for reseeding naturally degraded grasslands and its preparation method. The recommended usage method is direct spraying, with a dosage of 5.0-6.0 kg / mu. This conclusion is based on data from potted plant experiments conducted in an artificial climate chamber at the College of Grassland Science, Gansu Agricultural University (Ningbo Jiangnan Instrument Factory, Ningbo, China), under a 25°C light (16-hour) and 20°C dark (8-hour) cycle (light intensity 350 μE m-2 s-1), and 70-80% humidity. This is an indoor simulation and is not suitable for remediating degraded grasslands in alpine environments.

[0008] Microbial inoculants or microbial fertilizers are widely used in farmland or low-altitude areas, mainly developed for single function, which not only leads to the disconnection between development and actual work, but also greatly limits the activity of microorganisms and the ability to improve soil due to the different environment of high-cold pastoral area from farmland. Some studies have screened resource microorganisms in high-cold environment, but are limited to low-altitude farmland environment; some studies have been applied to degraded grassland, but the use of indoor simulation of high-cold environment greatly limits the practical application of microorganisms in high-cold degraded grassland.

[0009] Therefore, it is of great significance to use high-cold environment in-situ resource microorganisms to prepare microbial inoculants for the repair of high-cold degraded grassland.

[0010] Therefore, the present application provides a multifunctional microbial inoculant and a preparation method and application thereof to solve the above problems. SUMMARY

[0011] The present application aims to solve the technical problems raised in the background art and provide a multifunctional microbial inoculant and a preparation method and application thereof.

[0012] The above-mentioned purpose of the present application is achieved in the following manner:

[0013] The present application provides a multifunctional microbial inoculant, which comprises Bacillus subtilis MZS1, Rhodococcus sp.PG42 and an auxiliary synergist. Bacillus subtilis MZS1 was preserved in the China Center for Type Culture Collection on May 9, 2025, with the address of Wuhan University, Wuhan, China, and the preservation number of CCTCC NO:M20251009. Rhodococcus sp.PG42 was preserved in the China Center for Type Culture Collection on May 9, 2025, with the address of Wuhan University, Wuhan, China, and the preservation number of CCTCC NO:M20251008.

[0014] Further, the auxiliary synergist is an amino acid compound, which contains glutamic acid 9%, proline 3.5%, glycine 6.5%, alanine 7%, valine 7%, methionine 0.5%, isoleucine 0.5%, leucine 9%, tyrosine 2%, phenylalanine 5%, lysine 8%, histidine 6% and arginine 3%.

[0015] The present application provides a preparation method of a multifunctional microbial inoculant, which comprises the following steps:

[0016] S1. Culture medium preparation: solid culture medium for bacterial activation: 10g peptone, 3g beef extract, 5g sodium chloride, 15g agar, pH 7.0, 1L distilled water; culture medium for bacterial seed: 10g peptone, 3g beef extract, 5g sodium chloride, pH 7.0, 1L distilled water; fermentation medium: 3‰ yeast powder, 3‰ peptone, 0.5‰ magnesium sulfate heptahydrate, 0.1‰ calcium chloride, pH 6.8-7.0. Sterilize at 118-121°C for 20 minutes.

[0017] S2. Use an inoculating loop to pick up the two bacteria stored on the slant solid culture medium and culture them on a strain activation solid culture medium at 28°C. When visible colonies grow, they are used as primary strains.

[0018] The activated primary strains were inoculated into the seed culture medium at a rate of 1%, and cultured on a shaker at 28°C and 180 rpm for 24 hours. The strains were then inoculated into FJ culture medium at a ratio of 7:3 of Bacillus subtilis MZS1 and Rhodococcus PG42, and cultured at room temperature under 3000 lx incandescent light at night for 5 days to obtain the basic microbial agent.

[0019] S3. Add 100 g of amino acid complex to every liter of basic microbial agent and then use it.

[0020] The solution of the present invention also provides an application of a multifunctional microbial agent or the above-mentioned preparation method in preparing a repair agent for repairing alpine degraded grassland.

