Bacillus tequilensis QingTuHuS40 and application thereof

Abstract

The invention discloses bacillus tequilensis QingTuHuS40 and application of the bacillus tequilensis QingTuHuS40. The bacillus tequilensis QingTuHuS40 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on January 8, 2025, and the preservation number of the bacillus tequilensis QingTuHuS40 is CGMCC No.33325. The bacillus tequilensis QingTuHuS40 is named as QingTuHuS40. The bacillus tequilensis QingTuHuS40 and the application of the bacillus tequilensis QingTuHuS40 are named as QingTuHuS40. The bacillus tequilensis QingTuHuS40 can be planted at the rhizosphere of the red sand, and meanwhile, some other beneficial bacteria are also recruited, so that the bacterial community structure of the rhizosphere of the red sand is remodeled. Meanwhile, the soil fertility is improved, the soil salinization degree is improved, the capability of fully utilizing environmental nutrients of the plants is enhanced, the plant growth is promoted, and the salt tolerance is enhanced.

Description

Technical Field

[0001] The present invention relates to a Bacillus tequilensis QingTuHuS40 and its application, belonging to the field of microorganisms. Background Art

[0002] The Food and Agriculture Organization of the United Nations designated the theme of World Soil Day 2021 as "Halt soil salinization, boost soil productivity", implying the importance and necessity of the efficient utilization of saline soils. The total area of saline soils in China is approximately 0.369 billion hectares, accounting for 4.4% of the world's saline soils, mainly located in arid and semi-arid climate regions where rainfall is scarce and evaporation is high, which easily leads to soil salinization. Under salt stress, the massive accumulation of Na + and Cl - in plant cells causes ion imbalance, generates reactive oxygen species, leads to oxidative damage, affects plant metabolic disorders, and inhibits plant growth (Sunita, K., I. Mishra, J. Mishra, J. Prakash and N. K. Arora. "Secondary metabolites from halotolerant plant growth promoting rhizobacteria for ameliorating salinity stress in plants." Front. Microbiol 11 (2020): 567768.; Kibria, M. G., M. Hossain, Y. Murata and M. A. Hoque. "Antioxidant defense mechanisms of salinity tolerance in rice genotypes." Rice Sci 24 (2017): 155 - 62.). Meanwhile, salinization is accompanied by the deficiency of nitrogen (N) and phosphorus (P), which may affect the population structure and functions of soil microorganisms. In the past, much attention has been paid to the improvement and effective utilization of saline soils, and many strategies (including physical, chemical, and biological) have been developed to improve saline-alkali soils. In agricultural production, the soil properties can be effectively improved and crop productivity can be enhanced by planting salt-tolerant plants. Compared with other methods, bioremediation technology is an efficient, sustainable, and environmentally friendly soil remediation method.

[0003] Reaumuria soongorica is an extremely drought-tolerant and salt-tolerant small shrub belonging to the genus Reaumuria in the family Tamaricaceae. It is one of the key shrub species for wind prevention, sand fixation, and maintaining the stability of desert ecosystems in arid and semi-arid desert areas of northwestern China. During the process of maintaining the ecological balance in arid desert areas, Reaumuria soongorica has evolved special physiological characteristics to resist environmental factors such as drought and salinity (Shi, Y., X. Yan, P. Zhao, H. Yin, X. Zhao, H. Xiao, X. Li, G. Chen and X. Ma. "Transcriptomic analysis of a tertiary relict plant, extreme xerophyte reaumuria soongorica to identify genes related to drought adaptation." PLoS ONE 8(2013)), and it has important ecological significance in saline-alkali land improvement and ecological protection. However, studies have found that Reaumuria soongorica grows slowly, has weak stress resistance, and low survival rate at the seedling stage, resulting in its still not being widely used in saline-alkali land improvement and vegetation restoration (He, F. "Physiological effects of na + on improving the tolerances of drought, high temperature and wind-borne sand of salt-secreting xerophyte reaumuria soongorica." Lanzhou University: China(2019)). In recent years, researchers have studied the transcriptome and proteome characteristics of Reaumuria soongorica under salt stress (Liu, H., P. Chong, S. Yan, Z. Liu, X. Bao and B. Tan. "Transcriptome and proteome association analysis to screen candidate genes related to salt tolerance in reaumuria soongorica leaves under salt stress." Plants 12(2023); Yan, S., P. Chong, M. Zhao and H. Liu. "Physiological response and proteomics analysis of reaumuria soongorica under salt stress." Sci.Rep. 12(2022)). By adding H2 S (Liu, H., P. Chong, Z. Liu, X. Bao and B. Tan. "Exogenous hydrogen sulfide improves salt stress tolerance of reaumuria soongorica seedlings by regulating active oxygen metabolism." PeerJ 11 (2023)), NO, ABA, and Ca 2+ and NO compound treatment (Liu, Z., H. Liu, B. Tan, X. Wang and P. Chong. "Mitigation of salt stress in reaumuria soongarica seedlings by exogenous ca 2+ and no compound treatment." Agronomy 13 (2023)) and other methods to alleviate the impact of salt stress on Reaumuria soongorica seedlings. However, research on PGPR that can be used to promote the growth of Reaumuria soongorica and improve saline-alkali land in desert areas is relatively rare. Studies have confirmed that plant growth-promoting rhizobacteria (PGPR) can induce plants to recruit some microorganisms to colonize in the rhizosphere and interact with plants to enhance tolerance to salt stress. As an inoculant, PGPR alleviates plant salt stress by producing phytohormones, activating the plant antioxidant system, and increasing soil nutrient availability. For example, dual inoculation of ACC deaminase-producing Pseudomonas and Bradyrhizobium can more effectively improve the salt tolerance of soybeans by reducing salt-induced ethylene production and enhancing nutrient uptake (Win, K. T., S. Wasai-Hara, F. Tanaka, A. Z. Oo, K. Minamisawa, Y. Shimoda and H. Imaizumi-Anraku. "Synergistic n 2- Fixation and salt stress mitigation in soybean through dual inoculation of ACC deaminase - producing Pseudomonas and Bradyrhizobium. "Sci. Rep. 13 (2023)). Inoculation with Bacillus thuringiensis PM25 improves the oxidative damage of salt stress in maize by regulating growth, leaf pigments, antioxidant defense system, and stress - responsive gene expression (Ali, B., A. Hafeez, S. Ahmad, M. A. Javed, Sumaira, M. S. Afridi, T. M. Dawoud, K. S. Almaary, C. C. Muresan, R. A. Marc, et al. "Bacillus thuringiensis pm25 ameliorates oxidative damage of salinity stress in maize via regulating growth, leaf pigments, antioxidant defense system, and stress responsive gene expression." Front Plant Sci 13 (2022)). These studies mainly focus on agricultural crops, while there are few studies on PGPR of desert plants in saline - alkali areas. Therefore, it is of great significance to explore ecological strain resources suitable for desert saline - alkali land and to reveal the stress resistance enhancement of plants by PGPR. At the same time, it provides a theoretical basis for the combined remediation of saline - alkali land by salt - tolerant plants and PGPR.

