Arthrobacter sp. T64 capable of effectively promoting soybean growth and wheat yield increase in saline-alkali soil and application of arthrobacter sp. T64

Abstract

The invention discloses a pseudoarthrobacter sp. T64 for promoting soybean growth and wheat yield increase in saline-alkali soil and application of the pseudoarthrobacter sp. T64. The classification name of the pseudoarthrobacter sp. Is pseudoarthrobacter sp., the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on October 20, 2025, and the preservation number of the strain is CGMCC No.36271. The invention further discloses a preparation method of the pseudoarthrobacter sp. The addition of the pseudoarthrobacter T64 can effectively promote soybean growth and wheat yield increase in the saline-alkali soil.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology, specifically relating to a strain of *Arthrobacter* T64 that effectively promotes the growth of soybeans and increases wheat yield in saline-alkali land and its applications. Background Technology

[0002] *Pseudarthrobacter*, belonging to the phylum Actinobacteria, class Micrococci, order Micrococci, family Micrococciaceae, is a group of Gram-positive bacteria. While *Pseudarthrobacter* does not directly participate in nitrogen fixation in soybeans, its growth-promoting effect is significant. It secretes plant growth regulators, and some strains can synthesize plant hormones such as indoleacetic acid (IAA) and gibberellin (GA), promoting seed germination and root development. *Pseudarthrobacter* enhances the bioavailability of nutrients like phosphorus and iron in the soil by secreting organic acids or siderophores. It can also activate the jasmonic acid / ethylene signaling pathway in plants, enhancing their defense against diseases and abiotic stresses. *Pseudarthrobacter* can form symbiotic communities with rhizobia, Bacillus, and others, creating complementary growth-promoting bacterial communities. With the increasing salinization of arable land globally, screening for salt-tolerant growth-promoting bacteria has become crucial for sustainable agricultural development. *Pseudarthrobacter*, with its high salt tolerance, demonstrates unique advantages and immense potential in this field, but research on its role in soybean tolerance to salt stress remains relatively limited.

[0003] Saline-alkali land refers to land containing a large amount of soluble salt ions such as Na+. + K + Mg 2+ Cl - HCO 3- Land such as saline-alkali soils. Saline-alkali soils can be classified into saline soils (EC) according to their properties. SE ≥ 4 dS·m -1 And SAR < 13), saline-alkali soil (EC SE ≥ 4 dS·m -1 And SAR >13) and alkaline earth (EC SE < 4 dS·m -1 Based on salinity (SAR ≥ 13), saline-alkali soils can be classified into mildly saline-alkali soils (total salt content < 3‰), moderately to severely saline-alkali soils (3‰ < total salt content < 5‰), and severely saline-alkali soils (total salt content > 5‰). Saline-alkali soils with moderate to high salinity can significantly harm crop growth, primarily due to high osmotic pressure inhibiting water acquisition by crops, direct poisoning of crop cells by salt ions, and secondary damage caused by these two effects, such as oxidative stress, nutrient imbalance, and organ aging.

[0004] Soybeans are an important economic crop and one of the world's four major legumes. Their main functions include: 1) providing humans with nutrients such as protein, lipids, amino acids, and isoflavones, protecting the cardiovascular system, preventing osteoporosis, and improving immunity; 2) being processed into condiments and foods such as soybean oil, soy sauce, fermented black beans, tofu, and bean curd sheets; 3) using soybean straw as livestock feed, reducing breeding costs and risks; and 4) serving as a component in anti-cancer drugs. my country's domestic soybean production accounts for only about 20% of total consumption, with the remainder needing to be imported, which to some extent restricts my country's agricultural development and food security. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *Dendrobium pseudoarthritis* that effectively promotes the growth of soybeans and increases wheat yield in saline-alkali land, addressing practical problems and needs in production.

[0006] Another object of the present invention is to provide the application of this plant growth-promoting bacterium.

[0007] The objective of this invention can be achieved through the following technical solutions: Twenty-five different soybean varieties were collected from the salt-tolerant soybean breeding base in Kenli District, Dongying City, Shandong Province (118.89 E; 37.64 N). DNA was extracted from their rhizosphere soil and subjected to high-throughput sequencing. After obtaining the sequencing results, the differences in microbial communities among different soybean varieties were analyzed. Combined with yield data from different varieties, it was found that *Pseudomonas* bacteria are one of the key bacteria in the microbial community of high-yielding soybeans in saline-alkali land. Bacteria were isolated from the soybean rhizosphere soil using a spread plating method. After molecular biological identification, several *Pseudomonas* strains and other strains were obtained. Experimental verification showed that one *Pseudomonas* strain can effectively promote soybean growth and increase wheat yield, and this strain was named T64.

