Halotolerant glutamic acid bacillus CBGS-20 and application thereof

Abstract

This invention relates to a halophilic glutamate bacterium (Hydrogenophilus glutamate) Glutamicibacter halophytocola CBGS-20, with accession number CGMCC No. 34682, possesses not only the abilities to release silicon, dissolve phosphorus, solubilize potassium, and produce ferrophosphate and IAA, but also exhibits acid and alkali resistance, salt tolerance, heavy metal resistance, and the ability to control plant pathogens. This invention also discloses the application of this strain in promoting plant growth and improving soil quality. Based on the strain's growth and stress resistance characteristics, this strain shows great promise for applications in the production of novel bio-organic fertilizers, biopesticides, soil conditioners, and / or artificial substrates, as well as in the improvement of tailings areas, heavy metal-contaminated soils, and saline-alkali land.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, specifically relating to a halophilic glutamate bacterium CBGS-20 and its applications. Background Technology

[0002] Currently, due to intensive farming, industrial emissions, and climate change, approximately 33% of the world's arable land suffers from varying degrees of salinization, acidification, heavy metal pollution, and organic carbon deficit. While traditional chemical inputs can increase yields in the short term, they further exacerbate soil compaction, salinization, and microecological imbalance, creating a vicious cycle of "increased yield - degradation - increased yield - further degradation." Therefore, finding new microbial resources that combine nutrient supply, stress restoration, and ecological security functions has become crucial to resolving this contradiction.

[0003] Traditional fertilization methods struggle to address the core contradiction of "sufficient total amount but low availability" of elements: While the Earth's crust contains 28% silicon, it exists primarily as insoluble aluminosilicates, leaving crops with less than 1% available silicon. This significantly reduces the lodging and disease resistance of gramineous crops like rice and wheat. As for phosphorus and potassium, approximately 40% of the available phosphorus in global arable land is below the critical level, with chemical phosphate fertilizers achieving only 15%–20% utilization in the current season. The remaining portion is easily fixed or lost, leading to eutrophication in water bodies. Furthermore, less than 2% of the total potassium in soil is exchangeable, and long-term "nitrogen-phosphorus preference" fertilization exacerbates potassium depletion.

[0004] Current research on rhizosphere growth-promoting bacteria (PGPR) focuses on functions such as silicon release, phosphorus solubilization, potassium solubilization, and nitrogen fixation, but most of these are single-function strains. Therefore, screening for highly efficient functional microorganisms with strong stress resistance and multiple functions is of great significance for the creation of new biological products, the safe use of marginal land, and the promotion of sustainable development in agriculture. Summary of the Invention

[0005] The purpose of this invention is to provide a Halophilic Glutamate Bacillus CBGS-20 that can efficiently release silicon, dissolve phosphorus, and solubilize potassium, as well as produce ferrophilic compounds and IAA, and is also resistant to acids, alkalis, salts, heavy metals, and diseases. When applied to soil, especially saline-alkali land, this bacterium not only promotes plant growth but also improves soil quality.

[0006] The present invention adopts the following technical solution: A type of halophilic glutamate bacillus ( Glutamicibacter halophytocola CBGS-20 was deposited on May 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, with accession number CGMCC No. 34682.

[0007] The *Halophilic glutamate* CBGS-20 grew well on LB medium, exhibiting short rod-shaped cells, no spores, and Gram-positive staining. Colonies were round, white, opaque, and smooth with a moist surface. It could utilize sucrose, glucose, fructose, and cellobiose to produce acid, and could hydrolyze starch and cellulose. It did not produce H₂S, showed a negative indole test, and was positive for urea utilization.

[0008] The halophilic glutamate bacillus CBGS-20 has the ability to release silicon, dissolve phosphorus, and decompose potassium.

[0009] The halophilic glutamate bacterium CBGS-20 can produce ferrophilic acid and IAA.

[0010] The halophilic glutamate bacillus CBGS-20 can grow well at pH 6-10, NaCl concentration 0-10%, and temperature 15-35℃, and has the characteristics of acid and alkali resistance, salt resistance, and wide temperature range.

