Bacillus megaterium Q7 for promoting growth of leucaena leucocephala and improving abundance of probiotic microbial population

Abstract

The invention discloses a bacillus megaterium Q7 for promoting the growth of leucaena leucocephala and improving the abundance of a probiotic microbial population. The bacillus megaterium Q7 is a bacillus megaterium for promoting the growth of leucaena leucocephala. Belongs to the technical field of microorganisms. The bacillus megatherium Q7 provided by the invention improves the conversion from total-effect nutrients to effective nutrients, and enhances the consumption of the leucaena leucocephala on soil nutrients, thereby achieving the effect of promoting the growth of the leucaena leucocephala. After the bacillus megatherium Q7 provided by the invention is applied, the enzyme activity of soil is remarkably improved, the conversion of available nutrients in the soil is remarkably enhanced, and the nutrient consumption capacity of leucaena leucocephala is enhanced. The bacillus megaterium Q7 is applied to promote the growth and development of the leucaena leucocephala, so that the contents of soil hydrolytic nitrogen and available phosphorus are remarkably increased, and the conversion to soil nutrients is enhanced. In the genus level, the average relative abundance of bacillus treated by the bacillus atrophaeus Q7 is increased to 9.97% from 1.33%, the bacillus pseudobecomes the leucaena leucocephala soil community dominant bacteria genus, and the relative abundance of Fictibacillus is increased by about 6 times, which indicates that the inoculation of the strain Q7 enables the leucaena leucocephala soil community structure to be changed, and the leucaena leucocephala soil community dominant bacteria genus is changed. And the increase of the relative abundance of dominant bacteria is promoted. Wherein the bacillus and the Fictibacillus belong to obvious increase of relative abundance and are possibly an important factor for promoting plant growth.

Description

Technical Field

[0001] The invention relates to Bacillus megaterium Q7 (Bacillus megaterium) which can promote the growth of Leucaena leucaena and belongs to the technical field of microorganisms. Background Art

[0002] Fertilizers, as an important part of agricultural production, play a key role in promoting plant growth and increasing crop yields and income. However, due to the irrational use of chemical fertilizers and the lack of environmental awareness, soil compaction, decreased soil fertility and reduced soil microbial diversity have ultimately affected plant growth and development and the soil microenvironment. In recent years, the shortcomings of traditional chemical fertilizers have become increasingly apparent, and microbial fertilizers have been widely used due to their ecological benefits and excellent characteristics.

[0003] At this stage, existing studies have shown that microorganisms play an important role in enhancing plant characteristics, regulating microorganisms and plant growth, and have a positive impact on improving plant quality. As a crop with certain economic value, the roots, stems and leaves of Leucaena leucophylla are not only rich in nutrients such as protein and amino acids, but also have certain medicinal properties. They have the effects of promoting blood circulation and removing blood stasis, disinfecting and relieving swelling, and strengthening the kidneys and bones. They are widely used in human life. In addition, Leucaena leucophylla's high-quality stress resistance can be used as a pioneer tree species for afforestation. These qualities enhance the needs and economic returns of farmers and herdsmen around the world. Therefore, screening Leucaena leucophylla-adaptive microbial agents and understanding the growth-promoting mechanism of Leucaena leucophylla have become current research hotspots. At present, studies have used microorganisms on saline-alkali land to improve the growth quality of wolfberry by improving the soil environment.

[0004] The richness of soil microorganisms is usually accompanied by the diversity of soil microbial functions. Blindly applying non-host microorganisms to plant growth, whether plants can adapt to their microbial characteristics and whether they can thrive in such an environment remains to be verified. Therefore, the present invention screens out some rhizosphere bacteria from the root soil of Leucaena leucaena, thereby improving the adaptability of rhizosphere bacteria and Leucaena leucaena, achieving the effect of "suitable land and suitable tree", and further exploring the growth-promoting functional mechanism of rhizosphere bacteria on host plants, rather than blindly selecting microorganisms to match Leucaena leucaena.

