Bacillus tequilensis qingtuhush40 and application thereof

Abstract

The application discloses Bacillus tequilensis QingTuHuS40 and application thereof, wherein the Bacillus tequilensis QingTuHuS40 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on January 8, 2025, and the preservation number is CGMCC No. 33325. The Bacillus tequilensis QingTuHuS40 can be planted in rhizosphere of red sand, recruit some other beneficial bacteria, and reshape the bacterial community structure in the rhizosphere of the red sand. Meanwhile, the soil fertility is improved, the degree of soil salinization is improved, the ability of plants to fully utilize environmental nutrients is enhanced, the growth of plants is promoted, and the salt tolerance is enhanced.

Description

TECHNICAL FIELD

[0001] The present application relates to Bacillus tequilensis QingTuHuS40 and its application, and belongs to the field of microorganisms. BACKGROUND

[0002] The theme of World Soil Day 2021 by the Food and Agriculture Organization of the United Nations is "Combating soil salinization, improving soil productivity", which implies the importance and necessity of efficient use of saline soil. The total area of saline soil in China is about 36.9 million hectares, accounting for 4.4% of the world's saline soil, mainly located in arid and semiarid climate zones, where rainfall is less and evaporation is large, which easily leads to soil salinization. Under salt stress, Na + and Cl - accumulate in large quantities, causing ion imbalance, producing reactive oxygen, leading to oxidative damage, affecting plant metabolic function disorder, and inhibiting 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.). At the same time, salinization is accompanied by nitrogen (N) and phosphorus (P) deficiency, which may affect the population structure and function of soil microorganisms. In the past, people have paid great attention to the improvement and effective use of saline soil, and have developed many strategies (including physical, chemical and biological) to improve saline-alkali soil. In agricultural production, planting salt-tolerant plants can effectively improve soil properties and improve crop productivity. Compared with other methods, bioremediation technology is an efficient, sustainable and environmentally friendly soil remediation method.

[0003] Reaumuria soongorica is a super-xerophyte and salt-tolerant shrub of the Tamaricaceae family, and is one of the key shrub species for wind prevention, sand fixation and maintaining the stability of the desert ecosystem in arid and semi-arid desert regions of Northwest China. In the process of maintaining the ecological balance of arid desert regions, 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 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, which has led to its 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 red sand 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 inreaumuria soongorica leaves under salt stress." Plants 12 (2023); Yan, S., P. Chong, M. Zhao and H. Liu. "Physiological response and proteomics analysis ofreaumuria soongorica under salt stress." Sci. Rep. 12 (2022)). By adding H2S (Liu, H., P. Chong, Z. Liu, X. Bao and B. Tan. "Exogenous hydrogen sulfide improves saltstress tolerance of reaumuria soongorica seedlings by regulating activeoxygen metabolism." PeerJ 11(2023)), NO, ABA, and Ca 2+ Combined treatment with NO (Liu, Z., H. Liu, B. Tan, X. Wang and P. Chong). Mitigation of salt stress in reaumuria soongaricaseedlings by exogenous ca 2+and no compound treatment."Agronomy 13(2023)) and so on. However, the research on PGPR that can be used to promote the growth of H. negevensis and improve the saline-alkali land in desert areas is relatively rare. Studies have shown that plant growth-promoting rhizobacteria (PGPR) can induce plants to recruit some microorganisms to colonize the rhizosphere and interact with plants to enhance tolerance to salt stress. As an inoculant, PGPR can alleviate plant salt stress by producing plant hormones, activating plant antioxidant systems, 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 improving nutrient uptake (Win, K. T., S. Wasai-Hara, F. Tanaka, A. Z. Oo, K. Minamisawa, Y. Shimoda, and H. Imaizumi-Anraku. "Synergistic n2-fixation and salt stress mitigation in soybean through dual inoculation of acc deaminase-producing pseudomonas and bradyrhizobium." Sci. Rep. 13 (2023)). Inoculation of 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 crops, and there is less research on PGPR for desert plants in saline-alkali areas. Therefore, it is of great significance to explore PGPR strains suitable for saline-alkali land in desert areas and reveal the mechanisms of PGPR enhancing plant stress resistance. At the same time, it provides a theoretical basis for the joint repair of saline-alkali land by salt-tolerant plants and PGPR.