[0004] In addition, soil microorganisms play an important role in driving the biogeochemical cycles of carbon (C), nitrogen (N), and phosphorus (P) and responding to environmental changes. The C, N, and P that can be directly utilized by plants in the soil are very limited and must be transformed before being absorbed by plants. Rhizosphere microorganisms secrete various enzymes to drive the transformation of C, N, and P. Studies have found that PGPR - mediated increase in soil nutrient availability improves plant nutrient uptake, thus promoting plant growth. For example, nitrogen - fixing bacteria can convert atmospheric nitrogen into NO 3- or NH 4+(Mukherjee, S. and S. K. Sen. "Exploration of novel rhizospheric yeast isolate as fertilizing soil inoculant for improvement of maize cultivation." J. Sci. Food Agric. 95 (2015)), phosphate-solubilizing bacteria can hydrolyze phosphorus in a difficult-to-use form into an absorbable form (Amri, M., M. R. Rjeibi, M. Gatrouni, D. M. R. Mateus, N. Asses, H. J. O. Pinho and C. Abbes. "Isolation, identification, and characterization of phosphate-solubilizing bacteria from tunisian soils." Microorganisms 11 (2023)). Research has shown that inoculation with AM fungi significantly increased the copy numbers of nifH, amoA-AOA, narG, nirK, and nosZ genes under different plant diversity gradients (Wang, J., J. Wang, J. Z. He, Y. G. Zhu, N. H. Qiao and Y. Ge. "Arbuscular mycorrhizal fungi and plant diversity drive restoration of nitrogen-cycling microbial communities." Mol. Ecol 30 (2021): 4133-46.). The combined application of Bacillus sp. T28 with sea buckthorn pomace can increase the abundances of soil nitrogen functional genes nifH, narG, nirS, nirK, and nosZ (Yang, X., Q. Wan, D. Wu, J. Wang, T. Abbas and Q. Zhang. "The impact of novel azotobacter bacillus sp. T28 combined sea buckthorn pomace on microbial community structure in paddy soil." Environ. Res 224 (2023)). The accumulation of these beneficial bacteria increased the abundances of C, N, and P cycling functional genes in the soil, enhanced the ability of plants to fully utilize environmental nutrients, and promoted plant growth. However, the regulation of element cycling by soil microorganisms is a complex process, and only a few studies have investigated the effects of inoculated microorganisms on gene abundances and enzyme activities during element cycling processes.In particular, little is known about the interaction mechanisms among C, N, P cycling functional genes, rhizosphere microorganisms, and nutrients.

[0005] There is competition between foreign microorganisms and indigenous microorganisms, and PGPR can only function stably when adapted to local soil conditions. However, rhizosphere-promoting bacteria suitable for vegetation restoration in desert ecosystems and saline soil improvement are extremely rare, and there has been no public report on rhizosphere-promoting bacteria specifically for the desert plant Reaumuria soongorica. Summary of the Invention

[0006] Object of the Invention: To solve the above technical problems, the first object of the present invention is to provide a Bacillus tequilensis QingTuHuS40; the second object of the present invention is to provide a strain fermentation broth containing the Bacillus tequilensis QingTuHuS40; the third object of the present invention is to provide a microbial preparation containing the Bacillus tequilensis QingTuHuS40 or its strain fermentation broth; the fourth object of the present invention is to provide the application of the Bacillus tequilensis QingTuHuS40 or its strain fermentation broth or its biological preparation in improving saline-alkali soil or promoting plant growth.

[0007] Technical Solution: A Bacillus tequilensis QingTuHuS40 of the present invention was deposited with the China General Microbiological Culture Collection Center (CGMCC) on January 8, 2025, with the deposit number CGMCC No. 33325 and the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the taxonomic name being: Bacillus tequilensis QingTuHuS40.

[0008] Furthermore, the 16S rDNA sequence of the Bacillus tequilensis QingTuHuS40 is as shown in SEQ ID NO.1.

[0009] The strain fermentation broth of the present invention includes the Bacillus tequilensis QingTuHuS40 of the present invention.

