Mixed bacterial agent for adjusting haloxylon ammodendron soil rhizosphere soil microecosystem in desert and promoting growth of haloxylon ammodendron soil rhizosphere soil microecosystem

Abstract

The invention discloses a mixed bacterial agent ZL3 + 6 with effects of promoting growth of haloxylon ammodendron and regulating rhizosphere microflora and application of the mixed bacterial agent ZL3 + 6, and belongs to the technical field of microorganisms. According to the bacillus orientalis ZL3 and the bacillus cereus ZL6 provided by the invention, the conversion of soil nutrients to available nutrients is improved, and the rhizosphere microbial community structure is improved, so that the effect of promoting the growth of haloxylon ammodendron is achieved. When the ZL3 and the ZL6 are mixed and applied to a haloxylon ammodendron field, the contents of hydrolytic nitrogen, available phosphorus and rapidly available potassium in soil and the activity of sucrase are remarkably improved and are respectively improved by 49.27%, 80.48%, 12.65% and 64.40%. The plant height is increased by 51.75%, and the root length is increased by 262.18%. Meanwhile, the positive change of microbial communities is regulated and controlled, especially the enrichment of beneficial bacteria, the relative abundance of the prevotella and the vibrio is remarkably increased, and the relative abundance of the burkholderia is remarkably reduced. In general, the ZL3 + 6 inoculant is inoculated to promote the growth and development of haloxylon ammodendron by improving the soil fertility and improving the rhizosphere microflora structure.

Description

Technical Field

[0001] This invention relates to a mixed bacterial agent ZL3+6 that promotes growth and regulates the rhizosphere microbial community of Haloxylon ammodendron and its application, belonging to the field of microbial technology. Background Technology

[0002] Desertification is a global environmental problem that not only affects the balance of ecosystems but also poses a serious threat to human survival and development. Alxa League, located in the westernmost part of Inner Mongolia Autonomous Region, situated in the upper reaches of the Yellow River, is a crucial area for blocking wind and sand from the north and northwest. The Badain Jaran, Tengger, and Ulan Buh deserts traverse the entire region, with desertified land accounting for 72.93% of the league's total land area. Therefore, efficient ecological restoration of the Alxa desert region and improving soil fertility are critical issues that urgently need to be addressed.

[0003] Microbial inoculants can degrade and inhibit pathogen secretions in the soil, effectively reducing the proliferation and spread of pathogens and providing a healthy growing environment for crops. They can also promote the proliferation of beneficial microorganisms in the soil, which compete with pathogens for nutrients and space, further inhibiting pathogen growth and thus achieving biological control. Simultaneously, the microorganisms in microbial inoculants can decompose organic matter in the soil, increasing soil organic matter and porosity, breaking up soil compaction, and promoting the formation of aggregates, thereby improving soil structure and enhancing its water and heat retention capacity. Furthermore, some inoculants with phosphorus-solubilizing properties can increase the content of phosphorus and other nutrients in the soil that are available for plant absorption. Therefore, in-depth research into microbial inoculants that positively promote plant growth and development is indispensable for enriching the research on microbial fertilizers.

[0004] Soil microorganisms are among the most diverse and species-rich groups of organisms on land, exerting a significant influence on ecosystems such as forests, grasslands, wetlands, and farmlands, and playing a crucial role in the entire biogeochemical cycle. Applying microbial inoculants to plant roots may be an effective strategy to improve plant growth. However, conventional strain screening usually involves obtaining strains from non-target habitats such as agricultural soils or the rhizosphere of model plants. When these strains are introduced into harsh and highly specific habitats, they face challenges such as low colonization efficiency, competitive disadvantage with native microorganisms, and insufficient interaction compatibility with the target host plant. Therefore, screening for microorganisms that share the same habitat as the target plant and are naturally adapted to local extreme environmental pressures is particularly important.

[0005] Haloxylon ammodendron possesses excellent characteristics such as cold resistance, drought resistance, salt and alkali resistance, and wind and sand resistance. Its well-developed root system maximizes the absorption of groundwater and firmly anchors the above-ground plants from being carried away by wind and sand. When used to create shelterbelts, it plays a role in preventing wind erosion, conserving water resources, and curbing desertification. However, deserts suffer from scarce water resources and nutrient-poor soil, making plant growth difficult. While traditional chemical fertilizer application methods may show short-term effects, they cause long-term damage to the ecological environment. In contrast, PGPR microbial fertilizer, as a new type of environmentally friendly fertilizer developed in recent years, is pollution-free and has gained considerable popularity.