[0004] Furthermore, soil microbes 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, P in soil that can be directly utilized by plants is very limited and must be transformed to be absorbed by plants. Rhizosphere microbes secrete various enzymes to drive the transformation of C, N, P. Studies have found that PGPR mediates the increase in soil nutrient availability to improve plant nutrient absorption, thereby promoting plant growth. For example, nitrogen-fixing bacteria can convert atmospheric nitrogen into plant-usable 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 are able to hydrolyze forms of phosphorus that are difficult to use into forms that can be absorbed (Amri, M., M. R. Rjeibi, M. Gatrouni, D. M. R. Mateus, N. Asses, H. J. O. Pinho, and C. Abbès. "Isolation, identification, and characterization of phosphate-solubilizing bacteria from tunisian soils." Microorganisms 11 (2023)). Studies have shown that inoculation with AM fungi significantly increases the copy number 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 combination of T28 Bacillus with sea buckthorn pomace can increase the abundance 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 increases the abundance of C, N, P cycle functional genes in the soil, enhances the ability of plants to fully utilize environmental nutrients, and promotes plant growth. However, the regulation of soil microorganisms on element cycling is a complex process, and only a few studies have investigated the effects of microbial inoculation on gene abundance and enzyme activity in the element cycling process.Especially, the interaction mechanism between C, N, P cycle functional genes, rhizosphere microorganisms and nutrients is poorly understood.

[0005] There is competition between foreign microorganisms and indigenous microorganisms, and PGPR can only play a stable role under the condition of adapting to local soil conditions. However, rhizosphere growth promoting bacteria suitable for desert ecosystem vegetation restoration and saline soil improvement are extremely rare, and there is no public report of rhizosphere growth promoting bacteria specially used for desert plants. SUMMARY

[0006] The present application aims at solving the above technical problems, the first object of the present application is to provide a Bacillus tequilensis QingTuHuS40, the second object of the present application is to provide a strain fermentation liquor containing the Bacillus tequilensis QingTuHuS40, the third object of the present application is to provide a microbial preparation containing the Bacillus tequilensis QingTuHuS40 or the strain fermentation liquor thereof, and the fourth object of the present application is to provide the application of the Bacillus tequilensis QingTuHuS40 or the strain fermentation liquor thereof or the biological preparation thereof in improving saline soil or promoting plant growth.

[0007] The Bacillus tequilensis QingTuHuS40 of the present application is preserved in the China General Microbiological Culture Collection Center (CGMCC) on January 8, 2025, the preservation number is CGMCC No. 33325, the preservation address is No. 1, Xiliujia, Beichen West Road, Chaoyang District, Beijing, and the classification and naming is Bacillus tequilensis QingTuHuS40.

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

[0009] The strain fermentation liquor of the present application comprises the Bacillus tequilensis QingTuHuS40 of the present application.

[0010] The microbial preparation has the characteristics that the microbial preparation comprises the Bacillus tequilensis QingTuHuS40 or the fermentation liquor of the strain.

[0011] The Bacillus tequilensis QingTuHuS40, the fermentation liquor of the strain or the microbial preparation can be applied to improving soil fertility.

[0012] The Bacillus tequilensis QingTuHuS40, the fermentation liquor of the strain or the microbial preparation can be applied to improving saline soil.

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

[0014] The Bacillus tequilensis QingTuHuS40, the fermentation liquor of the strain or the microbial preparation can be applied to promoting plant growth.

[0015] The Bacillus tequilensis QingTuHuS40, the fermentation liquor of the strain or the microbial preparation can be applied to reshaping a rhizosphere bacterial community structure.

[0016] Further, the plant is a red sand, a Caroxylon passerinum, a Lycium ruthenicum or a Atraphaxis bracteata A.Los.

[0017] A mechanism of the Bacillus tequilensis QingTuHS40 for promoting growth of red sand seedlings and enhancing salt tolerance: the Bacillus tequilensis QingTuHS40 improves soil fertility and relieves soil salinization by reshaping a rhizosphere bacterial community structure and adjusting functional gene abundance, thereby promoting growth of the red sand and enhancing salt tolerance of the red sand. Figure 10 As shown in

[0018] Beneficial effects: compared with the prior art, the present application has the following obvious advantages:

[0019] This invention relates to Bacillus tequilensis QingTuHS40, which can colonize the rhizosphere of red sand and recruit other beneficial bacteria, thus reshaping the bacterial community structure of the red sand rhizosphere. Simultaneously, it improves soil fertility, mitigates soil salinity, enhances the plant's ability to fully utilize environmental nutrients, promotes plant growth, and strengthens salt tolerance. This invention's Bacillus tequilensis QingTuHS40 is primarily used for the cultivation of desert plants and afforestation in arid and saline-alkali desert areas. Attached Figure Description

[0020] Figure 1 Images show the colony morphology and Gram staining microscopic observation results of Bacillus tequilensis QingTuHuS40. In the images, A is the colony morphology image and B is the Gram staining microscopic image.