[0008] Pseudarthrobacter T64, classified as Pseudarthrobacter sp., was deposited on October 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36271. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The application of the *Pseudomonas pyrenoidosa* T64 described in this invention in promoting soybean growth and increasing wheat yield in saline-alkali land.

[0010] The application of the *Pseudomonas pyrenoidosa* T64 described in this invention in the preparation of products that promote the growth of soybeans and increase wheat yield in saline-alkali land.

[0011] The bacterial agent prepared from Pseudomonas T64 described in this invention.

[0012] As a preferred embodiment of the present invention, the bacterial agent is prepared by the following method: a single colony of *Pseudomonas pseudoarthritis* T64 is inoculated into TSB liquid medium and placed at 28°C and 170 r·min. -1 The bacteria were shaken on a shaker for about 48 hours to obtain the fermentation broth of *Arthrobacter* T64, which is the bacterial agent described above.

[0013] As a further preferred embodiment of the present invention, the single colony of *Arthrobacter pseudoarthritis* T64 is a colony that grows from *Arthrobacter pseudoarthritis* T64 glycerol culture activated by streaking on 1 / 2 TSB solid medium and then incubated overnight at 28°C.

[0014] The application of the microbial agent described in this invention in soybean plant cultivation.

[0015] A method for cultivating soybean plants involves burying soybean seeds in a seedling substrate, thoroughly watering them, and transplanting the seedlings into natural saline-alkali soil after germination and the emergence of new leaves. After transplanting, the seedlings are inoculated with a microbial agent prepared from T64 fermentation broth at an inoculation ratio of 1×10⁻⁶. 6 CFU·g -1 The soil was irrigated with the fungicide twice, with a 10-day interval between each application.

[0016] The application effects of this invention are as follows: This invention screened a strain of *Arthrobacter pseudoarthritis* T64 that can promote soybean growth and increase wheat yield in saline-alkali soil. Compared with the control group with added water, adding 1×10⁻⁶ bacteria per gram of saline-alkali soil significantly improved the growth rate. 6 CFU·g -1 After fermentation with T64, soybean plant growth was significantly promoted, as evidenced by good results in biomass measurements (plant height, aboveground fresh weight, and aboveground dry weight). Germination experiments showed that inoculation with *Pseudomonas pseudoartemisinus* T64 significantly improved the germination rate of soybean seeds under salt stress. Field trials also showed that the addition of *Pseudomonas pseudoartemisinus* T64 inoculant could effectively increase wheat yield. Attached Figure Description

[0017] Figure 1 Photo of bacterial strain on plate Figure 2 16S rRNA phylogenetic tree of strains Figure 3 Effects of inoculation with *Arthrobacter pseudoarthritis* T64 on the growth of potted soybeans Figure 4 Effects of inoculation with *Arthrobacter pseudoarthritis* T64 on wheat yield in coastal saline-alkali land Information on the preservation of biological materials

[0018] T64, classified as Pseudarthrobacter sp., was deposited on October 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36271. Detailed Implementation

[0019] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0020] Unless otherwise specified, the raw materials used in the following examples are as follows: The strain: The pseudoarthrobacter T64 was obtained and preserved by the Soil Microbiology and Organic Fertilizer Team of the College of Resources and Environmental Sciences of Nanjing Agricultural University from soybean rhizosphere soil in saline-alkali land.

[0021] Seedling substrate: Xingxing Xiangnong brand general-purpose special substrate is used. This seedling substrate is made from imported peat and coconut coir as the main raw materials, with the addition of natural minerals and humic acid organic fertilizer. It is produced after biological fermentation and harmless treatment and is purchased from Jiangsu Xingnong Substrate Technology Co., Ltd.

[0022] Natural saline-alkali soil: collected from the salt-tolerant soybean variety breeding base in Kenli District, Dongying City, Shandong Province (118.89 E; 37.64 N). The total salt content of this plot is between 3‰ and 5‰.

[0023] Soybean seeds: The soybean seeds used for the germination test were TZX-746, one of 25 varieties, provided by Professor Tian Zhixi's team at Yazhou Bay National Laboratory.

[0024] Solid culture medium formulation: TSB solid medium, i.e., tryptone soybean broth agar medium. The formulation is (g·L⁻¹) -1 ): 30 g tryptone soybean broth medium, 20 g agar powder, and deionized water to a final volume of 1000 mL, sterilized at 115℃ for 30 min. 1 / 2 TSB solid medium is prepared by diluting TSB solid medium twice with water.

[0025] Liquid culture medium formulation: TSB medium, i.e., tryptone soybean broth medium. The formulation is (g·L⁻¹) -1 ): 30 g of tryptone soybean broth medium was diluted to 1000 mL with deionized water and sterilized at 115°C for 30 min. 1 / 2 TSB medium was prepared by diluting TSB medium twice with water.