[0010] The microbial agent of the present invention is characterized by comprising Bacillus tequilensis QingTuHuS40 described in the present invention or the fermentation broth of the strain described in the present invention.

[0011] Use of Bacillus tequilensis QingTuHuS40 described in the present invention, the fermentation broth of the strain described in the present invention or the microbial agent described in the present invention in improving soil fertility.

[0012] Use of Bacillus tequilensis QingTuHuS40 described in the present invention, the fermentation broth of the strain described in the present invention or the microbial agent described in the present invention in improving saline-alkali soil.

[0013] Further, the saline-alkali soil is saline-alkali soil in desert areas.

[0014] Use of Bacillus tequilensis QingTuHuS40 described in the present invention, the fermentation broth of the strain described in the present invention or the microbial agent described in the present invention in promoting plant growth.

[0015] Use of Bacillus tequilensis QingTuHuS40 described in the present invention, the fermentation broth of the strain described in the present invention or the microbial agent described in the present invention in reshaping the structure of the plant rhizosphere bacterial community.

[0016] Further, the plant is Reaumuria soongorica, Caroxylon passerinum, Lycium ruthenicum or Atraphaxis bracteata A. Los.

[0017] Mechanism of Bacillus tequilensis QingTuHS40 in promoting the growth of Reaumuria soongorica seedlings and enhancing salt tolerance: Bacillus tequilensis QingTuHS40 improves soil fertility and alleviates soil salinization by reshaping the structure of the rhizosphere bacterial community and regulating the abundance of functional genes, thereby promoting the growth of Reaumuria soongorica and enhancing its salt tolerance. As Figure 10 shown,

[0018] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0019] The Bacillus tequilensis QingTuHS40 of the present invention can colonize in the rhizosphere of Reaumuria soongorica, and at the same time recruit some other beneficial bacteria to reshape the bacterial community structure in the rhizosphere of Reaumuria soongorica. At the same time, it improves soil fertility, ameliorates soil salinization, enhances the ability of plants to fully utilize environmental nutrients, promotes plant growth, and enhances salt tolerance. The Bacillus tequilensis QingTuHS40 of the present invention is mainly used for the cultivation of desert plants and afforestation in desert saline areas, etc. Brief Description of the Drawings

[0020] Figure 1 It is a diagram of the colony morphological characteristics of Bacillus tequilensis QingTuHuS40 and the microscopic observation results of Gram staining. Among them, A is the diagram of colony morphological characteristics, and B is the Gram staining microscope diagram.

[0021] Figure 2 It is a phylogenetic tree of the 16S rDNA gene sequence of Bacillus tequilensis QingTuHuS40. Among them, the superscript T represents the type strain, and the scale bar length is 2% base difference;

[0022] Figure 3 It is a diagram of the effects of NaCl stress and Bacillus tequilensis QingTuHuS40 on the growth and biomass of Reaumuria soongorica seedlings. Among them, A is a photo of Reaumuria soongorica under different treatments, B is plant height, C is total root length, and D is the dry weight of roots, stems and leaves;

[0023] Figure 4 It is a diagram of the effects of NaCl stress and Bacillus tequilensis QingTuHuS40 on the bacterial community structure in the rhizosphere of Reaumuria soongorica. Among them, A - B are stacked bar charts of species composition (at phylum level and genus level); C - E are α - diversity indices (Chao1, Shannon_2, Simpson) respectively; D is the β - diversity index (NMDS); is a cluster analysis chart of species abundance; G is a phylogenetic branching chart of species; I is a stacked bar chart of LDA value distribution;

[0024] Figure 5 It is an NMDS analysis and stacked chart of gene absolute abundances of soil samples functional genes. Among them, A is the NMDS analysis, and B is the stacked chart of gene absolute abundances;

[0025] Figure 6It is a graph showing the changes in the abundances of C-cycle related functional genes under different treatments. Among them, A is a schematic diagram of the C-cycle process, B is the absolute abundances of C-degradation functional genes (Top 6), and C is the absolute abundances of C-fixation functional genes (Top 6);

[0026] Figure 7 It is a graph showing the changes in the abundances of N-cycle related functional genes under different treatments. Among them, A is a schematic diagram of the N-cycle process, and B is the absolute abundances of N-cycle functional genes (Top 6);

[0027] Figure 8 It is a graph showing the changes in the abundances of P-cycle related functional genes under different treatments. Among them, A is a schematic diagram of the P-cycle process, and B is the absolute abundances of P-cycle functional genes (Top 6);

[0028] Figure 9 It is a graph showing the correlation analysis of the growth indicators of Reaumuria soongorica, environmental factors, and functional genes by Mantel test;

[0029] Figure 10 It is the mechanism by which Bacillus tequilensis QingTuHS40 promotes the growth of Reaumuria soongorica seedlings and enhances salt tolerance. Specific implementation manners

[0030] The technical solutions of the present invention will be further described below with reference to the accompanying drawings.

[0031] The materials, reagents, etc. used in the following examples can be obtained from commercial channels without special instructions. The experimental methods without specific conditions in the examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0032] Example 1

[0033] 1. Isolation and screening of Bacillus tequilensis QingTuHuS40

[0034] Bacillus tequilensis QingTuHuS40 was purified and isolated from the soil collected from the gobi around Qingtuhu Lake in the Shiyang River Basin in the northwest region.