[0021] Figure 2 Phylogenetic tree of 16S rDNA gene sequence of Bacillus tequilensis QingTuHuS40, where the superscript T indicates the type strain, and the bar length represents 2% base difference;

[0022] Figure 3 The figure shows the effects of NaCl stress and Bacillus tequilensis QingTuHuS40 on the growth, development and biomass of *Symplocos rubra* seedlings. In the figure, A is a photograph of *Symplocos rubra* under different treatments, B is the plant height, C is the total root length, and D is the dry weight of roots, stems and leaves.

[0023] Figure 4 Figure 1 shows the effects of NaCl stress and Bacillus tequilensis QingTuHuS40 on the rhizosphere bacterial community structure of red sand. AB represents the species composition stacking bar chart (phylum and genus levels); CE represents the α diversity indices (Chao1, Shannon_2, Simpson); D represents the β diversity index (NMDS); is the species abundance clustering analysis chart; G is the species clade diagram; and I is the LDA value distribution bar chart.

[0024] Figure 5 The images show NMDS analysis and gene abundance stacking plots of functional genes in soil samples. In the image, A represents NMDS analysis and B represents gene abundance stacking plots.

[0025] Figure 6Figure for the change of the abundance of C cycle related functional genes under different treatments, wherein A is a schematic diagram of the C cycle process, B is the absolute abundance of C degradation functional genes (Top 6), and C is the absolute abundance of C fixation functional genes (Top 6);

[0026] Figure 7 Figure for the change of the abundance of N cycle related functional genes under different treatments, wherein A is a schematic diagram of the N cycle process, and B is the absolute abundance of N cycle functional genes (Top 6);

[0027] Figure 8 Figure for the change of the abundance of P cycle related functional genes under different treatments, wherein A is a schematic diagram of the P cycle process, and B is the absolute abundance of P cycle functional genes (Top 6);

[0028] Figure 9 Figure for the analysis of the correlation between the growth index of Reaumuria soongorica and environmental factors and functional genes by Mantel test;

[0029] Figure 10 Mechanism of Bacillus tequilensis QingTuHS40 for promoting the growth of Reaumuria soongorica seedlings and enhancing the salt tolerance of Reaumuria soongorica seedlings. DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be further described below in combination with the drawings.

[0031] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified. The experimental methods not specified in the examples are usually performed according to 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 surrounding Gobi of Qingtu Lake in the Shiyang River Basin in the northwest region.

[0035] Isolation and screening process: high-throughput bacterial cultivation and identification method (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.) was used to isolate and screen PGPR producing IAA. 10 g of collected soil was dissolved in 90 ml of sterilized normal saline, and cultured at 30°C, 180 r / min on a shaker for 30 min. Gradient dilution was used to prepare 10 -3 , 10 -4 and 10 -5 dilutions. Different concentrations of dilutions were inoculated into 96-well Lauria Bertani (LB) liquid culture plates, and 30%-40% of the wells in the culture plates showed visible bacterial growth after incubation, which was considered to be derived from single cell culture, which could produce a large proportion of pure culture. The pure culture was then transferred to two 96-well LB liquid culture plates containing 0.1% L-tryptophan and cultured at 30°C for 2 days. Then, an equal volume of Salkowski reagent (prepared from 50 ml of 35% perchloric acid and 1 ml of 0.5 M FeCl3 solution) was added to one of the culture plates, and a pink color appeared after 30 min of color development in the dark, indicating the ability to produce IAA. The culture in the corresponding well with deeper color development was selected from the other culture plate and streaked for purification, then cultured at 30°C for 3 days, and single colonies were picked and isolated by streaking method and stored at -80°C.