[0026] Example 1: Analysis of Rhizosphere Microbial Community Characteristics of Different Soybean Varieties Twenty-five different soybean varieties were collected from the salt-tolerant soybean breeding base in Kenli District, Dongying City, Shandong Province (118.89 E; 37.64 N). DNA was extracted from the rhizosphere soil of different soybean varieties and subjected to high-throughput sequencing. OTUs with a relative abundance greater than 0.0001 and a frequency greater than 80% in all samples were defined as core taxa. Spearman's rank correlation coefficient and random forest model were used to predict potential functional bacteria in the core taxa, and 13 OTUs significantly enriched in high-yielding field varieties were identified (P < 0.05; R > 0), defined as potential key rhizosphere bacterial OTUs. Among the key OTUs, one OTU was classified as a genus of bacteria, indicating that *Pseudomonas* bacteria are one of the key rhizosphere bacteria of high-yielding soybean varieties in saline-alkali soil.

[0027] Example 2: Isolation and Identification of Functional Strains Take 1 g of soybean rhizosphere soil and place it in a centrifuge tube containing 9 mL of sterile water. Incubate at 28℃ and 170 r·min. -1 After shaking for 30 minutes, remove the sample and perform serial dilutions. Take 100 µL of soil suspensions of different concentration gradients and spread them on 1 / 2 TSB solid medium. Then, place the spread plates in an incubator at 28°C for 2 days. After colonies grow, select differentially expressed colonies for purification. Inoculate the purified strains onto 1 / 2 TSB solid plates and store them at 4°C for later use. Separately store the purified single colonies in glycerol tubes at -80°C.

[0028] Remove the glycerol tube from the -80°C freezer and allow it to thaw naturally at room temperature. Use an inoculation loop to collect the bacterial culture and streak it onto a 1 / 2 TSB solid medium plate. Incubate the streaked plate at 28°C for 2 days. Once single bacteria have grown, send the plate to a sequencing company for sequencing. Sequence comparison with the NCBI database revealed one strain of *Pseudomonas* (numbered T64) and one strain of *Acinetobacter* (numbered T113). The 16S rRNA sequence of T64 is as follows: Example 3: Liquid propagation culture of functional strains The *Pseudomonas pseudoarbus* T64 glycerol tubes isolated from soybean rhizosphere soil were removed from a -80°C freezer. A sterile inoculation loop was used to dip the ice residue inside the glycerol tubes, followed by streaking on 1 / 2 TSB solid medium for activation. The tubes were then incubated overnight at 28°C. After colonies appeared, single colonies were inoculated into 1 / 2 TSB liquid medium and incubated at 28°C and 170 rpm. -1 The fermentation broth of *Pseudomonas pseudoarthritis* T64 was obtained by shaking on a shaker for approximately 48 hours. The fermentation broth was centrifuged at 5500 rpm for 5 minutes, the supernatant was discarded, and the broth was resuspended in sterile buffer until the OD value of the resuspended bacterial solution was 1 (the concentration of this strain at an OD value of 1 is approximately 10⁻⁶). 9 CFU·mL -1 The culture methods for the remaining strains are the same as above.

[0029] Example 4: Effect of functional bacteria on soybean germination under salt stress The germination of soybean seeds under the action of functional bacteria at NaCl concentrations of 0 mM, 100 mM, and 200 mM was determined. Seeds were surface-sterilized by soaking in 70% alcohol for 2 min, followed by rinsing five times with sterile water to remove residual alcohol. Gauze was moistened with sterile water and laid flat in the germination box. The surface-sterilized seeds were placed on the gauze. Control and T64 (10) germination tests were conducted. 6 CFU·mL -1 Three treatments were used: seed soaking in bacterial solution, T113, and T25 seeds were placed in each seed box. The seeds were germinated in the dark at 25°C. The germination status of the seeds was recorded every 24 hours for 5 consecutive days.

[0030] As shown in Table 1, for TZX-746, after treatment with Bacillus pseudoartemisinus T64 bacterial solution, the germination rate of soybean seeds increased by 35.29%, 16.67%, and 1100% compared with the CK treatment (120 h) at 0 mM, 100 mM, and 200 mM NaCl concentrations, respectively; compared with the T113 treatment (120 h) at 0 mM and 100 mM NaCl concentrations, it increased by 27.78% and 75%, respectively. However, at a 200 mM NaCl concentration, the germination rate of seeds treated with T64 was 48%, while the germination rate of seeds treated with T113 was only 0%. For TZX-1736, treatment with *Pseudomonas pseudoarbus* T64 bacterial solution increased soybean seed germination rate by 26.32%, 140%, and 633.33% compared to the control (CK) treatment (120 h) at 0 mM, 100 mM, and 200 mM NaCl concentrations, respectively; and by 118.18%, 200%, and 175% compared to the T113 treatment (120 h) at 0 mM, 100 mM, and 200 mM NaCl concentrations, respectively. In conclusion, the results indicate that *Pseudomonas pseudoarbus* T64 can promote soybean seed germination, especially under salt stress.