[0005] If such rhizospheric bacteria exist, it will first ensure the adaptability of the microorganisms themselves to the soil environment in which Leucaena leucophylla grows, thereby exploring their important role in the growth-promoting mechanism of Leucaena leucophylla.

[0006] Therefore, screening highly efficient growth-promoting rhizosphere microorganisms is the key to promoting plant growth and development and green agricultural development. Studying the growth-promoting ability and mechanism of rhizosphere bacteria on plants, clarifying how soil fungal communities evolve under long-term rhizosphere bacteria application conditions, and their relationship with soil nutrients, can provide a theoretical basis and practical guide for the development of green agriculture and animal husbandry. Summary of the invention

[0007] [Purpose of the invention]

[0008] The present invention screened excellent PGPR strains with the ability to solubilize insoluble phosphates from the soil of the Qinghai-Tibet Plateau, and explored their effects on the growth and development of Leucaena leucophylla and soil improvement and their mechanism of action. The research results will provide a theoretical basis and practical guide for the development of Leucaena leucophylla planting industry and green agriculture and animal husbandry, and provide a basis for beneficial strains.

[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, using Bacillus megaterium Q7 as bacterial fertilizer, and the deposit number is CCTCC M 20241236.

[0011] The Bacillus megaterium Q7 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 M 20241236, and the deposit date is June 17, 2024.

[0012] The invention also provides an application of Bacillus megaterium Q7 in promoting the production of Leucaena leucaena plants. The Bacillus megaterium Q7 is applied as a bacterial fertilizer, and the deposit number is CCTCC M 20241236.

[0013] The present invention also provides an application of Bacillus megaterium Q7 in enhancing the utilization of soil nutrients by Leucaena leucophylla and improving the abundance of beneficial microbial populations, and Bacillus megaterium Q7 is applied as a bacterial fertilizer.

[0014] Preferably: after being treated with Bacillus megaterium Q7, the upper leaf area of ​​Leucaena leucophylla changed by -21.98% to 49.48% compared with the CK group, the middle leaf area changed by -21.61% to 15.71%, and the lower leaf area changed by -24.56% to 32.25%. The leaf thickness also showed different growth trends with the addition of the inoculant. Compared with the CK group, the upper leaf thickness of the group inoculated with three PGPR inoculants increased by 3.50% to 18.88%, the middle leaf thickness increased by 9.37% to 17.97%, and the lower leaf thickness increased by 3.82% to 13.74%.

[0015] Preferred: After treatment with Bacillus megaterium Q7, compared with CK, the Q7 bacterial agent treatment group significantly increased (P<0.05) the activities of soil catalase (S-CAT) (82.16%), urease (S-UE) (64.47%), and dehydrogenase (S-DHA) (502.20%) in Leucaena leucophylla seedlings. Compared with CK, the activity of alkaline phosphatase (S-ALP) increased by 70.06% under Q7 treatment, and the activity of sucrase (S-SC) increased by 280.58%. Inoculation of bacteria can significantly (P<0.05) increase the activity of soil enzymes in Leucaena leucophylla.

[0016] Beneficial Effects

[0017] After treatment with Bacillus megaterium Q7, compared with CK, Q7 treatment significantly increased the relative abundance of the top ten species of dominant soil microbial communities. Except for Pseuadomonas and Chryseolinea, the relative abundance of other genera increased significantly. Among them, the average relative abundance of Bacillus increased from 1.33% to 9.97%, becoming the dominant genus in the soil community of Leucaena leucaena. The relative abundance of Fictibacillus increased by about 6 times, indicating that the inoculation of strain Q7 changed the structure of the soil bacterial community of Leucaena leucaena and promoted the increase in the relative abundance of the dominant genera.

[0018] After treatment with Bacillus megaterium Q7, the inoculation of PGPR accelerated the growth of Leucaena leucaena seedlings, and the plant height and ground diameter were significantly increased (p<0.05). Compared with the CK group, the plant height of the Q7 group increased by 136.55% and the ground diameter increased by 135.10%.