[0011] The halophilic glutamate bacillus CBGS-20 can antagonize potato late blight and corn pyridae.

[0012] The halophilic glutamate bacillus CBGS-20 can tolerate heavy metal ions.

[0013] Specifically, the *Halophilus glutamate* CBGS-20 can grow normally in a culture medium containing 2 mM copper ions, 5 mM lead ions, 1 mM hexavalent chromium ions, and zinc ions.

[0014] Application of the above-mentioned halophilic glutamate bacterium CBGS-20 in the control of potato late blight and maize pyridaben.

[0015] An application of the above-mentioned halophilic glutamate bacillus CBGS-20 in soil ecological restoration.

[0016] Furthermore, the soil ecological restoration includes the remediation of tailings area soil, acidic soil, alkaline soil, saline-alkali soil and / or heavy metal contaminated soil.

[0017] Application of the above-mentioned halophilic glutamate bacterium CBGS-20 in promoting the growth of maize and wheat grown in saline-alkali soil.

[0018] An application of the above-mentioned halophilic glutamate bacterium CBGS-20 in the preparation of soil remediation agents, biofertilizers, biopesticides and / or artificial substrates.

[0019] A bio-fertilizer comprising the aforementioned *Halophilic glutamate* CBGS-20, with a viable count of not less than 2 × 10⁻⁶. 8 CFU / g.

[0020] A soil remediation agent comprising the aforementioned *Hydroxyglutamate* CBGS-20, with a viable count of not less than 2 × 10⁻⁶. 8 CFU / g.

[0021] Furthermore, the soil remediation agent is a heavy metal contaminated soil remediation agent.

[0022] Furthermore, the soil remediation agent is an acidic, alkaline, or saline-alkali soil remediation agent.

[0023] Furthermore, the soil remediation agent is a tailings area soil ecological remediation agent.

[0024] A biological pesticide comprising the aforementioned *Hydrogentodoxa citrate* CBGS-20.

[0025] Specifically, the biological pesticide is used to control potato late blight and corn pyrophyte.

[0026] The beneficial effects of this invention are as follows: (1) The CBGS-20 strain of halophilic glutamate of the present invention has comprehensive functions. It can not only provide key nutrients to plants through silicon release, phosphorus solubilization and potassium solubilization, thereby improving plant stress resistance and soil fertility; it can also secrete iron ferritin and IAA (indoleacetic acid) to help plants efficiently acquire iron and promote plant root development and growth metabolism; at the same time, it also has the function of preventing and controlling potato late blight and corn pyridus, thereby enhancing plant disease resistance.

[0027] (2) The *Halophilic Glutamate Bacillus* strain CBGS-20 of this invention exhibits extremely strong salt and alkali tolerance, with an optimal NaCl concentration range of 0-8% and a maximum tolerance concentration of up to 10%, far exceeding the adaptation threshold of ordinary microorganisms. Simultaneously, this bacterium also possesses heavy metal tolerance characteristics, enabling it to survive and function in extreme environments such as iron tailings areas and heavy metal-contaminated soils, as well as saline-alkali lands, effectively improving soil physicochemical properties, enhancing soil quality, and providing technical support for ecological restoration in extreme environments.

[0028] (3) The *Halophilic Glutamate Bacillus* CBGS-20 strain of the present invention provides an excellent strain resource for the research and development of novel bio-organic fertilizers, soil conditioners, and artificial substrates. Its multifunctional characteristics can significantly improve the application effect of related products. At the same time, as a microorganism with functions of releasing silicon, phosphorus, and potassium, it opens up a new path for the development and utilization of such functional microorganisms, and has practical economic and social benefits in agricultural production, ecological restoration, and other fields, with broad application prospects. Attached Figure Description

[0029] Figure 1 Phylogenetic tree of *Hydrogenobacterium glutamicum* CBGS-20.

[0030] Figure 2The growth curve of strain CBGS-20 is shown.

[0031] Figure 3 The effect of initial pH of the culture medium on the growth of *Hydroxyglutamate* CBGS-20.