[0036] 2. Biological characteristics of Bacillus tequilensis QingTuHuS40 are described Plate streaking method was used to isolate single colonies, and the morphological characteristics of the bacteria were observed. A small amount of bacterial solution was smeared and stained with Gram's stain, and the bacterial morphology and staining characteristics were observed under a light microscope. The morphological characteristics of Bacillus tequilensis QingTuHuS40 are shown in Figure 1 . The results of morphological observation and Gram's staining of the strain: Gram's staining was positive, short rod-shaped, blunt at both ends, paired or in a string, yellow colonies on LB plates, smooth and wet surface, irregular edge. The colony was opaque, with irregular edge, and was preliminarily determined as Bacillus.

[0037] Gram staining was positive, short rod-shaped, blunt round at both ends, in pairs or in a string, on LB plates, colonies were yellow, smooth, wet, irregular edge. The colony was opaque, the edge was irregular (see Figure 1 ), initially 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 bacterial 16S rDNA universal primers, primer 27F (SEQ ID NO. 2), 1492R (SEQ ID NO. 3). The amplification product was sent to Guangdong Meige Gene Technology Co., Ltd. for sequencing. The following sequence SEQ ID NO. 1 was obtained through 16S rDNA sequencing:

[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, and a phylogenetic tree was constructed using Maximum Likelihood Estimate method on MEGA 11.0, with General Time Reversible (GTR) parameter model and bootstrap sampling 500 times. Comparison found that the sequence was closest to the strain Bacillus tequilensis KCTC 13622 T , with Query Cover value of 100%, E value of 0.0, and Per. Ident value of 100%. The phylogenetic tree (see Figure 2 ) showed that the isolated strain was closest to Bacillus tequilensis KCTC 13622 T , and was on the same branch, and was second closest to Cetobacterium sp. ZF1NH01. It was preliminarily identified as Bacillus tequilensis, named Bacillus tequilensis QingTuHuS40. The strain was sent to the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, and was preserved on January 8, 2025, with the preservation number of CGMCC No. 33325.

[0044] Example 2

[0045] 1. Red sand seedling cultivation and growth conditions

[0046] The experiment of Bacillus tequilensis QingTuHuS40 regulating rhizosphere bacterial community structure and function of Reaumuria soongorica was carried out in the experimental base of Gansu Agricultural University from April to November 2023. The seeds of Reaumuria soongorica with uniform size and full grains were disinfected with 1% sodium hypochlorite solution for 30 min, rinsed with sterile water for 5 times, and sowed in plastic 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 of 1:2:1). After the seedlings grew stably (plant height reached 5 cm), they were thinned, and 1 plant was retained in each pot, totaling 30 pots. The Reaumuria soongorica seedlings grew naturally in the experimental base of Gansu Agricultural University, located in Anning District of Lanzhou City (36°5′N, 103°42′E), belonging to the mid-temperate climate zone with obvious continental climate characteristics, distinct four seasons, sufficient light, and dry climate.

[0047] 2. Experimental design and treatment

[0048] The application method of Bacillus tequilensis QingTuHuS40 in promoting the growth of Reaumuria soongorica, wherein the Bacillus tequilensis QingTuHuS40 bacterial suspension can be prepared, which can include the following steps: inoculating Bacillus tequilensis QingTuHuS40 in LB, culturing at 28°C for 48 h, harvesting the culture, diluting with sterile saline (0.9% NaCl), preparing the bacterial inoculum, and diluting to a final OD of 1.0 at 600 nm (~109CFU 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 to the rhizosphere of Reaumuria soongorica is irrigation.

[0049] The test set control (CK, no addition of NaCl and Bacillus tequilensis QingTuHuS40), salt treatment (S, only 400 mM NaCl is added), Bacillus tequilensis QingTuHuS40 single treatment (S40) and salt and Bacillus tequilensis QingTuHuS40 strain co-treatment (S40+S). Each treatment has 3 replicates. Sodium chloride (NaCl) is one of the main salt types in saline soil (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 reached 18-20 cm) were selected for treatment. Each pot was irrigated with 400 mM NaCl solution 500 mL once a day, continuously irrigated for 3 days, and the total amount reached 1500 mL. The control and single bacteria treatment were treated with the same amount of water. After 2 days of NaCl stress treatment, Bacillus tequilensis KCTC 13622 T The roots of the plants were inoculated with 100 mL of Bacillus tequilensis QingTuHS40 inoculum, and the control was inoculated with an equal amount of sterile saline.