[0021] The difficulty and significance of the technical problem solved by the present invention are:

[0022] Existing technologies that screen resource microorganisms for alpine environments are limited to use in low-altitude farmland environments. Although some have been applied to degraded grasslands, the use of indoor simulated alpine environments greatly limits their practical application in alpine degraded grasslands. This present invention, through research, directly uses and colonizes microorganisms, allowing their functional effects to be sustained. Furthermore, the experimental data from the direct application of microbial agents studied in this invention on alpine grasslands can overcome the errors caused by the inconsistency between natural environments (cold, drought, high altitude) and artificial climate chambers (25°C / 20°C), providing stronger practical support for their widespread application.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The multifunctional microbial agent of the present invention directly uses and colonizes microorganisms, which enables the effects of their functions to be continuously exerted;

[0025] 2. The microbial agent of the present invention has low dosage, good effect and reduces the cost of use;

[0026] 3. The microbial agent of the present invention is suitable for alpine grasslands, overcoming the errors caused by the inconsistency between the natural environment (cold, drought, high altitude) and the conditions of the artificial climate chamber (25°C / 20°C). DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] The implementation of the present invention is described in detail below with reference to specific embodiments.

[0029] The following are preferred embodiments of the present invention.

[0030] Example: This example provides a multifunctional microbial agent and applies it to alpine degraded grasslands, solving the problems of the existing technology, such as high cost, unclear effect, and inability to meet the needs of alpine environmental ecological restoration and development.

[0031] 1. A multifunctional microbial agent of the present invention

[0032] The bacterial agent comprises Bacillus subtilis MZS1 and Rhodococcus sp. PG42, and also comprises an auxiliary synergist; wherein, Bacillus subtilis MZS1 (Bacillus subtilis MZS1) is deposited in the China Center for Type Culture Collection, the deposit address is: Wuhan University, Wuhan, China; the deposit number is: CCTCC NO: M20251009; Rhodococcus sp. PG42 (Rhodococcus sp. PG42) is deposited in the China Center for Type Culture Collection, the deposit address is: Wuhan University, Wuhan, China; the deposit number is: CCTCC NO: M20251008.

[0033] Solid culture medium for bacterial activation: 10g peptone, 3g beef extract, 5g sodium chloride, 15g agar, pH 7.0, 1L distilled water. Seed culture medium: 10g peptone, 3g beef extract, 5g sodium chloride, pH 7.0, 1L distilled water. Fermentation medium (FJ medium): 3‰ yeast powder, 3‰ peptone, 0.5‰ magnesium sulfate heptahydrate, 0.1‰ calcium chloride, pH 6.8-7.0. Sterilize at 118-121°C for 20 min.

[0034] The two bacteria stored on the slant solid culture medium were dipped with an inoculating loop and cultured on a strain activation solid culture medium at a culture temperature of 28°C. After colonies visible to the naked eye grew, they were used as primary strains; the activated primary strains were inoculated into the seed culture medium at an inoculum rate of 1%, and cultured on a shaker at 28°C and a speed of 180 rpm for 24 hours. They were then inoculated into FJ culture medium at a ratio of 7:3 of Bacillus subtilis MZS1:Rhodococcus PG42, and cultured at room temperature with 3000 lx incandescent lamp illumination at night. The basic microbial agent was obtained after about 5 days of culture.

[0035] The auxiliary agent and synergist is amino acid. 100g of amino acid complex is added to each liter of basic microbial inoculant. The amino acid complex contains 9% glutamic acid, 3.5% proline, 6.5% glycine, 7% alanine, 7% valine, 0.5% methionine, 0.5% isoleucine, 9% leucine, 2% tyrosine, 5% phenylalanine, 8% lysine, 6% histidine, and 3% arginine.

[0036] 2. Application

[0037] The sampling site was located in the alpine degraded grassland of Jiajiongke Village, Maixi Township, Zoige County, Aba Prefecture, Sichuan Province (E102°56′, N33°39′, approximately 3469 m above sea level). The area is influenced by a tropical and marine monsoon climate, with an average annual temperature of 1.1°C, a maximum of 11.2°C, and a minimum of -9.3°C. The average annual precipitation is approximately 753 mm, with a rainy season from June to September. A 1000 m × 1000 m monitoring site was established within the degraded grassland, with six 30 m × 30 m sampling plots spaced at least 50 m apart.