[0031] Table 1. Effects of functional bacteria on soybean germination under salt stress

[0032] Example 5: Experiment on the effect of functional bacteria on the growth of soybeans planted in saline-alkali soil Add seedling substrate to 50-cell seedling trays, bury soybean seeds in the substrate, water thoroughly, and place in a greenhouse to germinate. Once the seeds have germinated and grown two new leaves, transplant the seedlings into pots, each containing 500g of natural saline-alkali soil. After the seedlings have stabilized in the pots, treat them with inoculum solution at a rate of 10g / L. 6 CFU·g -1 The soil was inoculated again after 7 days, while the control treatment was irrigated with an equal volume of sterile water. Each treatment was replicated 8 times. Ten days after the second bacterial inoculation, the plant height, aboveground fresh weight, and aboveground dry weight of soybeans were measured.

[0033] Table 2 shows that for TZX-746, although there was no significant difference in plant height among different treatments after inoculation with *Pseudomonas pseudoartemisinus* T64, the aboveground fresh weight and aboveground dry weight were significantly higher than those of the CK treatment, exceeding them by 46.43% and 40.00%, respectively. For TZX-1736, after inoculation with *Pseudomonas pseudoartemisinus* T64, the plant height, aboveground fresh weight, and aboveground dry weight were significantly higher among different treatments than those of the CK treatment, exceeding them by 22.47%, 33.85%, and 48%, respectively. In conclusion, the results indicate that *Pseudomonas pseudoartemisinus* T64 can significantly promote the growth of soybeans in saline-alkali land.

[0034] Table 2. Effects of functional bacteria on soybean growth in saline-alkali soil.

[0035] Example 6: Experiment on the effects of functional bacteria on wheat growth in the field The coastal saline-alkali land experimental field planted with wheat was divided into 9 plots, each 4 meters long and 3 meters wide. Three plots were treated with T64 inoculant (BFT64, 3 replicates), another three plots were treated with T64 inactivated by gamma rays (SBFT64, 3 replicates), and the last three plots served as the control (CK, 3 replicates). The inoculant solution was applied by root irrigation, with 300 mL of the original inoculant solution (10) applied at the wheat seedling stage. 9 CFU·mL -1 The inoculant was diluted 10 times and applied to the roots of each BFT64 and SBFT64 plot. A second application was made half a month later. Wheat from different treatments was threshed and yields were measured at harvest. The results showed that the T64 inoculant significantly increased wheat yield in saline-alkali land. The BFT64 treatment showed a 28.30% increase in yield compared to the control (CK), and a 19.30% increase compared to SBFT64.

[0036] Table 3. Effects of functional bacteria on wheat yield in saline-alkali land.

Claims

1. A strain of Arthrobacter pseudoarthritis ( Pseudarthrobacter sp.) T64, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36271, deposited on October 20, 2025, at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The application of the *Pseudomonas* T64 as described in claim 1 in promoting soybean growth and increasing wheat yield in saline-alkali land.

3. The application of the *Pseudomonas* T64 as described in claim 1 in the preparation of products that promote the growth of soybeans and increase wheat yield in saline-alkali land.

4. The bacterial agent prepared from *Pseudomonas* T64 according to claim 1.

5. The microbial agent according to claim 4, characterized in that, The bacteria were prepared by the following method: a single colony of *Pseudomonas* T64 was inoculated into 1 / 2 TSB liquid medium and placed at 28°C and 170 r·min. -1 The bacteria were shaken on a shaker for approximately 48 hours to obtain the fermentation broth of *Pseudomonas pseudoarthritis* T64, with a bacterial concentration ≥ 10⁻⁶. 9 CFU·mL -1 This refers to the aforementioned microbial agent.

6. The microbial agent according to claim 5, characterized in that, The aforementioned single colony of *Arthrobacter* T64 is a colony that grows from *Arthrobacter* T64 glycerol culture activated by streaking on 1 / 2 TSB solid medium and incubated overnight at 28°C.

7. The application of the microbial agent according to any one of claims 4 to 6 in soybean plant cultivation.

8. A method for cultivating soybean plants, characterized in that, Soybean seeds are buried in seedling substrate and thoroughly watered. After germination and the emergence of new leaves, the seedlings are transplanted into natural saline-alkali soil. After transplanting, the seedlings are inoculated with the inoculant as described in any one of claims 4 to 6 at an inoculation ratio of 1×10⁻⁶. 6 CFU·g -1 The soil was irrigated with the fungicide twice, with a 10-day interval between each application.

Images