[0050] 3. Sample collection and growth index determination

[0051] After 56 days of treatment of the Rehderia argentea seedlings, sampling was performed. Before sampling, a digital camera (canon EOS 6D, canon Inc., Japan) was used to take photos, and a ruler was used to measure the plant height of the Rehderia argentea seedlings; the soil and plant roots in each pot were carefully poured out, the large pieces of soil were shaken off, the surface attached soil of the roots was gently shaken and mixed, and placed in a 15 mL sterile and enzyme-free cryogenic storage tube, immediately frozen in liquid nitrogen, and transferred to a laboratory -80°C refrigerator for storage for analysis of rhizosphere soil microbial community structure and evaluation of element cycling functional potential. The above and underground parts of the plants were collected and taken back to the laboratory for washing with pure water, then the surface moisture was absorbed with filter paper, and the fresh weight was measured. The total root length was determined using the Wanpen LA-S plant root analysis system. The roots, stems and leaves were separated, killed at 105°C for 30 min, and dried at 80°C to constant weight. Then, the root, stem and leaf biomass was measured using a digital balance with a precision of 0.0001 g. All the soil in the pot was mixed evenly, and sampling was performed using the four-part method, air-dried, ground and sieved (100 mesh) for subsequent determination of soil basic physical and chemical indicators.

[0052] Effects of NaCl stress and Bacillus tequilensis QingTuHS40 on the growth and biomass of P. rubra seedlings Figure 3 . It was found that Figure 3 under NaCl stress, the plant height, total root length, and dry weight of stems, leaves, and roots of P. rubra seedlings were lower than those of the control group (CK). The plant height, total root length, and dry weight of stems, leaves, and roots of P. rubra seedlings inoculated with Bacillus tequilensis QingTuHS40 were higher than those of the control group (CK), among which the total root length and leaf dry weight increased by 29.62% and 37.71% (P<0.05), respectively. The plant height, total root length, and dry weight of stems, leaves, and roots of P. rubra seedlings treated with Bacillus tequilensis QingTuHS40 and NaCl were significantly higher than those of the NaCl treatment group (S), with an increase of 13.36%, 20.93%, 246.30%, 142.38%, and 42.33%, respectively, except for the plant height, which was restored to the control level.

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

[0054] The air-dried soil collected from the pot experiment was used to determine the soil pH value according to the instructions of Sartorius PB-10 pH meter, and the conductivity was determined using a DDSJ308F conductivity meter. The total carbon (TC) and total nitrogen (TN) were determined using an elemental analyzer. The contents of soil organic matter (SOM), total phosphorus (TP), total potassium (TK), available phosphorus (AP), and available potassium (AK) in the rhizosphere soil were determined using the potassium dichromate volumetric method, NaOH fusion-molybdenum antimony anti-colorimetric method, NaOH fusion-flame photometry method, sodium bicarbonate method, and ammonium acetate extraction-flame photometry method, respectively (Bao, Soil Agro-chemical Analysis, 3rd Edition, Beijing: China Agricultural Press, 2000). The activities of soil urease, alkaline phosphatase, invertase, and catalase were determined using the phenol sodium-sodium hypochlorite colorimetric method, phosphoric acid benzenesodium colorimetric method, 3,5-dinitrosalicylic acid colorimetric method, and potassium permanganate titration method, respectively (Guan, 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 QingTuHS40 had a significant effect on most soil physicochemical properties and soil enzyme activities (see Table 1). Under NaCl stress, the soil conductivity, Na + content, alkaline phosphatase, and catalase activities were significantly higher than those of the control group, while the contents of organic matter, available phosphorus, and available potassium, and urease activity were significantly lower than those of the control group. NaCl stress and Bacillus tequilensis QingTuHS40 treatment significantly increased the plant height, total root length, and dry weight of stems, leaves, and roots of P. rubra seedlings, with an increase of 13.36%, 20.93%, 246.30%, 142.38%, and 42.33%, respectively, except for the plant height, which was restored to the control level.

