Bacillus orientalis YG1 with growth promoting effect on leucaena leucocephala and lespedeza bicolor and application of bacillus orientalis YG1

Abstract

The invention discloses bacillus toonensis YG1 with a growth promoting effect on leucaena leucocephala and lespedeza bicolor, and belongs to the technical field of microorganisms. The bacillus toonensis YG1 has the growth promoting effect on leucaena leucocephala and lespedeza bicolor. The bacillus orientalis YG1 provided by the invention improves the conversion from total-effect nutrients to effective nutrients, and enhances the consumption of leucaena leucocephala and lespedeza bicolor on soil nutrients, thereby achieving the effect of promoting the growth of leucaena leucocephala and lespedeza bicolor. The bacillus orientalis YG1 is applied, the biomass and available phosphorus of lespedeza leucocephala are increased by 181.73% and 50.32% respectively most obviously, and the content of hydrolytic nitrogen and the content of organic matter of lespedeza are increased by 45.18% and 48.90% respectively most obviously. Other physicochemical indexes in leucaena leucocephala soil and lespedeza soil are obviously increased, and meanwhile, the soil enzyme activities of the leucaena leucocephala soil and the lespedeza soil are In general, the improvement of soil physicochemical properties and the significant increase of soil enzyme activity by inoculating the YG1 microbial inoculum may be an important factor for promoting plant growth.

Description

Technical Field

[0001] The invention relates to a Bacillus yonensis YG1 (Bacillus toyonensis) capable of promoting the growth of Leucaena leucaena and Lespedeza leucaena, belonging to the technical field of microorganisms. Background Art

[0002] With the accelerating pace of infrastructure construction such as mineral resource development, road network construction, water conservancy projects and industrial bases, the problem of exposed rock slopes has become increasingly serious, which has aggravated soil erosion, air pollution and biodiversity reduction in the local ecological environment. If environmental governance is not strengthened in time, these damages will have irreversible effects on the environment. The problem of exposed rock slopes is one of the environmental challenges that need to be solved urgently. Ecological environmental restoration is an important measure to protect the environment and maintain social stability, and it is a necessary condition for improving the ecological environment. At present, in order to improve the efficiency of restoration and consolidate the results of restoration, people inevitably need to use pesticides and fertilizers during plant growth. However, due to the long-term application of fertilizers, certain elements in the soil continue to accumulate, causing changes in its physical and chemical properties, thereby endangering food security. Therefore, efficient and greening restoration of exposed rock slopes is a major issue that urgently needs to be solved.

[0003] Microbial agents can degrade and inhibit the secretions of pathogens in the soil, effectively reduce the growth and spread of pathogens, provide a healthy growth environment for crops, and promote the proliferation of beneficial microorganisms in the soil. These beneficial microorganisms can compete with pathogens for nutrients and space, further inhibit the growth of pathogens, and thus achieve the effect of biological control; at the same time, the microorganisms in microbial agents can decompose organic matter in the soil, increase soil organic matter and porosity, break soil compaction, and promote the formation of aggregate structure, thereby improving soil structure and improving soil water retention and heat preservation capacity; in addition, some agents with phosphorus dissolving effect can increase the content of nutrients such as phosphorus in the soil that can be absorbed and utilized by plants. Therefore, in-depth exploration of microbial agents that have a positive role in promoting plant growth and development is of indispensable importance for enriching the research on microbial fertilizers.

[0004] Soil microorganisms are one of the most diverse and species-rich biological groups on land. They have a significant impact on ecosystems such as forests, grasslands, wetlands and farmlands, and play an important role in the entire biogeochemical cycle. The Qinghai-Tibet Plateau is one of the key areas for global biodiversity research due to its high altitude, strong radiation, low oxygen content, large temperature difference between day and night, and unique water and heat distribution pattern. Its strong topographic changes have formed a unique and diverse climate in the region, nurtured unique microbial resources, and promoted the research and development of microorganisms in extreme habitats.