[0019] Through the above technical solutions, it can be seen that compared with the traditional technology, the relative abundance of the present invention under the treatment of Q7 microbial agent shows an increasing trend. Welch's t test found that the abundance of secondary functional genes such as carbohydrate metabolism, amino acid metabolism, and metabolism of cofactors and vitamins under Q7 treatment was significantly different from that under CK treatment, indicating that these metabolic pathways may be closely related to the metabolic mechanism of Leucaena leucophylla in response to Q7 microbial agent inoculation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 This is an evolutionary tree diagram of the Bacillus megaterium Q7 strain provided by the present invention.

[0022] Figure 2 This is a colony diagram of Bacillus megaterium Q7 (LB solid culture medium) provided by the present invention.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] Figure 6 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.

[0027] Figure 7 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.

[0028] Figure 8 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.

[0029] Fig. 9 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.

[0030] Fig.10 This is a stacked diagram of species distribution at the phylum Q7 level of Bacillus megaterium of the present invention. DETAILED DESCRIPTION

[0031] 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.

[0032] The embodiment of the present invention discloses that the present invention provides a Bacillus megaterium Q7 which promotes the growth of Leucaena leucophylla and increases the abundance of probiotic microbial population.

[0033] The raw materials and reagents involved in the examples are all obtained from commercial channels, and no requirements are made on their brands. All methods not mentioned are commonly used experimental methods. Bacillus megaterium Q7 was isolated and screened by this laboratory from the soil in the Qinghai-Tibet Plateau, and is 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 M 20241236, and the preservation date is June 17, 2024.

[0034] Example 1

[0035] 1. Sample source

[0036] 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.

[0037] 2Separation and screening

[0038] (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.

[0039] (2) LB liquid medium: peptone 10 g, yeast extract powder 5 g, sodium chloride 5 g, deionized water 1000 mL, pH 7.2.

[0040] (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.

[0041] (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.

[0042] Take 0.1g soil, 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, store them in a 4℃ refrigerator, and obtain pure strains. According to the main biological characteristics, bacteria Q4, Q7 and YG1 were obtained.

[0043] Example 2

[0044] 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.

[0045] 1. Preparation of inoculants

[0046] 1.1 Culture medium

[0047] (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.

[0048] (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.

[0049] 1.2 Preparation of inoculants

[0050] (1) One cell of Bacillus megaterium Q7 was selected from each well-preserved slant and inoculated into nutrient agar solid medium and activated at 25°C for 48 h.

[0051] (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.

[0052] (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.

[0053] (4) The control group was treated with an equal volume of sterile water diluted 100 times.

[0054] 2. Planting of Leucaena leucaena seedlings

[0055] The potted plants were tested with the leguminous plant Leucaena leucophylla. After soaking the seeds 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, with one seedling planted in each pot. The soil used for potted plants came from Baqing County, Nagqu City, Tibet Autonomous Region, China. After the Leucaena leucophylla 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.

[0056] Determination and methods of potted plant indicators

[0057] For plants: Use vernier calipers and tape measures to measure the diameter at breast height and plant height of the seedlings; and dry the Leucaena leucaena to determine its dry weight.

[0058] 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).

[0059] The results show that:

[0060] Effects of Bacterial Agent Treatment on the Growth of Leucaena Leucocephala

[0061] Note: P<0.05.

[0062] Depend on Figure 5 and Figure 6 It can be seen that compared with the control group, Q7 treatment increased the plant height of Leucaena leucophylla (P < 0.05, the same below), the plant height increased by 136.55%, the ground diameter increased by 135.10%, and the total biomass increased significantly by 326.40%. Therefore, Q7 bacterial agent treatment can increase the plant height, ground diameter and biomass of Leucaena leucophylla.

[0063] Effects of bacterial strains on soil physical and chemical properties

[0064] Note: P<0.05.