[0056] Under the co-treatment of QingTuHuS40, the contents of soil organic matter, total carbon, available phosphorus, available potassium and Na + increased significantly, while the conductivity and catalase activity decreased significantly. The soil pH value decreased slightly under the inoculation of Bacillus tequilensis QingTuHuS40, but there was no significant difference. There was no significant difference in total nitrogen, total phosphorus and total potassium among the treatments. It was indicated that the short-term NaCl stress and QingTuHuS40 treatment had a significant effect on soil organic matter, available potassium, available phosphorus and soil enzyme activity, but had no significant effect 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 high-throughput sequencing technology to amplify and sequence the 16S rRNA V4-V5 variable region genes of rhizosphere soil bacteria. The library construction and sequencing were completed by Guangdong Meigene Gene Technology Co., Ltd. The raw data was analyzed by the EasyAmyplicon Amplicon Analysis Platform developed by Liu Yongxin team of Chinese Academy of Agricultural Sciences Genome Institute (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 Meigene Cloud Platform (http: / / cloud.magigene.com / ). QIIME2 (2022.8) software DADA2 was used for quality control, denoising, splicing, chimeric removal and generation of OTUs feature table. The Silva database (Release 132) was used for bacterial 16S rRNA gene, and the UNITE database (Release 8.0) was used for fungal ITS sequence. The classify-sklearn algorithm of QIIME2 was used for species annotation of each OTUs feature sequence. The results are shown in Figure 4 ​

[0062] NaCl stress and Bacillus tequilensis QingTuHuS40 had a significant impact on the rhizosphere bacterial community structure of Reaumuria soongorica seedlings (see Figure 4 ). Proteobacteria, Bacteroidota, Planctomycetota, Acidobacteriota and Chloroflexi were dominant in the rhizosphere soil of different treatment groups, with a cumulative relative abundance of more than 80% (see Figure 4 A). Under the combined treatment of NaCl and Bacillus tequilensis QingTuHuS40, the relative abundance of Proteobacteria (45.47%), Bacteroidota (20.44%) and Planctomycetota (13.12%) increased significantly compared with the control and NaCl treatment groups, while the relative abundance of Acidobacteriota (6.83%) and Chloroflexi (5.51%) decreased. At the genus level, the abundance of bacterial species in different treatment groups differed greatly (see Figure 4 B). Under NaCl stress, the abundance of Pseudomonas increased significantly compared with the control group. Under the combined treatment of NaCl and Bacillus tequilensis QingTuHuS40, the relative abundance of Stenotrophomonas, Planctomicrobium, Pseudomonas and SJA-28 increased significantly 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, but increased significantly compared with the control group after inoculation with Bacillus tequilensis QingTuHuS40 (p<0.05). Compared with the control, both NaCl stress and inoculation with Bacillus tequilensis QingTuHuS40 reduced the richness and diversity of rhizosphere soil bacteria (see Figure 4 C, Figure 4 D, Figure 4Figure 6. The chao1 index of the control group was higher than other treatment groups, indicating that the control group had the highest species richness. In the presence of NaCl, the Shannon index of the community decreased, and the Simpson index increased, with no significant differences between groups. NMDS separated the rhizosphere bacteria in different treatment groups of soil, with the closest proximity to the inoculation of Bacillus tequilensis QingTuHuS40, the control group in the middle (see Figure 6A). The PCoA plot showed that the rhizosphere bacteria in the control group were closer to the NaCl treatment group than to the Bacillus tequilensis QingTuHuS40 treatment group (see Figure 6B). Figure 4 Figure 7. LEfSe (Linear discriminant analysis Effect Size) analysis results showed that there were 20 biomarkers with LDA scores > 3.5 (see Figure 7A). The control group had 12 biomarkers, the NaCl treatment group had 6 biomarkers, and the Bacillus tequilensis QingTuHuS40 treatment group had 2 biomarkers (see Figure 7B). Figure 4 Figure 8. The results of LEfSe (Linear discriminant analysis Effect Size) analysis showed that there were 20 biomarkers with LDA scores > 3.5 (see Figure 8A). The control group had 12 biomarkers, the NaCl treatment group had 6 biomarkers, and the Bacillus tequilensis QingTuHuS40 treatment group had 2 biomarkers (see Figure 8B). Figure 5 Figure 9. Among these biomarkers, 12 taxa were present in the rhizosphere soil of the control group, with the most significant contributions from Acidobacteriota, Vicinamibacteria, and Vicinamibacterales. There were 6 biomarker taxa in the rhizosphere soil of the NaCl and Bacillus tequilensis QingTuHuS40 co-treatment group, with the most significant contributions from Stenotrophomonas, Flavobacteriales, and Devosiaceae. There was 1 biomarker taxon in the rhizosphere soil of the NaCl treatment and the Bacillus tequilensis QingTuHuS40 single treatment group, respectively, which were Armatimonadota and Microgenomatia. The results showed that NaCl and Bacillus tequilensis QingTuHuS40 could significantly change the species composition and distribution of bacterial communities in the rhizosphere soil of Rehderodendron psodo-chameneri seedlings, reduce the richness and diversity of species, and the impact on species composition was higher than that on species richness and diversity.