[0005] Leucaena leucaena and Lespedeza leucaena are important candidate plants for ecological restoration of exposed slopes due to their strong tolerance and biological nitrogen fixation characteristics. However, the slopes have poor three-dimensional conditions, insufficient nitrogen, phosphorus and potassium in the soil, and it is difficult for plants to grow. Although the traditional method of applying chemical fertilizers can see results in the short term, it will damage the ecological environment in the long run, and excessive fertilization may also reduce the quality of forests. In contrast, PGPR microbial fertilizer, as a new type of environmentally friendly fertilizer in recent years, is pollution-free and quite popular.

[0006] Therefore, in-depth research and screening and configuration of PGPR microbial agents with significant growth-promoting effects on Leucaena leucaena and Lespedeza from the soil of the Qinghai-Tibet Plateau have important practical significance for promoting the development of pollution-free biological fertilizers, helping to give full play to the potential advantages of microbial fertilizers and thus promote the sustainable development of agricultural and forestry production. Summary of the invention

[0007] [Purpose of the invention]

[0008] The present invention aims to screen out a bacterial agent made from a strain that promotes the growth of Leucaena leucaena and Lespedeza from the Qinghai-Tibet Plateau, so as to provide a solution for improving the ecology of exposed slopes in my country.

[0009] [Technical solution]

[0010] In order to solve the above technical problems, the present invention discloses a method for promoting the growth of Leucaena leucaena and Lespedeza chinensis, using Bacillus toyonensis YG1 as bacterial fertilizer, and the preservation number is CCTCC NO:M 20241524.

[0011] The Bacillus toyonensis YG1 of the present invention is isolated from a soil sample in Baqing County, Nagqu City, Tibet Autonomous Region, and is stored in a -80 degree Celsius refrigerator in the Soil and Water Conservation Laboratory of Nanjing Forestry University, and is deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China. The deposit number is CCTCC NO: M20241524, and the deposit date is July 9, 2024.

[0012] The present invention also provides an application of Bacillus orientalis YG1 in promoting the production of Leucaena leucaena and Lespedeza leucaena plants. Bacillus orientalis YG1 is applied as a bacterial fertilizer, and the preservation number is CCTCC NO:M 20241524.

[0013] The present invention also provides an application of Bacillus orientalis YG1 in enhancing the utilization of soil nutrients by Leucaena leucaena and Lespedeza leucaena, and the Bacillus orientalis YG1 is applied as a bacterial fertilizer.

[0014] Furthermore, the microbial agent of the present invention is prepared according to the following steps and applied to Leucaena leucaena and Lespedeza potted plants:

[0015] 1) Pick a cell of Bacillus toyota YG1 from the well-preserved slant and inoculate it into LB agar solid medium, and activate it at 28°C for 48 hours.

[0016] 2) Use an inoculation loop to pick up a loop of bacterial slurry from the activated Bacillus subtilis YG1 strain and add it to LB liquid culture medium. Keep the culture medium at 28°C and 200 rpm for 24 hours to prepare the seed solution.

[0017] 3) Inoculate the seed solution at a 5% inoculum into LB liquid medium for amplification culture, and culture on a shaker at 28°C and 200 rpm for 2-3 days. Dilute with sterile water or continue fermentation to ensure that the bacterial solution OD 600 When the pH is 0.8-1.2, the fermentation liquid is obtained.

[0018] 4) Dilute the above fermentation liquid 100 times with sterile water and add it to the soil at the root of Lespedeza potted plants, with 100 mL of the diluted liquid per pot.

[0019] 5) The control was treated with an equal volume of sterile water.