[0035] Separation and screening process: High-throughput bacterial cultivation and identification methods (Zhang, J.Y.; Liu, Y.X.; Guo, X.X. et al. (2021) High-throughput cultivation and identification of bacteria from the plant root microbiota. Nature Protocols 16, (2021): 988-1012.) were used to separate and screen PGPR that produce IAA. Weigh 10 g of the collected soil and dissolve it in 90 ml of sterilized physiological saline, shake and culture it in a shaker at 30 °C and 180 r / min for 30 min, and sequentially prepare dilutions of 10 -3 , 10 -4 and 10 -5 by serial dilution. Inoculate different concentrations of the dilution into a 96-well Lauria Bertani (LB) liquid culture plate. If 30%-40% of the wells in the culture plate show visible bacterial growth after the incubation period, it can be considered that each culture is derived from a single cell, and thus a large proportion of pure cultures can be produced. Then transfer the pure cultures to two 96-well LB liquid culture plates containing 0.1% L-tryptophan and culture them at 30 °C for 2 d. Then, add an equal volume of Salkowski reagent (prepared from 50 mL of 35% perchloric acid and 1 mL of 0.5 M FeCl 3 solution) to one of the culture plates. After 30 min of color development in the dark, a pink color indicates the ability to produce IAA. Select the cultures in the corresponding wells with darker color development from the other culture plate for streak purification, then culture them at 30 °C for 3 d, pick single colonies, and use the streak method for isolation and purification, and store them in a -80 °C refrigerator.

[0036] 2. Biological characteristics description of Bacillus tequilensis QingTuHuS40: Isolate single colonies by the streak plate method and observe the morphological characteristics of the single colonies of bacteria. Dip a small amount of bacterial liquid for smear and perform Gram staining, and observe the bacterial morphology and staining characteristics under an optical microscope. The morphological characteristics of Bacillus tequilensis QingTuHuS40 are shown in Figure 1 . Results of strain morphological observation and Gram staining: All Gram stainings were positive, short rod-shaped, with blunt ends at both ends, appearing in pairs or in strings. On the LB plate, the colonies were yellow, smooth, moist on the surface, and irregular at the edges. The colonies were opaque and the edges were uneven, and it was preliminarily determined to be the genus Bacillus.

[0037] All were Gram-positive, short rod-shaped, with blunt ends, occurring in pairs or in strings. On LB plates, the colonies were yellow, smooth, moist on the surface, and irregular at the edges. The colonies were opaque and had an uneven edge (see Figure 1 ), and were preliminarily identified as Bacillus.

[0038] 2. Molecular biology identification information and construction of phylogenetic tree

[0039] The genomic DNA of Bacillus tequilensis was extracted using a bacterial DNA rapid extraction kit (Fangzhou DZ314-03, Guangzhou Fangzhou Biosafety Technology Co., Ltd.). PCR amplification was performed using universal primers for bacterial 16S rDNA, primer 27F (SEQ ID NO.2), 1492R (SEQ ID NO.3). The amplified product was sent to Guangdong Megagenomics Technology Co., Ltd. for sequencing. Through 16S rDNA sequencing, the following sequence SEQ ID NO.1 was obtained:

[0040]

[0041] SEQ ID NO.2: 27F (5'-AGAGTTTGATCMTGGCTCAG-3');

[0042] SEQ ID NO.3: 1492R (5'-TACGGYTACCTTGTTACGACTT-3');

[0043] The 16S rDNA sequences of 7 strains in the same family (Bacillaceae) as Bacillus tequilensis QingTuHuS40 in the GenBank database were selected. A phylogenetic tree was constructed using the Maximum Likelihood Estimate method on MEGA 11.0. The General Time Reversible (GTR) parameter model was used for the analysis, and bootstrap sampling was performed 500 times. By comparison, this sequence was found to be T similar to the strain Bacillus tequilensis KCTC 13622 with known genus and species information, with a Query Cover value of 100%, an E value of 0.0, and a Per.Ident value of 100%. The phylogenetic tree (see Figure 2 ) results showed that the isolated strain was closest to Bacillus tequilensis KCTC 13622 T and was on the same branch. The distance to Cetobacterium sp. ZF1NH01 was the second closest. It was preliminarily identified as Bacillus tequilensis and named Bacillus tequilensis QingTuHuS40. This strain was sent to the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, for preservation on January 8, 2025, with the preservation number CGMCC No. 33325.

[0044] Example 2

[0045] 1. Cultivation and growth conditions of Reaumuria soongorica seedlings

[0046] From April to November 2023, an experiment on regulating the rhizosphere bacterial community structure and function of Reaumuria soongorica by Bacillus tequilensis QingTuHuS40 was carried out in the experimental base of Gansu Agricultural University. Reaumuria soongorica seeds of the same size and plump grains were selected and disinfected with 1% sodium hypochlorite solution for 30 min, rinsed 5 times with sterile water, and sown in plastic flower pots with a diameter of 24.8 cm, a height of 28 cm, and containing 5 kg of substrate in April 2023. The potting substrate was a mixture of soil, sandy soil, and humus (weight ratio 1:2:1). After the seedlings grew stably (plant height reached 5 cm), thinning was carried out, and 1 plant was retained in each pot, for a total of 30 pots. The Reaumuria soongorica seedlings grew under natural conditions in the experimental field of Gansu Agricultural University, located in Anning District, Lanzhou City, Gansu Province (36°5′N, 103°42′E), belonging to the mid-temperate climate zone, with obvious continental climate characteristics, distinct seasons, sufficient sunlight, and dry climate.