[0006] Therefore, in-depth research and screening of PGPR microbial agents that have a significant growth-promoting effect on Haloxylon ammodendron from desert soils is of great practical significance for promoting the development of pollution-free bio-fertilizers. It helps to fully leverage the potential advantages of microbial fertilizers and thus provide a theoretical basis and technical guidance for the sustainable development of desertified areas. Summary of the Invention

[0007] [Purpose of the Invention]

[0008] This invention aims to screen strains from deserts that promote the growth of Haloxylon ammodendron and regulate the rhizosphere microbial community, providing a solution for efficient ecological restoration in desertified areas of my country.

[0009] [Technical Solution]

[0010] To address the aforementioned technical problems, this invention discloses a method for promoting the growth of Haloxylon ammodendron and regulating the rhizosphere microbial community. Bacillus toyonensis ZL3 and Bacillus cereus ZL6 are used as microbial fertilizers, with preservation numbers CCTCCNO:M 2025419 and 2025420, respectively.

[0011] The *Bacillus toyonensis* ZL3 and *Bacillus cereus* ZL6 described in this invention were isolated from the rhizosphere of *Achnatherum gracile* in Alxa League, Inner Mongolia Autonomous Region, China. They were preserved at -80°C in the Soil and Water Conservation Laboratory of Nanjing Forestry University and deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China. The accession numbers are CCTCC NO: M 2025419 and 2025420, respectively, and the deposit date is March 10, 2025.

[0012] This invention also provides the application of Bacillus toyonensis ZL3 and Bacillus cereus ZL6 in promoting the production of Haloxylon ammodendron plants. Bacillus toyonensis ZL3 and Bacillus cereus ZL6 are used as microbial fertilizers, with preservation numbers CCTCC NO:M 2025419 and 2025420.

[0013] This invention also provides an application of Bacillus oryzae ZL3 and Bacillus cereus ZL6 in enhancing the utilization of soil nutrients by Haloxylon ammodendron and regulating the rhizosphere microbial community, using Bacillus oryzae ZL3 and Bacillus cereus ZL6 as microbial fertilizer.

[0014] Furthermore, the microbial inoculant of the present invention is prepared according to the following steps and applied to the field of Haloxylon ammodendron:

[0015] 1) Select cells of Bacillus cereus ZL3 and Bacillus cereus ZL6 from the well-preserved slant and inoculate them into LB agar solid medium and activate at 28°C for 48 hours.

[0016] 2) The activated Bacillus cereus ZL3 and Bacillus cereus ZL6 strains were picked up with an inoculation loop and added to LB liquid medium. Seed culture was prepared by constant temperature shaking at 28℃ and 200r / min for 24h.

[0017] 3) Inoculate the seed culture at a rate of 5% into LB liquid medium for amplification culture. Incubate at 28℃ and 200 rpm on a shaker for 2-3 days. Dilute with sterile water or continue fermentation to ensure the OD of the culture. 600 The concentration is 0.8 to 1.2, which yields the fermentation broth.

[0018] 4) Weigh 500g of wheat bran and put it into a resealable bag. Add 200mL of water and 150mL of the original liquid, mix well, and let it ferment fully.

[0019] 5) Add the wheat bran with bacterial solution to the area around the roots of the seedlings (5cm away from the roots), applying 2kg per seedling each time.

[0020] 6) The control group was treated with an equal volume of wheat bran without sterilizing agent.

[0021] Beneficial effects

[0022] This invention provides a mixed microbial inoculant that promotes the growth of Haloxylon ammodendron. Applying this inoculant to Haloxylon ammodendron plants can effectively increase plant height, root length, and diameter at breast height, improve physicochemical properties and enzyme activity, and regulate the soil rhizosphere microbial community, showing promising development prospects. Specifically:

[0023] In terms of Haloxylon ammodendron growth indicators

[0024] After inoculating Haloxylon ammodendron seedlings with PGPR inoculant, compared with the CK group, the plant height increased by 17.59%, 68.11%, and 51.75%, respectively; the ground diameter increased by -40.6%, 48.66%, and 90.91%, respectively; and the root length of Haloxylon ammodendron increased by 109.45%, 206.55%, and 262.18% compared with the CK treatment, respectively.