[0020] Beneficial Effects

[0021] The present invention provides a microbial agent that can promote the growth of Leucaena leucaena and Lespedeza leucaena. The microbial agent is applied to Leucaena leucaena and Lespedeza leucaena plants to effectively increase the biomass of the underground part of Leucaena leucaena and promote the rooting and development of Leucaena leucaena, and the development prospect is promising. Specifically:

[0022] On the growth indicators of Leucaena leucaena and Lespedeza

[0023] After the seedlings of Leucaena leucaena and Lespedeza were inoculated with PGPR fungicide, compared with the CK group, the plant height increased by 53.07% and 20.84%, the ground diameter increased by 79.07% and 43.25%, the fresh weight of the aboveground part increased by 308.27% and 37.50%, the dry weight of the aboveground part increased by 237.35% and 44.33%, the fresh weight of the underground part increased by 3.25% and 133.33%, the dry weight of the underground part increased by 77.37% and 10.00%, and the total biomass increased by 181.73% and 29.94%, respectively; the root length, root surface area, average diameter, root volume, number of root tips and number of tillers of Leucaena leucaena increased by 121.37%, 70.69%, 49.02%, 151.16%, 168.52% and 191.11%, respectively compared with the CK treatment. The root length, root surface area, average diameter, root volume, root tip number and tiller number of Lespedeza bicolor increased by 67.90%, 61.76%, 5.88%, 58.33%, 89.51% and 88.68% respectively compared with the CK treatment.

[0024] In terms of rhizosphere soil physical and chemical properties

[0025] Compared with the CK group, the YG1 bacterial agent treatment group had an increase of 9.94% in soil hydrolyzed nitrogen, 50.32% in available phosphorus, 18.15% in available potassium, 49.01% in soil electrical conductivity, and 12.99% in organic matter. The Lespedeza chinensis soil hydrolyzed nitrogen increased by 45.18%, 43.57% in available phosphorus, 8.84% in available potassium, 15.87% in soil electrical conductivity, and 48.90% in organic matter.

[0026] It can be seen from the above technical scheme that the present invention discloses a microbial agent for promoting the growth of Leucaena leucaena and Lespedeza. The technical effect achieved is that the microbial agent provided by the present invention promotes the growth of Leucaena leucaena and Lespedeza plants after application, especially promotes the underground biomass and root system of Leucaena leucaena and Lespedeza plants, so that the plant's rooting and soil fixing ability is effectively exerted. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the present invention are briefly introduced below.

[0028] Figure 1 This is the evolutionary tree diagram of the Bacillus subtilis YG1 strain provided by the present invention.

[0029] Figure 2 This is a colony diagram of Bacillus subtilis YG1 (LB solid culture medium) provided by the present invention.

[0030] Figure 3 A schematic diagram of the phosphorus solubilization ability of the strains to be screened provided by the present invention for organic phosphorus and inorganic phosphorus, from left to right are Bacillus atrophaeus Q4, Bacillus megaterium Q7, and Bacillus toyotii YG1.

[0031] FIG4 is a schematic diagram of the phosphorus solubilization ability of the strains to be screened provided by the present invention for organic phosphorus and inorganic phosphorus at different temperatures, from left to right are Q4, Q7, and YG1.

[0032] Figure 5 This is a schematic diagram of the changes in plant height and ground diameter of Leucaena leucaena after being treated with the control group and the strain to be screened provided by the present invention.

[0033] Figure 6 This is a schematic diagram of the changes in plant height and ground diameter of Lespedeza after being treated with the control group and the strain to be screened provided by the present invention.

[0034] Figure 7 A schematic diagram of changes in biomass indicators of Leucaena leucaena after treatment with the control group and the strain to be screened provided by the present invention, from left to right are CK, Q4, Q7, and YG1.

[0035] Figure 8 This is a schematic diagram of changes in biomass indicators of Lespedeza bicolor after treatment with the control group and the strain to be screened provided by the present invention, from left to right are CK, Q4, Q7, and YG1.

[0036] Fig. 9 This is a schematic diagram of the changes in the content of hydrolyzed nitrogen, available phosphorus and organic matter of the strain Leucaena leucophylla to be screened provided by the present invention.