[0047] 2. Experimental design and treatment

[0048] The application method of Bacillus tequilensis QingTuHuS40 in promoting the growth of Reaumuria soongorica. Among them, it can be applied by preparing a bacterial suspension of Bacillus tequilensis QingTuHuS40, and its preparation may include the following steps: Inoculate Bacillus tequilensis QingTuHuS40 in LB, culture at 28 °C for 48 h, harvest the culture, dilute it with sterile physiological saline (0.9% NaCl) to prepare a bacterial inoculum, and dilute it to a final OD of 1.0 at 600 nm (~109 CFU mL -1 )). The above-mentioned application method for promoting the growth of Reaumuria soongorica is to apply the bacterial inoculum to the rhizosphere of Reaumuria soongorica. The application method of applying it to the rhizosphere of Reaumuria soongorica is irrigation.

[0049] The experiment set up a control (CK, without adding NaCl and the growth-promoting bacterium Bacillus tequilensis QingTuHuS40), a salt treatment (S, only adding 400 mM NaCl), a single treatment with Bacillus tequilensis QingTuHuS40 (S40), and a combined treatment with salt and Bacillus tequilensis QingTuHuS40 strain (S40+S). There were 3 replicates for each treatment. Sodium chloride (NaCl) is one of the main salts in saline soils (see Chen, J.-T. et al. (2021). Molecular aspects of plant salinity stress and tolerance). Therefore, 400 mM NaCl was selected for salt stress treatment in this experiment. In July 2023, seedlings with consistent growth (plant height reaching 18 - 20 cm) were selected for treatment. Each pot was watered once a day with 500 mL of 400 mM NaCl solution for 3 consecutive days, with a total amount of 1500 mL. The control and single-bacterium treatments were watered with an equal amount of water. After 2 days of NaCl stress treatment, at Bacillus tequilensis KCTC 13622 T 100 mL of the inoculum of Bacillus tequilensis QingTuHS40 was inoculated into the roots of the plants, and the control was inoculated with an equal amount of sterile saline.

[0050] 3. Sample collection and determination of growth indices

[0051] Samples were taken after 56 days of treatment of Reaumuria soongorica seedlings. Before sampling, photos were taken with a digital camera (canon EOS 6D, canon Inc., Japan), and the plant height of Reaumuria soongorica seedlings was measured with a scale; the soil and plant roots in each pot were carefully poured out, large pieces of soil were shaken off, the soil attached to the root surface was gently shaken and mixed evenly, placed in a 15 mL sterile and enzyme-free cryotube, immediately frozen in liquid nitrogen, and transferred to a -80 °C refrigerator in the laboratory for storage to analyze the rhizosphere soil microbial community structure and evaluate the potential of element cycling function. The aboveground and underground parts of the plants were collected separately, brought back to the laboratory in a low-temperature sampling box, washed with pure water, and then the surface moisture was blotted dry with filter paper, and their fresh weights were weighed respectively. The total root length was determined using the Wanshen LA-S plant root analysis system. The roots, stems, and leaves were separated, blanched at 105 °C for 30 min, and dried to a constant weight at 80 °C. Then, the biomasses of the roots, stems, and leaves were measured with a digital balance with a precision of 0.0001 g. All the soil in the pot was mixed evenly, sampled by the quartering method, air-dried, ground, and sieved (100 mesh) for subsequent determination of basic soil physical and chemical indices.

[0052] The effects of NaCl stress and Bacillus tequilensis QingTuHS40 on the growth and biomass of Reaumuria soongorica seedlings are shown in Figure 3 . As can be seen from Figure 3 , under NaCl stress, the plant height, total root length, and dry weights of roots, stems, and leaves of Reaumuria soongorica seedlings were lower than those of the control group (CK). The plant height, total root length, and dry weights of roots, stems, and leaves of Reaumuria soongorica seedlings inoculated with Bacillus tequilensis QingTuHS40 were higher than those of the control group (CK). Among them, the total root length and dry weight of leaves increased significantly by 29.62% and 37.71% (P<0.05). When Bacillus tequilensis QingTuHuS40 and NaCl were co-treated, the plant height, total root length, and dry weights of leaves, stems, and roots of Reaumuria soongorica seedlings increased significantly by 13.36%, 20.93%, 246.30%, 142.38%, and 42.33% compared with the NaCl treatment group (S), and all recovered to the control level except for the plant height.

[0053] 4. Determination of soil physical and chemical properties and enzyme activities

[0054] Air-dried soil collected from the pot experiment was taken respectively, and the soil pH value was measured according to the instructions of the Sartorius PB-10 pH meter, and the conductivity was measured using a Leici DDSJ308F conductivity meter; total carbon (TC) and total nitrogen (TN) were measured using an elemental analyzer; the content of soil organic matter (SOM), total phosphorus (TP), total potassium (TK), available phosphorus (AP), and available potassium (AK) in rhizosphere soil were measured by dichromate volumetric method, NaOH fusion-molybdenum antimony anti-colorimetry, NaOH fusion-flame photometry, sodium bicarbonate method, and ammonium acetate extraction-flame photometer method respectively (Bao Shidan, Soil Agricultural Chemistry Analysis, Third Edition, Beijing: China Agricultural Press, 2000); the activities of soil urease, alkaline phosphatase, sucrase, and catalase were measured by phenol sodium-sodium hypochlorite colorimetry, disodium phenyl phosphate colorimetry, 3,5-dinitrosalicylic acid colorimetry, and potassium permanganate titration method respectively (Guan Songyin, Soil Enzymes and Their Research Methods, Beijing: Agricultural Press, 1986). The results are shown in Table 1.