[0038] Usage: During the grass growth period in May, June, and July, three plots were sprayed with a dilution of 1 L of microbial inoculant plus 100 g of amino acids per mu (3 plots were sprayed with the same amount of water as a control). At the end of August, when the grass was mature, the grass height, cover, number of species, above- and below-ground biomass, soil physical and chemical characteristics (pH, moisture content, organic carbon, total nitrogen, total phosphorus), and enzyme activities (urease, sucrase, catalase) were measured.

[0039] The results are shown in Table 1 below.

[0040] Table 1

[0041]

[0042] As can be seen from the results in Table 1 above, the application of the multifunctional microbial agent of the present invention can fix and convert nitrogen in the air in an in-situ high-altitude and cold environment, activate insoluble phosphorus in the degraded grassland soil, provide nutrients for vegetation, and effectively promote the above- and underground biomass of the grassland by improving the soil pH, increase soil enzyme activity, and achieve the restoration of degraded grassland in a high-altitude and cold environment.

[0043] Low-temperature-resistant functional microorganisms meet the needs of soil restoration with "high organic residues" in alpine grasslands. In addition to acting as fertilizers themselves, bacterial agents are also functionally based on improving soil nutrients, especially solving the problem of high organic matter and low nutrients in alpine pastoral areas, and coordinating and balancing the plant's nitrogen absorption capacity and absorption form preferences, and grasping its regulation and influence on various key biochemical processes in the material cycle.

[0044] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A multifunctional microbial agent, characterized in that: The bacterial agent comprises Bacillus subtilis MZS1, Rhodococcus sp. PG42 and an auxiliary synergist; wherein, Bacillus subtilis MZS1 is deposited in the China Center for Type Culture Collection, the deposit address is: Wuhan University, Wuhan, China; the deposit number is: CCTCC NO: M20251009; Rhodococcus sp. PG42 is deposited in the China Center for Type Culture Collection, the deposit address is: Wuhan University, Wuhan, China; the deposit number is: CCTCC NO: M20251008.

2. A multifunctional microbial agent according to claim 1, characterized in that The auxiliary synergist is an amino acid complex, which contains 9% glutamic acid, 3.5% proline, 6.5% glycine, 7% alanine, 7% valine, 0.5% methionine, 0.5% isoleucine, 9% leucine, 2% tyrosine, 5% phenylalanine, 8% lysine, 6% histidine and 3% arginine.

3. The method for preparing a multifunctional microbial agent according to claim 1, characterized in that: The following steps are involved: S1. Culture medium preparation: solid culture medium for bacterial activation: 10g peptone, 3g beef extract, 5g sodium chloride, 15g agar, pH 7.0, 1L distilled water; culture medium for bacterial seed: 10g peptone, 3g beef extract, 5g sodium chloride, pH 7.0, 1L distilled water; fermentation medium: 3‰ yeast powder, 3‰ peptone, 0.5‰ magnesium sulfate heptahydrate, 0.1‰ calcium chloride, pH 6.8-7.

0. Sterilize at 118-121°C for 20 minutes. S2. Use an inoculating loop to pick up the two bacteria stored on the slant solid culture medium and culture them on a strain activation solid culture medium at 28°C. When visible colonies grow, they are used as primary strains. The activated primary strains were inoculated into the seed culture medium at a rate of 1%, and cultured on a shaker at 28°C and 180 rpm for 24 hours. The strains were then inoculated into FJ culture medium at a ratio of 7:3 of Bacillus subtilis MZS1 and Rhodococcus PG42, and cultured at room temperature under 3000 lx incandescent light at night for 5 days to obtain the basic microbial agent. S3. Add 100 g of amino acid complex to every liter of basic microbial agent and then use it.

4. Use of a multifunctional microbial agent according to claims 1-2 or a preparation method according to claim 3 in preparing a repair agent for repairing alpine degraded grassland.

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