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

[0064] QMEC is a high-throughput qPCR-based functional gene chip method for evaluating and quantifying the genetic potential of microorganisms for mineralizing soil organic matter and releasing C, N, and P. (Zheng, B., Y. Zhu, J. Sardans, J. Peñuelas, and R. S. Xu, 2018. Quantitative Microbial Elemental Cycling (QMEC): A high-throughput qPCR-based functional gene chip for assessing and quantifying the genetic potential of microorganisms for mineralizing soil organic matter and releasing C, N, and P. Soil Biology and Biochemistry 123, 105-115.) 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.). The present experiment used QMEC to quantify a total of 63 genes related to C, N, P cycling, including 31 C cycling, 22 N cycling, and 9 P cycling related functional genes and one 16S rRNA gene (Table 2). Details of the measurements and primers were as described in (Zheng, B., Y. Zhu, J. Sardans, J. The same as described in 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. The sample DNA was extracted according to the instructions of the magnetic bead method soil DNA extraction kit (FINOROP DC306-03, Guangzhou Fangzhou Biosafety Technology Co., Ltd.), and the total amount and purity of the DNA were detected using a Qubit 4.0 (Thermo Fisher Scientific, Waltham, USA) instrument after extraction, to ensure that the DNA concentration was uniformly diluted to 20 ng / μl. qPCR reaction and fluorescence signal detection were performed 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: fluorescent dye (Roche LightCycler 480 SYBR Green I Master) 10 μl, primers 0.4 μl each, nuclease-free water 7.2 μl, sample DNA 2 μl, a total of 20 μl; initial enzyme activation at 95°C for 5 minutes, followed by 40 cycles of 95°C denaturation for 15 seconds, 60°C annealing for 60 seconds, and 72°C extension for 20 seconds, and finally 95°C for 15 seconds, 60°C for 60 seconds. All qPCR reactions were performed three times, and each run contained a non-template negative control. After standardization of the data using 16S rRNA as the internal reference, the relative quantitative information of each gene in each sample was collected. The absolute value of the 16S rRNA gene was obtained according to the quantitative information of the Roche instrument. The results are shown in Figure 5 .

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

[0066]

[0067] Composition of rhizosphere soil functional genes:

[0068] The differences in functional gene composition under different treatments were evaluated using NMDS Figure 5(A) Functional gene composition under NaCl stress was significantly different from other groups on axis 1. The functional gene composition of the NaCl stress and Bacillus tequilensis QingTuHuS40 co-treatment groups was closer to the control group on axis 1, but separated on axis 2. Under NaCl stress, the abundance of functional genes related to C, N, and P element cycling was lower than the control, while after Bacillus tequilensis QingTuHuS40 inoculation, the abundance of functional genes related to C, N, and P element cycling was significantly increased compared to the control. Figure 6 (B). The results showed that Bacillus tequilensis QingTuHuS40 can mediate the expression of functional genes in the rhizosphere soil of red sand seedlings.

[0069] Carbon cycle processes and key gene abundance:

[0070] Functional genes involved in the carbon cycle include C fixation and C degradation genes (see Table 2); Figure 6 (See section A). The expression of these genes differed significantly across different treatment groups. Of the 31 genes, 23 showed significant differences. Among them, 12 genes are involved in carbon degradation (see the Top 6 for absolute abundance). Figure 6 The abundance of genes in the B group includes those involved in starch hydrolysis (sga), hemicellulose hydrolysis (abfA, xylA, and manB), and lignin hydrolysis (mnp and glx). There are 11 genes involved in carbon fixation (see Top 6 for absolute abundance). Figure 7 Genes involved in carbon degradation (C) include acetyl-CoA synthase (acsA), succinyl-CoA synthase (smtA), α-subunit formyltransferase (mct), 5-methyltetrahydrofolate convertase (acsE), ribulose-1,2-bisphosphate carboxylase (rbcL), and fumarate reductase flavoprotein (frdA). Under NaCl salt stress, the abundance of C degradation genes sga, mnp, manB, glx, and mct was significantly lower than that in the control group. The C fixation gene mct was significantly lower than that in the control group, while the gene frdA was significantly higher than that in the control group. Under the co-treatment of NaCl and Bacillus subtilis, the abundance of genes sga, abfA, xylA, mnp, manB, glx, acsA, smtA, mct, acsE, and rbcL was significantly increased compared with NaCl treatment. In summary, regardless of the presence or absence of NaCl, inoculation with Bacillus tequilensis QingTuHuS40 increases the abundance of genes related to C degradation and C fixation.