[0055] NaCl stress and inoculation with Bacillus tequilensis QingTuHuS40 had significant effects on most soil physical and chemical properties and soil enzyme activities (see Table 1). Under NaCl stress, the soil conductivity, Na + content, alkaline phosphatase, and catalase activities increased significantly compared with the control group, while the contents of organic matter, available phosphorus, and available potassium, and urease activity decreased significantly compared with the control group. NaCl stress and Bacillus tequilensis

[0056] Under the combined treatment of QingTuHuS40, the contents of soil organic matter, total carbon, available phosphorus, available potassium and Na + content, urease and sucrase activities increased significantly, while the electrical conductivity and catalase activity decreased significantly. After inoculating Bacillus tequilensis QingTuHuS40, the soil pH value decreased, but there was no significant difference. There was no significant difference in soil total nitrogen, total phosphorus and total potassium among treatments. It shows that short-term NaCl stress and Bacillus tequilensis QingTuHuS40 treatment have obvious effects on organic matter, available potassium, available phosphorus and soil enzyme activities, but have no obvious effects on total nitrogen, total phosphorus and total potassium.

[0057] Table 1 Soil physical and chemical properties and soil enzyme activities

[0058]

[0059]

[0060] 5. Analysis of rhizosphere microbial composition and diversity

[0061] The composition and diversity of soil microorganisms were analyzed by amplifying and sequencing the 16S rRNA V4-V5 variable region genes of rhizosphere soil bacteria using high-throughput sequencing technology. The library construction and sequencing work were entrusted to Guangdong Megagene Biotechnology Co., Ltd. The down-stream data was analyzed using the EasyAmyplicon amplicon analysis platform developed by the team of Yong-Xin Liu from the Institute of Genomics, Chinese Academy of Agricultural Sciences (Liu, Y.X., L. Chen, T. Ma, X., et al. "Easyamplicon: An easy-to-use, open-source, reproducible, and community-based pipeline for amplicon data analysis in microbiome research." iMeta 2(2023)) and the Megagene Cloud platform (http: / / cloud.magigene.com / ). The DADA2 in QIIME2 (2022.8) software was used for quality control, denoising, splicing, chimera removal and generation of OTUs feature table. The Silva database (Release132) was used for bacterial 16S rRNA genes, and the UNITE database (Release8.0) was used for fungal ITS sequences. The classify-sklearn algorithm of QIIME2 was used to annotate the species of the characteristic sequences of each OTUs. The results are as Figure 4 shown.

[0062] NaCl stress and Bacillus tequilensis QingTuHuS40 had significant effects on the rhizosphere bacterial community structure of Reaumuria soongorica seedlings (see Figure 4 ). Five phyla, Proteobacteria, Bacteroidota, Planctomycetota, Acidobacteriota, and Chloroflexi, were dominant in the rhizosphere soil of different treatment groups, with a cumulative relative abundance exceeding 80% ( Figure 4 A in). Under the combined treatment of NaCl and Bacillus tequilensis QingTuHuS40, the relative abundances of Proteobacteria (45.47%), Bacteroidota (20.44%), and Planctomycetota (13.12%) were significantly increased compared with the control group and the NaCl treatment group, while the relative abundances of Acidobacteriota (6.83%) and Chloroflexi (5.51%) were decreased. At the genus level, there were significant differences in the abundances of bacterial species among different treatment groups (see Figure 4 B in). Under NaCl stress, the abundance of Pseudomonas was significantly increased compared with the control group. Under the combined treatment of NaCl and Bacillus tequilensis QingTuHuS40, the relative abundances of four genera, Stenotrophomonas, Planctomicrobium, Pseudomonas, and SJA-28, were significantly increased compared with the control group, while Vicinamibacteraceae, MND1, and Pirellula decreased significantly (p<0.05). In addition, the abundance of Firmicutes species decreased under NaCl stress, while after inoculation with Bacillus tequilensis QingTuHuS40, the abundance of Firmicutes species was significantly increased compared with the control group (p<0.05). Compared with the control, both NaCl stress and inoculation with Bacillus tequilensis QingTuHuS40 decreased the richness and diversity of rhizosphere soil bacteria ( Figure 4 C in, Figure 4 D in, Figure 4In E). The Chao1 index of the control group was higher than that of other treatment groups, indicating that the species richness was the largest in the control group. In the presence of NaCl, the Shannon index of the inter-group community decreased, and the Simpson index increased, both without significant differences. NMDS separated the rhizosphere bacteria in the soils of different treatment groups, and those inoculated with Bacillus tequilensis QingTuHuS40 were close, with the control group in the middle (see Figure 4 In F). The results of LEfSe (Linear discriminant analysis Effect Size) analysis showed that there were 20 biomarkers with an LDA score > 3.5 ( Figure 4 In G, Figure 4 In I). Among these biomarkers, 12 taxa were present in the rhizosphere soil of the control group, and the most contributing bacterial groups were Acidobacteriota, Vicinamibacteria, and Vicinamibacterales. Six biomarker taxa were present in the rhizosphere soil of the co-treatment group of NaCl and Bacillus tequilensis QingTuHuS40, and the most contributing bacterial groups were Stenotrophomonas, Flavobacteriales, and Devosiaceae. One biomarker taxon was present in the rhizosphere soil of the NaCl treatment group and the Bacillus tequilensis QingTuHuS40 single-treatment group, which were Armatimonadota and Microgenomatia, respectively. The results showed that NaCl and Bacillus tequilensis QingTuHuS40 could significantly change the species composition and distribution of the rhizosphere soil bacteria of Reaumuria soongorica seedlings, reduce the species richness and diversity, and the impact on the species composition was higher than that on the species richness and diversity.