[0037] Fig.10 This is a schematic diagram of the changes in the content of hydrolyzed nitrogen, available phosphorus and organic matter of the strain Lespedeza to be screened provided by the present invention.

[0038] Fig.11 This is a schematic diagram of the effects of the control group and the strain to be screened provided by the present invention on the photosynthetic pigments of Leucaena leucaena plants, from left to right: CK, Q4, Q7, and YG1.

[0039] Fig.12 This is a schematic diagram of the effects of the control group and the strain to be screened on the photosynthetic pigments of Lespedeza plants provided by the present invention, which are CK, Q4, Q7, and YG1 from left to right.

[0040] Fig.13 Effects of the control group and the strain to be screened provided by the present invention on the enzyme activity of the soil of Leucaena leucaena potted plants, from left to right are CK, Q4, Q7, and YG1.

[0041] Fig.14 Effects of the control group and the strain to be screened provided by the present invention on soil enzyme activity in Lespedeza potted plants, from left to right: CK, Q4, Q7, YG1. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in 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 creative work are within the scope of protection of the present invention.

[0043] The raw materials and reagents involved in the examples are all obtained from commercial channels, and no requirements are made on their brands. All the methods not mentioned are commonly used experimental methods. Bacillus toyonensis YG1 was isolated and screened by this laboratory from the soil in the Qinghai-Tibet Plateau, and was preserved by the China Center for Type Culture Collection (CCTCC). The address of the preservation unit is Wuhan University, Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. The preservation number is CCTCC NO: M 20241524, and the preservation date is July 9, 2024.

[0044] Example 1

[0045] 1. Sample source

[0046] The strains were screened from soil samples from Baqing County, Nagqu City, Tibet Autonomous Region, China. After the samples were collected, they were placed in a 4°C incubator and brought back to the laboratory for storage in a -80°C refrigerator, and the soil microorganisms were isolated within 48 hours.

[0047] 2Separation and screening

[0048] (1) NA solid medium: peptone 10 g, beef extract powder 3 g, sodium chloride 5 g, agar 15 g, 1000 ml deionized water, pH 7.2-7.4.

[0049] (2) LB liquid medium: peptone 10 g, yeast extract powder 5 g, sodium chloride 5 g, deionized water 1000 mL, pH 7.2.

[0050] (3) Montana organophosphorus medium: glucose 10 g, ammonium sulfate 0.5 g, sodium chloride 0.3 g, potassium chloride 0.3 g, magnesium sulfate heptahydrate 0.3 g, ferrous sulfate heptahydrate 0.03 g, manganese sulfate tetrahydrate 0.03 g, calcium carbonate 5.0 g, lecithin 0.3 g, agar 20 g, pH 7.0-7.5, distilled water 1000 mL.

[0051] (4) Montana inorganic phosphate medium: glucose 10 g, ammonium sulfate 0.5 g, sodium chloride 0.3 g, potassium chloride 0.3 g, magnesium sulfate heptahydrate 0.3 g, ferrous sulfate heptahydrate 0.03 g, manganese sulfate tetrahydrate 0.03 g, tricalcium phosphate 5.0 g, agar 20 g, pH 7.0-7.5, distilled water 1000 mL.

[0052] Take 0.1g of soil and place it in a conical flask containing 100ml LB liquid medium and place it on a shaker. After 3 days, use the plate dilution method to culture in a 28℃ incubator with NA solid medium for about 2 to 5 days, pick a single colony, repeatedly streak and purify, and inoculate the obtained strains on solid medium plates and store them in a 4℃ refrigerator to obtain pure strains. According to the main biological characteristics, bacteria Q4, Q7 and YG1 were obtained.

[0053] Example 2

[0054] Investigate the growth-promoting effect of the fungus. Combined with potted plant experiments, the selected fungi were applied to the seedling soil to explore their potential effects in production applications.