[0071] Nitrogen cycle processes and key gene abundance:

[0072] Functional genes involved in N cycle are related to nitrogen fixation, nitrification, denitrification, ammonium assimilation and organic nitrogen mineralization (see Table 2; Figure 7 In different treatment groups, 18 of the 22 N cycle-related genes were significantly different. In Figure 8 The absolute abundance of the top 6 genes in different treatment groups is shown in Table B, including urea synthase gene (ureC), nitrous oxide reductase gene (nosZl, nosZ2), nitrite reductase gene (nirK3, nirSl) and glutamate dehydrogenase gene (gdhA). The ureC and gdhA genes are involved in the process of urea conversion to ammonia and organic nitrogen mineralization, respectively. NosZl, nosZ2, nirK3 and nirSl are all involved in the denitrification process. Therefore, after inoculation of Bacillus, denitrification is the dominant pathway of nitrogen cycle. Under NaCl salt stress, the absolute abundance of nosZl and nosZ2 genes increased significantly compared with the control group, while the abundance of nirK3 and gdhA decreased significantly compared with the control group. Salt stress has a significant impact on N cycle-related genes. After inoculation of Bacillus, the absolute abundance of the six genes increased significantly compared with the control. The results show that Bacillus tequilensis QingTuHuS40 plays an important role in the regulation of bacterial N metabolism in the rhizosphere of H. mongolicum.

[0073] Phosphorus cycle processes and key gene abundance:

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

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

[0076] Mentel test results showed the correlations between soil properties and bacterial community composition and element cycling-related functional genes (see ​ ). Red sand seedling growth indicators (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). Planctomycetota community composition 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). Bacteroidota community composition was significantly correlated with PH, EC, and Na (p < 0.05). Proteobacteria and Acidobacteriota community compositions were significantly correlated with SA (p < 0.05). Chloroflexi community composition was significantly correlated with AP (p < 0.05). C, N, and P element cycling-related functional genes were extremely significantly correlated with LBM, SOM, AP, and SA (p < 0.01), significantly correlated with SBM, RBM, and UA (p < 0.05), and not significantly correlated with pH, EC, TP, TK, and Na. C degradation and P cycling functional genes were significantly correlated with PH and CA (p < 0.05). C fixation functional genes were significantly correlated with APA (p < 0.05), and P cycling functional genes were extremely significantly correlated with APA (p < 0.01).

Claims

1. A Bacillus tekirae species for reshaping the structure of plant rhizosphere bacterial communities ( Bacillus tequilensis QingTuHuS40, characterized in that, The Bacillus tergenta ( Bacillus tequilensis QingTuHuS40 was deposited on January 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33325. The described Bacillus tergentii (…) Bacillus tequilensis The 16S rDNA sequence of QingTuHuS40 is shown in SEQ ID NO.

1. The plant species are red sand, pearl hairy grass, black goji berry or sand wood knotweed.

2. A fermentation broth for a bacterial strain, characterized in that, Including the Bacillus tekirae as described in claim 1 ( Bacillus tequilensis )QingTuHuS40.

3. A microbial preparation, characterized in that, Including the Bacillus tekirae as described in claim 1 ( Bacillus tequilensis The fermentation broth of the strain described in claim 2 or QingTuHuS40.

4. The Bacillus tergentii according to claim 1 ( Bacillus tequilensis Application of the fermentation broth of the strain described in claim 2 or the microbial preparation described in claim 3 in improving saline-alkali soil in desert areas.

5. The Bacillus tergentii according to claim 1 ( Bacillus tequilensis The application of the fermentation broth of the strain described in claim 2 or the microbial preparation described in claim 3 in reshaping the structure of plant rhizosphere bacterial communities.

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