[0065] The application of the Bacillus megaterium Q7 provided by the present invention significantly enhances the transformation of soil fast-acting nutrients and increases the nutrient consumption capacity of Leucaena leucaena. Figure 7As shown in Table 1, the application of Bacillus megaterium Q7 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 48.99%. The pot experiment showed that Bacillus megaterium Q7 can effectively utilize nitrogen, phosphorus and potassium in the soil without changing the soil pH, increase the content of soil organic carbon, thereby promoting the growth of Leucaena leucaena and improving soil fertility.

[0066] Table 1 Effects of bacterial agent treatment on the physical and chemical properties of Leucaena leucophylla soil

[0067] Effects of strains on soil enzyme activities

[0068] Note: P<0.05.

[0069] Table 2 Effects of bacterial agent treatment on soil enzyme activity of Leucaena leucophylla

[0070] Example 3

[0071] Investigate the effect of strains on the composition of microbial communities.

[0072] The collected and sorted rhizosphere soil samples of Leucaena leucaena were sent to Guangzhou Kidio Development Co., Ltd. for high-throughput sequencing using the Omicsmart platform.

[0073] The microbial diversity in potting soil was detected by high-throughput sequencing, and the composition of the microbial community in potting soil was analyzed. Fig.10 The top ten dominant bacterial groups in terms of phylum abundance in the soil of Leucaena leucaena were shown, including Proteobacteria, Bacteroidota, Firmicutes, Acidobacteriota, Chloroflexi, Verrucomicrobiota, Planctomycetota, Actinobacteriota, Gemmatimonadota, and Cyanobacteria. Compared with the CK group, the abundance of Proteobacteria (27.80%), Patellar bacteria (47.33%), Firmicutes (175.70%), and Actinobacteria (39.58%) increased in the Q7 bacterial agent treatment group, while the abundance of the remaining phyla decreased.

[0074] Spearman analysis was performed at the genus level. The Q7 treatment significantly increased the relative abundance of the top ten species of the dominant soil microbial community. Except for Pseudomonas and Chryseolinea, the relative abundance of the remaining genera increased significantly. Among them, the average relative abundance of Bacillus increased from 1.33% to 9.97%, becoming the dominant genus in the Leucaena soil community. The relative abundance of Fictibacillus increased by about 6 times, indicating that the inoculation of strain Q7 changed the structure of the soil bacterial community of Leucaena and promoted the increase in the relative abundance of the dominant genera.

[0075] Table 3 Alpha diversity index of rhizosphere soil bacterial community in potted plants

[0076] 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.

[0077] 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, characterized in that: Bacillus megaterium Q7 was used as a bacterial fertilizer, and its deposit number is CCTCC NO: M20241236.

2. A method for reducing the abundance of pathogenic microorganisms, characterized in that: Bacillus megaterium.Q7 was used as a bacterial fertilizer, and its deposit number is CCTCC NO: M20241236.

3. An application of Bacillus megaterium Q7 in promoting the production of Leucaena leucaena plants, characterized in that: Bacillus megaterium Q7 was used as bacterial fertilizer, and its deposit number was CCTCC NO: M20241236.

4. An application of Bacillus megaterium Q7 in promoting the growth of Leucaena leucophylla and increasing the abundance of probiotic microbial populations. The invention is characterized in that: Bacillus megaterium Q7 was used as bacterial fertilizer.

5. The use according to claim 4, characterized in that: After treatment with Bacillus megaterium Q7, plant height, ground diameter, total biomass and root surface area increased by 136.55%, 135.10%, 326.40% and 167.10%, respectively, compared with the control group.

6. The use according to claim 4, characterized in that: After treatment with Bacillus megaterium Q7, the average relative abundance of Bacillus in Leucaena leucaena soil increased from 1.33% to 9.97%. CCA analysis found that the Bacillus genus was positively correlated with the samples treated with the inoculant; it was positively correlated with soil HN, AP, AK, EC, and SOC, indicating that Bacillus had a certain causal relationship with the increase in soil nutrient content and the promotion of plant growth, that is, the application of inoculants increased the relative abundance of Bacillus to varying degrees, thereby increasing the soil nutrient content and promoting plant growth.

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