[0032] Figure 4 The effect of NaCl concentration on the growth of *Hydroxyglutamate* CBGS-20.

[0033] Figure 5 The effect of temperature on the growth of *Hydrogenospora halophila* CBGS-20. Detailed Implementation

[0034] The present invention will be further described below with reference to the embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the embodiments, and any modifications made by those skilled in the art within the scope defined by the claims also fall within the scope of protection of the present invention.

[0035] Example 1: Isolation, identification and preservation of *Haloxyglutamate* CBGS-20 The isolated samples were obtained from the rhizosphere soil (0-40cm soil layer) of a tailings mine in Chengde, Hebei Province. Freshly collected samples were collected using sterile bags and diluted with sterile water to prepare 10... -4 10 -5 10 -6 Three dilutions of soil suspension were evenly spread on Alexandrov medium and incubated at 30°C for 2-3 days. Twelve single colonies with a clear zone were picked and streaked 2-3 times until a pure culture was obtained. The culture was then transferred to LB slant and stored at 4°C for later use.

[0036] The Alexandrov medium formula is as follows: 5.0 g sucrose, 2.0 g Na₂HPO₄, 0.5 g MgSO₄·7H₂O, 0.005 g FeCl₃, 0.1 g CaCO₃, 1.0 g magnesium trisilicate, 15-20 g agar, and 1000 mL water, with the pH adjusted to 7.0-7.4. The LB medium formula is as follows: 10 g tryptone, 5 g yeast extract, 10 g NaCl, and 1000 mL water, with a pH of 7.2-7.5 (solid medium requires the addition of 15-20 g agar).

[0037] Using iron tailings sand as a substrate and the increase in available silicon content in the fermentation broth as a standard, the silicon release capacity of single colonies obtained from the initial screening was re-screened using a leaching desilication medium. The leaching desilication medium formula was: 10 g glucose, 0.2 g KH₂PO₄, 0.2 g MgSO₄·7H₂O, 0.2 g NaCl, 0.2 g CaCl₂·2H₂O, 1.0 g iron tailings sand, 5 g CaCO₃, 1000 mL water, pH 7.0–7.2. The iron tailings sand sample was obtained from an iron tailings dam in Chengde, Hebei Province, and was ground through a 200-mesh sieve.

[0038] During the secondary screening process, the seed culture was fermented in LB medium to the logarithmic growth phase, and then inoculated at 10% by volume into leaching and desilication medium. A control group inoculated with an equal volume of LB blank medium was used. The cultures were incubated at 30℃ on a shaker at 180 r / min. Samples were taken on day 5, and the supernatant from the centrifuged fermentation broth was collected. The available silicon content was determined using the silicomolybdenum blue colorimetric method. The secondary screening results showed that strain CBGS-20 exhibited the best silicon release effect, with its fermentation broth containing 2.05 times the available silicon content of the control.

[0039] Physiological and biochemical characteristics of the screened strain CBGS-20 were investigated. It grew well on LB medium, exhibiting short rod-shaped cells, no spores, and Gram-positive staining. Colonies were round, white, opaque, and smooth with a moist surface. It could utilize sucrose, glucose, fructose, and cellobiose to produce acid, and could hydrolyze starch and cellulose. It did not produce H2S, showed a negative indole test, and was positive for urea utilization. Specific results are shown in Table 1.

[0040] Table 1. Physiological and biochemical characteristics of strain CBGS-20 .

[0041] Total DNA was extracted from strain CBGS-20 using a bacterial genomic DNA extraction kit. Using this DNA as a template, amplification was performed using universal 16S rDNA primers: 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). Agarose gel electrophoresis revealed a band of approximately 1500 bp. The PCR amplification products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0042] The sequencing results were submitted to NCBI for homology analysis with existing 16S rDNA sequences in the database. 16S rDNA gene sequences from closely related strains were selected from Genebank, and a phylogenetic tree was constructed using the neighbor-joining method with MEGA 11.0 software. The results are as follows: Figure 1 As shown, it is similar to Glutamicibacter halophytocolaThe strain CBGS-20 is most closely related to KLBMP 5180, with a similarity of 99.86%. Based on morphological observation and physiological and biochemical reaction results, strain CBGS-20 was identified as *Haloxyglutamate*. Glutamicibacter halophytocola ).