[0055] 1. Preparation of inoculants

[0056] 1.1 Culture medium

[0057] (1) The composition and content of nutrient agar solid medium are as follows: peptone 10 g, beef extract powder 3 g, sodium chloride 5 g, agar 15 g, deionized water 1000 ml, pH 7.2-7.4.

[0058] (2) The composition and proportion of LB liquid culture medium are as follows: add 950 ml of deionized water, 10 g of tryptone, 5 g of yeast extract powder, and 5 g of sodium chloride into a container, shake the container until the solute is dissolved, adjust the pH of the solution to 7.4 with 5 mol / L sodium hydroxide, and make up to 1 L.

[0059] 1.2 Preparation of inoculants

[0060] (1) One cell of Bacillus subtilis YG1 was selected from each well-preserved slant and inoculated into nutrient agar solid medium and activated at 25°C for 48 h.

[0061] (2) Use an inoculation loop to pick up a loop of bacterial slurry from the activated strain and add it to LB liquid culture medium. Keep the culture medium at 25°C and 200 rpm for 24 h to prepare the seed solution.

[0062] (3) Dilute the seed solution by adding sterile water or continue fermenting until the fermentation liquid OD 600 The concentration is in the range of 0.8 to 1.2, and then sealed and stored in a refrigerator at 4°C for later use.

[0063] (4) The control group was treated with an equal volume of sterile water diluted 100 times.

[0064] 2. Planting of Leucaena leucaena and Lespedeza seedlings

[0065] The potted plants were tested with the leguminous plant Lespedeza as the test object. After soaking the seeds of Leucaena leucophylla and Lespedeza in pure water for 12 hours, the water was filtered and the seeds were soaked in 5% sodium hypochlorite solution for 10 minutes for disinfection, then washed with pure water until odorless, and placed in a seedling cup for germination for a week. After the seeds germinated into seedlings, the seedlings with similar height and growth were transplanted into potted plants, and one seedling was planted in each pot. The soil used for potted plants came from Baqing County, Nagqu City, Tibet Autonomous Region, China. After the Leucaena leucophylla and Lespedeza seedlings grew well, the prepared bacterial agent was applied, with the application of the bacterial agent as the first day. After three months, the samples were destroyed and used for experiments.

[0066] Determination and methods of potted plant indicators

[0067] For plants: vernier calipers and tape measures were used to measure the diameter at breast height and plant height of the seedlings; the dry weight of Leucaena leucophylla and Lespedeza were measured by drying and sterilizing; the photosynthetic data of Lespedeza leaves were measured using a LI-6400 portable photosynthetic meter (Li-Cor Inc. USA), and the chlorophyll content of plant leaves was measured using a mixed liquid extraction method; the root morphological indicators of the tree seedlings, such as root length, root surface area, root volume and average root diameter, were measured and analyzed using a root analysis system (WinRHIZO 2013).

[0068] For potting soil: soil available phosphorus was determined by acid dissolution-molybdenum antimony countercolorimetry; soil alkaline nitrogen was determined by alkaline diffusion method; available potassium content was determined by flame photometry after extraction with ammonium acetate; soil organic matter was determined by potassium dichromate volumetric method; soil hydrolysis content was determined by alkaline diffusion method; soil pH was determined by mettlertoledo pH meter (water-soil ratio was 5:1).

[0069] The results show that:

[0070] Effects of fungal agent treatment on the growth of Leucaena leucaena and Lespedeza

[0071] Note: P<0.05.

[0072] Depend on Figure 5 and Figure 7 It can be seen that compared with the control group, the YG1 treatment increased the plant height of Leucaena leucophylla (P < 0.05, the same below), and its plant height increased by 53.07%; the total biomass increased significantly by 181.73%. Figure 6 and Figure 8 It can be seen that compared with the control group, YG1 treatment increased the plant height of Lespedeza (P < 0.05, the same below), its plant height increased by 20.84%, and the total biomass increased by 29.94%; overall, YG1 inoculant treatment can increase the plant height, ground diameter and biomass of Lespedeza and Lespedeza, and the promoting effect of YG1 on Lespedeza is stronger than that on Lespedeza.