[0063] 6. Quantitative Microbial Element Cycling (QMEC)

[0064] QMEC is a method based on high-throughput qPCR functional gene chips to evaluate and quantify the genetic potential of microbiota to mineralize soil organic matter and release C, N, and P (Zheng, B., Y. Zhu, J. Sardans, J. and J.Su."Qmec:A tool for high-throughput quantitative assessment of microbial functional potential in c,n,p,and s biogeochemical cycling."Sci.China Life Sci 61(2018):1451-62.)。In this experiment, QMEC was used to quantify a total of 63 genes related to C, N, and P cycling, including 31 functional genes related to C cycling, 22 related to N cycling, 9 related to P cycling, and one 16S rRNA gene (Table 2). Details of the measurements and primers are as in (Zheng, B., Y. Zhu, J. Sardans, J. The same as described in J. Su. "Qmec: A tool for high-throughput quantitative assessment of microbial functional potential in c, n, p, and s biogeochemical cycling." Sci. China Life Sci 61 (2018): 1451-62). Sample DNA was extracted according to the instructions of the magnetic bead method soil DNA extraction kit (FINOROPDC306-03, Guangzhou Ark Biosafety Technology Co., Ltd.). After extraction, the total amount and purity of DNA were detected using a Qubit 4.0 (Thermo Fisher Scientific, Waltham, USA) instrument to ensure that the DNA concentration was uniformly diluted to 20 ng / μl. qPCR reactions and fluorescence signal detections were carried out in a SmartChip Real-Time PCR System (WaferGen Biosystems USA), and amplification curves and melting curves were automatically generated. qPCR reaction system and reaction conditions: 10 μl of fluorescent dye (RocheLightCycler 480 SYBR Green I Maste), 0.4 μl of each primer, 7.2 μl of nuclease-free water, 2 μl of sample DNA, for a total of 20 μl; initial enzyme activation was carried out at 95 °C for 5 minutes, followed by 40 cycles of 95 °C denaturation for 15 seconds, 60 °C annealing for 60 s, and 72 °C extension for 20 seconds, and finally 95 °C for 15 s and 60 °C for 60 s. All qPCR reactions were carried out three times, and each run included a non-template negative control. After standardizing the data using 16S rRNA as an internal reference, the relative quantitative information of each gene in each sample was collected. The absolute value of the 16S rRNA gene was obtained based on the quantitative information of the Roche instrument. The results are as Figure 5 shown.

[0065] Table 2 Element cycling functional genes measured in the experiment

[0066]

[0067] Composition of rhizosphere soil functional genes:

[0068] The differences in the composition of functional genes under different treatments were evaluated using NMDS ( Figure 5In A). Under NaCl stress, the functional gene composition was significantly separated from other groups on axis 1. The functional gene composition of the co-treatment group with NaCl stress and inoculation of Bacillus tequilensis QingTuHuS40 was close to the control group on axis 1 and separated on axis 2. Under NaCl stress, the abundances of functional genes related to the cycling of C, N, and P elements were lower than those of the control, while after inoculation with Bacillus tequilensis QingTuHuS40, the abundances of functional genes related to the cycling of C, N, and P elements increased significantly compared with the control ( Figure 5 In B). The results showed that Bacillus tequilensis QingTuHuS40 could mediate the expression of functional genes in the rhizosphere soil of Reaumuria soongorica seedlings.

[0069] Carbon cycling process and key gene abundances:

[0070] Functional genes involved in carbon cycling include C fixation and C degradation genes (see Table 2; Figure 6 In A). Among different treatment groups, the expressions of these genes showed obvious differences. 23 out of 31 genes were significantly different. Among them, 12 genes were involved in carbon degradation (the top 6 in absolute abundance are shown in Figure 6 In B), including genes involved in starch hydrolysis (sga), hemicellulose hydrolysis genes (abfA, xylA, and manB), and lignin hydrolysis (mnp and glx) genes. 11 genes were involved in carbon fixation (the top 6 in absolute abundance are shown in Figure 6 In C), including acetyl-CoA synthase gene (acsA), gene for synthesizing succinyl-CoA (smtA), gene for the α-subunit of oxidoreductase formyltransferase (mct), 5-methyltetrahydrofolate convertase gene (acsE), gene for synthesizing ribulose bisphosphate carboxylase (rbcL), and fumarate reductase flavoprotein gene (frdA). Under NaCl salt stress, the abundances of C degradation genes sga, mnp, manB, glx, and mct were significantly lower than those of the control group. The gene mct involved in C fixation was significantly lower than that of the control group, while the gene frdA was significantly higher than that of the control. Under the co-treatment of NaCl and Bacillus, the abundances of genes sga, abfA, xylA, mnp, manB, glx, acsA, smtA, mct, acsE, rbcL were significantly increased compared with the NaCl treatment. Generally speaking, whether NaCl is present or not, inoculation with Bacillus tequilensis QingTuHuS40 will increase the abundances of genes related to C degradation and C fixation.

[0071] Nitrogen cycling process and key gene abundances:

[0072] The functional genes of the N cycle are involved in N metabolic pathways such as nitrogen fixation, nitrification, denitrification, ammonium assimilation, and organic nitrogen mineralization (see Table 2; Figure 7 in A). Among different treatment groups, 18 out of 22 N cycle-related genes showed significant differences. The Figure 7 absolute abundances of the top 6 genes in different treatment groups are shown in B, including the urease synthesis gene (ureC), the genes encoding nitrous oxide reductase (nosZ1, nosZ2), the genes encoding nitrite reductase (nirK3, nirS1), and the glutamate dehydrogenase gene (gdhA). The ureC and gdhA genes are involved in the conversion of urea to ammonia and the process of organic nitrogen mineralization, respectively. nosZ1, nosZ2, nirK3, and nirS1 are all involved in the denitrification process. Therefore, after inoculation with Bacillus, denitrification becomes the dominant pathway of the nitrogen cycle. Under NaCl salt stress, the absolute abundances of the nosZ1 and nosZ2 genes increased significantly compared with the control group, while the abundances of nirK3 and gdhA decreased significantly compared with the control group. Salt stress has a significant impact on N cycle-related genes. After inoculation with Bacillus, the absolute abundances of these six genes all increased significantly compared with the control. The results indicate that Bacillus tequilensis QingTuHuS40 plays an important role in regulating the N metabolism of rhizosphere bacteria of Reaumuria soongorica.