[0043] Strain CBGS-20 was deposited on May 27, 2025, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 34682.

[0044] Example 2 Growth curve of *Haloxyglutamate* CBGS-20 A small amount of bacterial cells was picked from the slant of a preserved CBGS-20 test tube using an inoculation loop and inoculated into LB liquid medium. The culture was incubated at 25°C and 180 rpm for 14 h using a shaker to obtain the seed culture. At an inoculation rate of 2% (v / v), the seed culture was inoculated into LB medium (30 mL in a 100 mL shake flask) and incubated at 25°C and 180 rpm. Samples were taken at 0 h, 0.5 h, 1 h, 1.5 h, 2 h, and 4 h, and then every 2 h until 46 h. The OD of the bacterial culture was measured using a visible spectrophotometer. 600 The value is used as an indicator of the cell concentration in the fermentation broth.

[0045] Under culture conditions of 25℃ and 180 r / min, the growth curve of strain CBGS-20 is as follows: Figure 2 As shown in the curve analysis, the growth process of the strain can be divided into four typical stages: lag phase, logarithmic growth phase, stationary phase, and death phase. Lag phase (0-0.5 h): In the early stage of culture, strain CBGS-20 adapts to the new environment, adjusts its metabolism, and prepares for rapid proliferation; at this time, bacterial growth is relatively slow. Logarithmic growth phase (0.5-14 h): Starting from 0.5 h, the strain enters the logarithmic growth phase, and the number of bacteria increases exponentially, indicating that the strain is metabolically active and reproduces rapidly. At 14 h, the bacterial concentration reaches its peak. Stationary phase (14-46 h): The bacterial concentration remains basically unchanged. In this stage, the microbial proliferation rate equals the mortality rate, and the number of viable bacteria is in dynamic equilibrium. Death phase (after 46 h): The number of bacteria shows a downward trend. In this stage, the microbial proliferation rate is less than the mortality rate, possibly due to excessive consumption of nutrients or feedback inhibition by metabolic products leading to a decrease in the number of bacteria.

[0046] To further analyze the growth characteristics of strain CBGS-20, the growth curve was fitted using the Logistic equation (see...). Figure 2 ), fitting coefficient R 2The coefficient of performance (COP) of 0.999 indicates an excellent fit, demonstrating that the growth behavior of strain CBGS-20 conforms to a typical Logistic growth model, and the fermentation process can be optimized by regulating relevant factors. Furthermore, the long stationary phase of strain CBGS-20 indicates its high stability during cultivation, a characteristic that significantly supports its potential in practical applications. For example, in industrial production, a longer stationary phase helps improve the strain's utilization efficiency and product yield; in environmental remediation, stable growth characteristics help the strain continue to function in complex environments, indicating that strain CBGS-20 has good environmental adaptability.

[0047] Example 3: Determination of Phosphorus and Potassium Solubilizing Abilities of *Haloxyglutamate* CBGS-20 NBRIP phosphate-solubilizing medium: glucose 10.0 g, Ca3(PO4)2 5.0 g, (NH4)2SO4 0.5 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, MgCl2·6H2O 5.0 g, agar 15.0 g, 0.4% bromophenol blue 6 mL, water 1000 mL, pH 7.0~7.2.

[0048] Potassium-solubilizing medium: 10.0 g sucrose, 1.0 g Na2HPO4, 0.5 g (NH4)2SO4, 1.0 g MgSO4·7H2O, 0.2 g yeast extract, 0.1 g NaCl, 0.1 g CaCO3, 0.005 g FeCl3, 5.0 g potassium feldspar, 15.0 g agar, 1000 mL water, pH 7.0~7.2.

[0049] Seed culture of strain CBGS-20 was prepared and inoculated onto NBRIP phosphate-solubilizing medium and potassium-solubilizing medium, respectively. After culturing in a constant temperature incubator at 30℃ for 2 days, its growth and the presence of a lysis zone were observed.