[0025] In terms of rhizosphere soil physicochemical properties and enzyme activity

[0026] Compared with the control group, the soil organic carbon content of Haloxylon ammodendron in the ZL3+6 inoculant treatment group increased by 43.75%; available phosphorus increased by 80.48%; available potassium increased by 12.65%; hydrolyzable nitrogen increased by 49.28%; soil sucrase activity increased significantly by 64.40%; and soil catalase activity increased by 19.90%.

[0027] On the rhizosphere microbial community

[0028] Compared with the control (CK), the ZL3+6 inoculant treatment group significantly reduced the relative abundance of Burkholderia and significantly increased the relative abundance of Curvibacter, Vibrionimonas and Streptomyces.

[0029] As can be seen from the above technical solution, the present invention discloses a mixed microbial agent for promoting the growth of Haloxylon ammodendron. The technical effect achieved is that the mixed microbial agent provided by the present invention promotes the growth of Haloxylon ammodendron plants after application, especially promotes the root development of Haloxylon ammodendron, and effectively exerts the plant's ability to root and fix sand. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the present invention will be briefly described below.

[0031] Figure 1 This is an phylogenetic tree diagram of the Bacillus oryzae ZL3 strain provided by the present invention.

[0032] Figure 2 This is an phylogenetic tree diagram of Bacillus cereus strain ZL6 provided by the present invention.

[0033] Figure 3 This is a colony diagram of Bacillus oryzae ZL3 (LB solid medium) provided by the present invention.

[0034] Figure 4 This is a colony diagram of Bacillus cereus ZL6 (LB solid medium) provided by the present invention.

[0035] Figure 5This diagram illustrates the changes in plant height, root length, and diameter at ground level of Haloxylon ammodendron after treatment with the control group and the strain provided by this invention.

[0036] Figure 6 This is a schematic diagram illustrating the changes in the rhizosphere soil microbial community of Haloxylon ammodendron after treatment with the control group and the strain provided in this invention. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] All raw materials and reagents used in the examples were obtained from commercially available sources, and no brand requirements were specified. Methods not mentioned were all commonly used experimental methods. *Bacillus toyonensis* ZL3 and *Bacillus cereus* ZL6 were isolated from the rhizosphere of *Achnatherum splendens* in Alxa League, Inner Mongolia Autonomous Region, China, and deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. The accession numbers are CCTCC NO: M 2025419 and 2025420, and the deposit date is March 10, 2025.

[0039] Example 1

[0040] 1. Sample Source

[0041] Strains were screened from soil samples taken from the rhizosphere of *Achnatherum sp.* in Alxa League, Inner Mongolia Autonomous Region, China. After collection, samples were placed in a 4°C incubator and then transported back to the laboratory for storage at -80°C. Soil microorganisms were isolated within 48 hours.

[0042] 2. Separation and Screening

[0043] (1) NA solid culture medium: 10g peptone, 3g beef extract powder, 5g sodium chloride, 15g agar, 1000ml deionized water, pH 7.2-7.4.

[0044] (2) LB liquid culture medium: 10g peptone, 5g yeast extract, 5g sodium chloride, 1000mL deionized water, pH 7.2.

[0045] (3) Mongkina Organic Phosphorus Medium: 10g glucose, 0.5g ammonium sulfate, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate heptahydrate, 0.03g ferrous sulfate heptahydrate, 0.03g manganese sulfate tetrahydrate, 5.0g calcium carbonate, 0.3g lecithin, 20g agar, pH 7.0-7.5, 1000mL distilled water.

[0046] (4) Mongkina Inorganic Phosphorus Culture Medium: 10g glucose, 0.5g ammonium sulfate, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate heptahydrate, 0.03g ferrous sulfate heptahydrate, 0.03g manganese sulfate tetrahydrate, 5.0g tricalcium phosphate, 20g agar, pH 7.0-7.5, 1000mL distilled water.

[0047] Take 0.1g of soil and place it in an Erlenmeyer flask containing 100ml of LB liquid medium. Place the flask on a shaker. After 3 days, use the plate dilution method to incubate on NA solid medium at 28℃ for about 2-5 days. Pick single colonies, repeatedly streak to purify, and inoculate the obtained strains onto solid medium plates. Store at 4℃ to obtain pure strains. Based on the main biological characteristics, bacteria ZL3 and ZL6 were obtained.