[0073] Effects of bacterial strains on soil physical and chemical properties

[0074] Note: P<0.05.

[0075] After application, the Bacillus japonii YG1 provided by the present invention significantly enhances the transformation of soil quick-acting nutrients and increases the nutrient consumption capacity of Leucaena leucaena and Lespedeza leucaena. Fig. 9 As shown in Table 1, the application of Bacillus yyoides YG1 promoted the growth and development of Leucaena leucaena, significantly increased the content of soil hydrolyzed nitrogen and available phosphorus, and increased the conversion to soil nutrients, among which the available phosphorus increased the most, significantly increasing by 50.32%. Fig.10 As shown in Table 2, the application of Bacillus orientalis YG1 also significantly increased the hydrolyzed nitrogen and available phosphorus content of Lespedeza soil, of which hydrolyzed nitrogen increased by 45.18%, available phosphorus increased by 43.57%, and organic carbon increased most significantly, by 48.9%. The pot experiment showed that Bacillus orientalis YG1 can effectively utilize nitrogen, phosphorus, and potassium in the soil without changing the soil pH, increase the soil organic carbon content, thereby promoting the growth of Leucaena and Lespedeza and improving soil fertility.

[0076] Table 1 Effects of bacterial agent treatment on the physical and chemical properties of Leucaena leucophylla soil

[0077] Table 2 Effects of bacterial agent treatment on the physical and chemical properties of Lespedeza soil

[0078] Effects of strains on soil enzyme activities

[0079] Note: P<0.05.

[0080] Table 3 Effects of bacterial agent treatment on soil enzyme activity of Leucaena leucophylla

[0081] Table 4 Effects of bacterial agent treatment on soil enzyme activities of Lespedeza chinensis

[0082] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0083] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for promoting the growth of Leucaena leucaena and Lespedeza leucaena, characterized in that: Bacillus toyonensis.YG1 was used as bacterial fertilizer, and its deposit number is CCTCC NO:M 20241524.

2. A method for improving soil physical and chemical properties and increasing soil enzyme activity, characterized in that: Bacillus toyonensis.YG1 was used as bacterial fertilizer, and its deposit number is CCTCC NO:M 20241524.

3. An application of Bacillus orientalis YG1 in promoting the production of Leucaena leucaena and Lespedeza chinensis plants, characterized in that: Bacillus toyota YG1 was used as bacterial fertilizer, and its preservation number was CCTCC NO:M 20241524.

4. Use of Bacillus orientalis YG1 in enhancing the utilization of soil nutrients by Leucaena leucaena and Lespedeza chinensis. Bacillus toyota YG1 was used as bacterial fertilizer.

5. The use according to claim 4, characterized in that: After treatment with Bacillus yyophyta YG1, the biomass and available phosphorus content of Leucaena leucophylla increased significantly by 181.73% and 50.32% respectively; the most obvious increase in Lespedeza was the content of hydrolyzed nitrogen and organic matter, which increased by 45.18% and 48.90% respectively. The transformation of soil available nutrients was strengthened, and the nutrient consumption capacity of Leucaena leucophylla and Lespedeza leucophylla was enhanced.

6. The use according to claim 4, characterized in that: After treatment with Bacillus yyophyta YG1, the conductivity and available phosphorus of Leucaena leucaena increased the most, by 49.01% and 50.32% respectively; the hydrolyzed nitrogen and organic matter content of Lespedeza leucaena increased the most, by 45.18% and 48.90% respectively. At the same time, soil enzyme activity also increased, and the activities of catalase (S-CAT), alkaline phosphatase (S-ALP), and sucrase (S-SC) all increased significantly. The improvement of soil physical and chemical properties and the significant increase in enzyme activity are important factors in promoting the growth of Leucaena leucaena and Lespedeza leucaena.

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