[0050] The calculation method is as follows: Solubility = Diameter of the solubility zone D / Diameter of the colony d.

[0051] The results showed that CBGS-20 colonies exhibited distinct transparent zones around their periphery in both NBRIP phosphate-solubilizing and potassium-solubilizing media, with D / d values ​​of 1.90 and 1.35, respectively, indicating that they possess certain phosphate-solubilizing and potassium-solubilizing abilities.

[0052] Example 4: Stress resistance of *Haloxyglutamate* CBGS-20 The stress resistance of strain CBGS-20 was investigated based on its growth under different pH, NaCl concentrations, and temperatures. The specific method was as follows: The pH of liquid LB medium was set to 3–12, the NaCl concentration to 0–15%, and the temperature to 15℃–45℃. Seed culture of strain CBGS-20 was inoculated into 30 mL LB liquid shake flasks at a 1% (v / v) inoculation rate. The flasks were then cultured in a shaker at 30℃ and 180 r / min for 24 h. Bacterial concentration was expressed as the OD value at 600 nm using a spectrophotometer.

[0053] The optimal growth pH for strain CBGS-20 is 6–9. At pH 10, the cell concentration decreases significantly, but the OD value remains greater than 1.50, indicating that the strain's growth is inhibited by alkalinity. It can grow well within the pH range of 6–10 (see...). Figure 3 When pH ≤ 5 or pH ≥ 11, the growth of the strain is significantly inhibited and it cannot grow normally, indicating that it has strong acid and alkali resistance and environmental adaptability. It can be applied to acidic or alkaline soils, especially showing significant advantages in alkaline soils. Since most soils in iron tailings areas are alkaline, it has a good application prospect in the ecological reclamation of tailings area soils.

[0054] Strain CBGS-20 grows well in the range of 0–8% NaCl (see [reference needed]). Figure 4 When the NaCl concentration was 10%, the number of strains decreased sharply, indicating that the growth of the strain was significantly inhibited. At higher salt concentrations, the osmotic pressure in the fermentation broth exceeded the osmotic pressure inside the bacteria, causing cell shrinkage due to water loss, thereby inhibiting the physiological and biochemical reactions within the microorganisms and suppressing their growth and reproduction. This strain, CBGS-20, can grow well in the range of 0-8% NaCl, indicating that it has good salt tolerance and can maintain normal growth and colonization during the remediation of saline-alkali soils.

[0055] Depend on Figure 5 It can be seen that strain CBGS-20 grows well in the temperature range of 10℃ to 35℃, exhibiting good thermostability. However, the number of strains drops sharply when the temperature is <15℃ or ≥40℃, indicating that the strain's growth is inhibited. When the temperature is too low, the activity of the biological enzymes in the cells decreases, leading to a slowdown in the rate of corresponding enzymatic reactions, and consequently, a decrease in the rate of metabolism and proliferation. Conversely, when the temperature is too high, proteins within the bacteria become inactive, causing cell death, resulting in slow or no growth. The optimal culture temperature for strain CBGS-20 is 20~30℃.

[0056] Example 5: Ability of Halophilic Glutamate Bacterium CBGS-20 to produce heptaphilin and IAA Prepare a seed culture of strain CBGS-20, inoculate it onto CAS detection medium, and incubate at 30℃ for 2 days. The appearance of a yellow-green halo indicates the production of heparin. Adjust the bacterial concentration to 1×10⁻⁶. 9 CFU / mL was inoculated into LB liquid medium (containing 100 mg / L L-tryptophan) at a volume ratio of 2%, and cultured on a shaker at 30℃ and 180 r / min for 1-2 days. 50 μL of the supernatant after centrifugation at 8000 r / min was taken and 50 μL of Salkowski colorimetric solution was added. After developing the color in the dark for 30 min, if a pink color appeared, it was positive, indicating that the strain could secrete IAA. The darker the color, the greater the secretion intensity. No color change was negative, indicating that the strain could not secrete IAA.