[0048] Example 2

[0049] To investigate the effects of microbial strains on growth promotion, a field experiment was conducted in the Tengger Desert of Alxa Left Banner, Inner Mongolia Autonomous Region (106°41′0″E, 39°54′6″N). The screened microbial agent was applied to the rhizosphere of seedlings to explore its potential efficacy in production applications.

[0050] 1. Preparation of microbial agents

[0051] 1.1 Culture medium

[0052] (1) The composition and content of the nutrient agar solid culture medium are: 10g peptone, 3g beef extract powder, 5g sodium chloride, 15g agar, 1000ml deionized water, pH 7.2-7.4.

[0053] (2) The composition and preparation method of LB liquid culture medium are as follows: Add 950ml of deionized water, 10g of tryptone, 5g of yeast extract and 5g of sodium chloride to the container, shake the container until the solute is dissolved, adjust the pH of the solution to 7.4 with 5mol / L sodium hydroxide, and make up to 1L.

[0054] 1.2 Preparation of microbial agents

[0055] (1) Select Bacillus cereus ZL3 and Bacillus cereus ZL6 from the well-preserved slant and inoculate them into LB agar solid medium and activate at 28°C for 48h.

[0056] 2) The activated Bacillus cereus ZL3 and Bacillus cereus ZL6 strains were picked up with an inoculation loop and added to LB liquid medium. Seed culture was prepared by constant temperature shaking at 28℃ and 200r / min for 24h.

[0057] 3) Inoculate the seed culture at a rate of 5% into LB liquid medium for amplification culture. Incubate at 28℃ and 200 rpm on a shaker for 2-3 days. Dilute with sterile water or continue fermentation to ensure the OD of the culture. 600 The concentration is 0.8 to 1.2, which yields the fermentation broth.

[0058] 4) Weigh 500g of wheat bran and put it into a resealable bag. Add 200mL of water and 150mL of the original liquid, mix well, and let it ferment fully.

[0059] 5) Add the wheat bran with bacterial solution to the area around the roots of the seedlings (5cm away from the roots), applying 2kg per seedling each time.

[0060] 6) The control group was treated with an equal volume of wheat bran without sterilizing agent.

[0061] 2 field trials

[0062] Four treatments were applied to Haloxylon ammodendron in the Alashan region: wheat bran treatment without inoculant (CK), wheat bran treatment inoculated with Bacillus cereus (ZL3), wheat bran treatment inoculated with Bacillus cereus (ZL6), and wheat bran treatment inoculated with a Bacillus cereus complex (ZL3+6), with 10 replicates for each treatment. A 40m² plot was selected. 2 The open space was arranged in a square shape and divided into 40 plots, each 1 meter long and 1 meter wide. In each plot, a pit with a length, width, and height of 30 cm was dug, and one-year-old Haloxylon ammodendron seedlings were planted in the pit.

[0063] 2. Determination and Methods of Field Indicators

[0064] For the plants: use vernier calipers and measuring tape to measure the seedling height, diameter at ground level, and root length.

[0065] For soil physicochemical and enzyme activities: available phosphorus in soil was determined by acid dissolution-molybdenum antimony colorimetric method; available nitrogen in soil was determined by alkaline hydrolysis diffusion method; available potassium content was determined by flame photometry after extraction with ammonium acetate; soil organic matter was determined by potassium dichromate titration method; soil hydrolysis content was determined by alkaline hydrolysis diffusion method; soil pH was determined by Mettler Toroledo pH meter; glucosidase was determined by fluorescence method; amylase was determined by colorimetric method; sucrase was determined by spectrophotometry; urease and catalase were determined by biochemical detection kits.

[0066] For rhizosphere microbial communities: use ez na A DNA kit was used to extract complete genomic DNA from soil samples, and the concentration of the extracted DNA was detected using a Nanodrop RND-2000. Primers were used to amplify the V3-V4 region of the 16S rRNA gene by PCR and followed by MiSeq sequencing.

[0067] The results show that:

[0068] Effects of fungicide treatment on the growth of Haloxylon ammodendron

[0069] Note: P<0.05.

[0070] Depend on Figure 5 It was found that, compared with the control group, the ZL3+6 treatment increased the height of Haloxylon ammodendron (P<0.05, the same below), with a 51.75% increase in height; a 262.18% increase in root length; and a 90.91% increase in diameter at root. Overall, the ZL3+6 inoculant treatment promoted the growth of Haloxylon ammodendron seedlings and significantly increased root length.

[0071] Effects of bacterial strains on soil physicochemical properties

[0072] Note: P<0.05.