[0073] Phosphorus cycle process and key gene abundances:

[0074] The functional genes related to the P cycle are involved in P metabolic pathways such as inorganic phosphorus solubilization, organic phosphorus mineralization, and phosphorus absorption and transport (see Table 2; Figure 8 in A). Among different treatment groups, 8 out of 9 P cycle-related genes showed significant differences. The genes with the highest absolute abundances in different treatment groups are phnK, phoD, phoX, ppx, gcd, and pqqC, which are the ATP-binding protein gene of the phosphate transport system (phnK), the alkaline phosphatase genes involved in organic phosphorus mineralization (phoD and phoX), the exopolyphosphatase gene involved in the conversion of polyphosphate to inorganic phosphorus (ppx), the quinoprotein glucose dehydrogenase gene involved in inorganic phosphorus solubilization (gcd), and the pyrroloquinoline-quinone synthase gene (pqqC). Under NaCl salt stress, except for phnK, the absolute abundances of the gcd, pqqC, phoX, ppx, and phoD genes decreased significantly compared with the control group (p < 0.05). After inoculation with Bacillus tequilensis QingTuHS40, the absolute abundances of the phnK, gcd, pqqC, phoX, ppx, and phoD genes all increased significantly. The results indicate that the functional genes related to the P cycle are relatively sensitive to the external environment. NaCl stress will inhibit the gene expression, while beneficial bacteria will activate the gene expression.

[0075] Correlation between soil properties and functional genes related to bacterial community composition and element cycling:

[0076] The results of the Mantel test showed the correlation between soil properties and functional genes related to bacterial community composition and element cycling (see Figure 9 ). The growth indexes of Reaumuria soongorica seedlings (PH, LBM, SBM, and RBM) were positively correlated with soil properties (SOM, AP, AK, UA) (r > 0.4), and negatively correlated with EC and CA (r < -0.2). EC and pH were negatively correlated with AP, AK, UA, and APA (r < -0.2), and EC was positively correlated with Na and pH (r > 0.3). SOM was positively correlated with AP, AK, Na, UA, SA, and APA (r > 0.3). Soil enzyme activities (UA, SA, and APA) were positively correlated with SOM, TP, AP, AK, and Na (r > 0.2), while CA was negatively correlated with pH, SOM, AP, and AK (r < -0.3). The community composition of Planctomycetota was extremely significantly correlated with LBM, SOM, and AP (p < 0.01), and significantly correlated with SBM, RBM, AK, UA, and SA (p < 0.05). The community composition of Bacteroidota was significantly correlated with PH, EC, and Na (p < 0.05). The community compositions of Proteobacteria and Acidobacteriota were significantly correlated with SA (p < 0.05). The community composition of Chloroflexi was significantly correlated with AP (p < 0.05). The functional genes related to C, N, and P element cycling were extremely significantly correlated with LBM, SOM, AP, and SA (p < 0.01), significantly correlated with SBM, RBM, and UA (p < 0.05), and had no obvious relationship with pH, EC, TP, TK, and Na. The C degradation and P cycling functional genes were significantly correlated with PH and CA (p < 0.05). The C fixation functional gene was significantly correlated with APA (p < 0.05), and the P cycling functional gene was extremely significantly correlated with APA (p < 0.01).

Claims

1. A Bacillus tequilensis QingTuHuS40, characterized in that: The Bacillus tequilensis QingTuHuS40 was deposited in the China General Microbiology Center (CGMCC) on January 8, 2025, with the deposit number CGMCC No.33325.

2. The Bacillus tequilensis QingTuHuS40 according to claim 1, characterized in that The 16SrDNA sequence of the Bacillus tequilensis QingTuHuS40 is shown in SEQ ID NO.

1.

3. A strain fermentation broth, characterized in that: It comprises the Bacillus equilensis QingTuHuS40 described in claim 1 or 2.

4. A microbial preparation, characterized in that: It comprises the Bacillus equilensis QingTuHuS40 described in claim 1 or 2 or the strain fermentation broth described in claim 3.

5. Use of the Bacillus tequilensis QingTuHuS40 according to claim 1 or 2, the strain fermentation liquid according to claim 3 or the microbial preparation according to claim 4 in improving soil fertility.

6. Use of the Bacillus tequilensis QingTuHuS40 according to claim 1 or 2, the strain fermentation liquid according to claim 3 or the microbial preparation according to claim 4 in improving salinized soil.

7. The use according to claim 6, characterized in that: Salinized soil is saline-alkali soil in desert areas.

8. Use of the Bacillus tequilensis QingTuHuS40 according to claim 1 or 2, the strain fermentation liquid according to claim 3 or the microbial preparation according to claim 4 in promoting plant growth.

9. Use of the Bacillus tequilensis QingTuHuS40 described in claim 1 or 2, the strain fermentation broth described in claim 3, or the microbial preparation described in claim 4 in reshaping the structure of plant rhizosphere bacterial communities.

10. The use according to claim 8 or 9, characterized in that: The plants are red sand, pearl hair grass, black wolfberry or sand wood polygonum.

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