[0057] The results showed that strain CBGS-20 had a solubility index of 1.95 in CAS medium, indicating that it had a good ability to produce ferrophosphate. At the same time, Salkowski colorimetric results showed that strain CBGS-20 also had the ability to produce IAA, further demonstrating its ability to promote plant growth.

[0058] Example 6: Antagonism of strain CBGS-20 against plant pathogens The antagonistic effect of strain CBGS-20 against plant pathogens was determined using the plate confrontation method. Pathogen fragments were inoculated at the center of PDA plates, and strain CBGS-20 was inoculated at equal intervals on both sides of the fragments. The plates were incubated at 25°C for 48 h, with three replicates. The absence of normal pathogen growth near strain CBGS-20 indicated an antagonistic effect. The results (see Table 2) showed that strain CBGS-20 had significant antagonistic effects against *Pythium spp.*, the causal agent of potato late blight, and *Pythium spp.*, the causal agent of corn rot.

[0059] Table 2. Antagonistic activity of strain CBGS-20 against some pathogenic fungi. .

[0060] The antibacterial activity of CBGS-20 against several plant pathogens was determined using the growth rate method. CBGS-20 was cultured in LB liquid medium for 24 h, centrifuged, and the supernatant was filtered through a 0.22 μm sterile filter. A certain amount of the sterile filtrate was added to PDA medium at 40–45 °C, mixed well, and poured into 9 cm petri dishes. A blank control was used without the addition of sterile filtrate. After the medium solidified, plant pathogen blocks were inoculated in the center of each plate, with three replicates, and incubated at 28 °C. The colony diameter was measured using the cross-cross method, and the inhibition rate was calculated. The results showed that CBGS-20 achieved a 53.8% inhibition rate against potato late blight and a 55.6% inhibition rate against Pythium spp., the causal agent of corn rot.

[0061] Inhibition rate (%) = (Coronavirus colony diameter of control bacteria - Coronavirus colony diameter of treated bacteria) / (Coronavirus colony diameter of control bacteria - Coronavirus block diameter) × 100.

[0062] Example 7: Tolerance of strain CBGS-20 to copper, lead, chromium, and zinc. Lead acetate (Pb) was separated using ultrapure water. 2+ Copper sulfate (Cu) 2+ ), potassium dichromate (Cr 6+ ), zinc sulfate (Zn 2+ A metal ion solution with a mass concentration of approximately 100 mM was prepared as a stock solution. In subsequent experiments, specific amounts of each metal ion stock solution were added to LB medium to produce media containing 0.5 mM, 1.0 mM, 2.0 mM, 5.0 mM, and 10.0 mM Cu, respectively. 2 + Pb 2+ Cr 6+ and Zn 2+ The strain CBGS-20 was sterilized at 121℃, plated, and inoculated onto different culture media. Its tolerance to heavy metal ions was determined based on the growth of the strain.

[0063] The results are shown in Table 3. Strains CBGS-20 were tested in a Cu2+ environment containing 2 mM copper ions. 2+ 5 mM lead ions Pb 2+ 1 mM hexavalent chromium ions Cr 6+ and zinc ions Zn 2+ Normal growth in the culture medium indicates that strain CBGS-20 is resistant to Cu. 2+ Pb 2+ Cr 6+ Zn 2+ The presence of a certain degree of tolerance indirectly indicates that strain CBGS-20 can be used for the remediation of soils contaminated with these four metal ions.

[0064] Table 3. Tolerance of strain CBGS-20 to heavy metal ions .

[0065] Example 8: Effects of Halophilic Glutamate Bacillus CBGS-20 on Maize Plant Growth and Soil Nutrients Test soil: Garden soil and vermiculite were mixed at a volume ratio of 1:1, and iron tailings sand (from an iron tailings mine in Chengde, Hebei Province) was added at a weight ratio of 8:2. The mixture was then used as the test soil.

[0066] Test plants: The maize variety was "Zhengdan 958", purchased from a seed company in Hebei Province. Mature, plump, and uniformly sized healthy seeds were selected, and the seed surface was disinfected with 0.1% potassium permanganate solution for 30 minutes. The seeds were then rinsed three times with deionized water and air-dried for later use.