[0073] The combined application of *Bacillus oryzae* ZL3 and *Bacillus cereus* ZL6 provided by this invention significantly enhanced the conversion of available nutrients in the soil and increased the nutrient consumption capacity of *Haloxylon ammodendron*. Table 1 shows that the application of *Bacillus oryzae* ZL3 and *Bacillus cereus* ZL6 promoted the growth and development of *Haloxylon ammodendron*, significantly increased the content of hydrolyzable nitrogen, available phosphorus, and available potassium in the soil, and increased the conversion of nutrients into soil nutrients. Available phosphorus saw the largest increase, significantly increasing by 80.48%. Field experiments demonstrate that *Bacillus oryzae* ZL3 and *Bacillus cereus* ZL6 can effectively utilize nitrogen, phosphorus, and potassium in the soil without altering soil pH, increasing soil organic carbon content, thereby promoting the growth of *Haloxylon ammodendron* and improving soil fertility.

[0074] Table 1. Effects of microbial inoculant treatment on the physical and chemical properties of Haloxylon ammodendron soil.

[0075] Effects of strains on soil enzyme activity

[0076] Note: P<0.05.

[0077] The mixed application of Bacillus cereus ZL3 and Bacillus cereus ZL6 provided by this invention significantly improved soil enzyme activity. As shown in Table 2, the activities of soil catalase and sucrase increased by 19.90% and 64.40%, respectively.

[0078] Table 2 Effects of microbial inoculant treatment on soil enzyme activity of Haloxylon ammodendron

[0079] The impact of strains on rhizosphere microbial communities

[0080] The present invention provides a method for altering the rhizosphere microbial community structure of Haloxylon ammodendron after the combined application of Bacillus cereus ZL3 and Bacillus cereus ZL6. Figure 6 It can be seen that, compared with the control group, the treatment group with ZL3+6 inoculant significantly reduced the relative abundance of Burkholderia and significantly increased the relative abundance of Curvibacter, Vibrionimonas and Streptomyces.

[0081] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0082] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for promoting the growth of Haloxylon ammodendron, characterized in that, Bacillus oryzae ZL3 and Bacillus cereus ZL6 were used as microbial fertilizers, with preservation numbers CCTCCNO:M 2025419 and 2025420, respectively.

2. The application of a mixed microbial agent ZL3+6 in promoting the growth and development of Haloxylon ammodendron, characterized in that, Bacillus toyonensis ZL3 and Bacillus cereus ZL6 were used as microbial fertilizers, with preservation numbers CCTCC NO:M 2025419 and 2025420, respectively.

3. A method for improving soil physicochemical properties and enhancing soil enzyme activity using a mixed microbial agent ZL3+6, characterized in that... Bacillus toyonensis ZL3 and Bacillus cereus ZL6 were used as microbial fertilizers, with preservation numbers CCTCCNO:M 2025419 and 2025420, respectively.

4. The application of a mixed microbial agent ZL3+6 in improving the rhizosphere soil microbial community of Haloxylon ammodendron. Its characteristics are: Bacillus toyonensis ZL3 and Bacillus cereus ZL6 were used as microbial fertilizers, with preservation numbers CCTCC NO:M2025419 and 2025420, respectively.

5. The application as described in claim 2, characterized in that: After treatment with ZL3+6, the plant height, root length, and ground diameter of Haloxylon ammodendron increased significantly, with plant height increasing by 51.75%, root length by 262.18%, and ground diameter by 90.91%. Overall, the ZL3+6 inoculant treatment promoted the growth of Haloxylon ammodendron seedlings and significantly increased root length.

6. The application as described in claim 3, characterized in that: After ZL3+6 treatment, the contents of hydrolyzable nitrogen, available phosphorus, and available potassium in the soil of Haloxylon ammodendron increased significantly, enhancing the conversion to available nutrients. Among these, available phosphorus saw the largest increase, rising by 80.48%. Simultaneously, soil enzyme activity improved, with catalase and sucrase activities increasing by 19.90% and 64.40%, respectively. The improvement in soil physicochemical properties and the significant increase in enzyme activity are important factors promoting the growth of Haloxylon ammodendron.

7. The application as described in claim 4, characterized in that: ZL3+6 treatment altered the rhizosphere microbial community structure of Haloxylon ammodendron. The relative abundance of Burkholderia decreased significantly, while the relative abundance of Curvibacter, Vibrionimonas, and Streptomyces increased significantly.

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