[0067] Seed culture of strain CBGS-20 was prepared using liquid LB medium, and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL, based on 100 mL·kg -1 Mix the soil into the test soil. For the control group, mix the same volume of LB medium into the soil and conduct a maize pot experiment. In a 25℃ greenhouse, after seed germination, retain the three best-growing seedlings from each pot. After 30 days, measure the above-ground indicators of the soil and plants, as well as the root growth status. Each experiment was conducted in triplicate.

[0068] Table 4. Effects of *Hydrogenobacterium glutamicum* CBGS-20 on maize plant growth .

[0069] The results are shown in Table 4. Strain CBGS-20 significantly promoted maize growth. Compared with the CK treatment group, the maize plant height, stem diameter, fresh weight, and dry weight of the treated group increased by 20.44%, 18.83%, 33.87%, and 35.48%, respectively. The chlorophyll content of the treatment group was significantly higher than that of the control group, increasing by 12.82%.

[0070] Table 5. Effects of *Hydrogenobacterium glutamicum* CBGS-20 on soil fertility (unit: %) .

[0071] Table 5 shows that applying CBGS-20 bacterial solution in the maize pot experiment significantly increased the contents of nitrate nitrogen, ammonia nitrogen, available silicon, available phosphorus, and available potassium in the soil, by 18.65%, 11.71%, 15.38%, 35.42%, and 20.55%, respectively, compared with the control. This further demonstrates that applying CBGS-20 bacterial solution can effectively increase soil nutrient content and improve soil quality.

[0072] Example 9: Effects of Halophilic Glutamate Bacterium CBGS-20 on Maize Growth in Saline-Alkali Land Test soil: Garden soil and vermiculite were mixed at a volume ratio of 1:1, and iron tailings sand (from Chengde, Hebei Province) was added at a weight ratio of 9:1. The mixture was then used as the test soil. NaCl, Na2SO4, NaHCO3, and Na2CO3 were mixed at a molar ratio of 1:1:1:1 to prepare a 12 g / kg saline-alkali solution. The prepared saline-alkali solution was used to soak the test soil, resulting in a soil salinity of 5.6 g / kg and a soil pH of 9.29.

[0073] Test plants: The maize variety was "Zhengdan 958", purchased from a seed company in Hebei Province. Mature, plump, and uniformly sized healthy seeds were selected, and the seed surface was disinfected with 0.1% potassium permanganate solution for 30 minutes. The seeds were then rinsed three times with deionized water and air-dried for later use.

[0074] Disinfected seeds were spread evenly on two layers of filter paper soaked in sterile water in a petri dish. After germination for two days, seedlings of uniform size were selected and transplanted into flowerpots, two seedlings per pot. After transplanting, the soil was inoculated with different concentrations of CBGS-20 bacterial culture solution by watering, so that the bacterial cell concentration in the soil was 1×10⁻⁶. 6 CFU / g, 1×10 7 CFU / g and 1×10 8 The three treatments, designated T1, T2, and T3, were calculated using CFU / g. A control group (CK) was established, consisting of no bacterial culture but an equal volume of deionized water. The treatments were administered under 12-hour light exposure daily, with regular water replenishment. Each treatment was replicated in five places. After 14 days of cultivation, plant height, root length, aboveground fresh and dry weight, and underground fresh and dry weight were measured.

[0075] Table 6. Effects of *Haloxyglutamate* CBGS-20 on maize plant growth .

[0076] The results (see Table 6) showed that low concentrations of bacterial solution promoted the growth of maize seedlings, while high concentrations inhibited their growth. Among them, group T1 showed the best growth-promoting effect, with plant height, aboveground fresh weight, underground fresh weight, aboveground dry weight, and underground dry weight increasing by 22.80%, 73.40%, 90.00%, 50.00%, and 66.67% respectively (P<0.05), and root length increasing by 21.23%.

[0077] Example 10: Effects of Halophilic Glutamate Bacterium CBGS-20 on Wheat Growth in Saline-Alkali Land Test soil: Garden soil and vermiculite were mixed at a volume ratio of 1:1, and iron tailings sand (from Chengde, Hebei Province) was added at a weight ratio of 9:1. The mixture was then used as the test soil. NaCl, Na2SO4, NaHCO3, and Na2CO3 were mixed at a molar ratio of 1:1:1:1 to prepare a 12 g / kg saline-alkali solution. The prepared saline-alkali solution was used to soak the test soil, resulting in a soil salinity of 5.6 g / kg and a soil pH of 9.29.

[0078] Test plant: Wheat variety Gaoyou 2018, purchased from a seed company in Hebei Province. Mature, plump, and uniformly sized healthy seeds were selected, and the seed surface was disinfected with 0.1% potassium permanganate solution for 30 min. The seeds were then rinsed three times with deionized water and air-dried for later use.

[0079] Disinfected seeds were spread evenly on two layers of filter paper soaked in sterile water in a petri dish. After germination for two days, seedlings of uniform size were selected and transplanted into flowerpots, with five seedlings per pot. After transplanting, the soil was inoculated with different concentrations of CBGS-20 bacterial culture solution by watering, so that the bacterial cell concentration in the soil was 1×10⁻⁶. 6 CFU / g, 1×10 7 CFU / g and 1×10 8 The three treatments, designated T1, T2, and T3, were calculated using CFU / g. A control group (CK) was established, consisting of no bacterial culture but an equal volume of deionized water. The treatments were administered under 12-hour light exposure daily, with regular water replenishment. Each treatment was replicated in five places. After 14 days of cultivation, plant height, root length, aboveground fresh and dry weight, and underground fresh and dry weight were measured.

[0080] Table 7. Effects of Halophilic Glutamate Bacterium CBGS-20 on wheat growth .

[0081] As shown in Table 7, wheat growth was best in group T2, with root length, fresh weight, and dry weight increasing by 44.24%, 43.14%, and 40.91% respectively compared to the control group (CK). However, plant height showed no significant difference compared to the CK group (P<0.05). Wheat growth was significantly better in group T1 than in group T3, and group T3 showed growth inhibition compared to the CK group. These results indicate that the addition of CBGS-20 bacterial solution had no significant effect on wheat plant height, and high concentrations of CBGS-20 bacterial solution inhibited wheat growth.

[0082] The present invention has been described in detail with reference to the above embodiments. It should be noted that the above embodiments are merely illustrative examples. Without departing from the spirit and essence of the present invention, those skilled in the art can devise various alternatives and improvements to the present invention, all of which should be understood to be within the scope of protection of the present invention.

Claims

1. A type of halophilic glutamate bacillus ( Glutamicibacter halophytocola CBGS-20, characterized in that, Its accession number is CGMCC No.34682.

2. The *Halophilic Glutamate Bacterium CBGS-20* according to claim 1, characterized in that, It has the ability to release silicon, dissolve phosphorus, and dissolve potassium, and it can also produce ferrophilic elements and IAA.

3. The *Halophilic Glutamate Bacterium CBGS-20* according to claim 1, characterized in that, It can grow well at pH 6-10, NaCl concentration 0-10%, and temperature 15-35℃.

4. The *Halophilic Glutamate Bacterium CBGS-20* according to claim 1, characterized in that, It can antagonize potato late blight and corn pyridae.

5. The application of the *Haloxyglutamate* CBGS-20 as described in claim 1 in the control of potato late blight and maize pyridaben.

6. The application of the *Halophilic Glutamate Bacillus CBGS-20* as described in claim 1 in soil ecological restoration.

7. The application according to claim 6, characterized in that, The soil ecological restoration includes the remediation of tailings area soil, acidic soil, alkaline soil, saline-alkali soil and / or heavy metal contaminated soil.

8. The application of the *Halophilic Glutamate Bacillus CBGS-20* as described in claim 1 in promoting the growth of maize and wheat grown in saline-alkali soil.

9. The use of the *Halophilic Glutamate Bacillus CBGS-20* as described in claim 1 in the preparation of soil remediation agents, biofertilizers, biopesticides, and / or artificial substrates.

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