Rhizosphere growth-promoting bacteria for enhancing salt tolerance of corn and application thereof

Abstract

The application provides a rhizosphere growth promoting bacterium capable of enhancing salt tolerance of corn and application thereof, and belongs to the technical field of agricultural microorganisms. The application provides a Pseudomonas bacterium TJ1-185, with a preservation number of CDMCC No.64979. The Pseudomonas bacterium TJ1-185 can improve salt stress tolerance of plants and relieve damage of salt stress to plants. The results of examples show that the Pseudomonas bacterium TJ1-185 can promote germination of corn under salt stress, improve plant height, root length, fresh weight of aboveground part, fresh weight of root, dry weight of aboveground part and dry weight of root of corn under salt stress, and also can reduce content of malondialdehyde, thereby promoting growth of corn under salt stress and relieving damage of salt stress to corn.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a rhizosphere growth-promoting bacterium that enhances the salt tolerance of maize and its application. Background Technology

[0002] Soil salinization refers to the phenomenon where salt-containing water in the subsoil rises to the surface due to capillary forces, and after evaporation, soluble salts precipitate and accumulate on the soil surface. Soil salinization can be further classified into natural and secondary salinization based on its causes. Natural salinization is mainly distributed around salt lakes, while secondary salinization is mainly distributed in the low-lying areas of river and lake terraces. The causes of soil salinization are divided into natural and anthropogenic factors. Natural factors mainly refer to the accumulation of soluble salts in the soil surface after natural circulation, including climate change, topography, geology, and hydrological conditions. Anthropogenic factors mainly include excessive deforestation, improper irrigation, and large-scale land reclamation. Soil salinization is a widespread problem worldwide. It adversely affects the physical and chemical properties of soil. High salt concentrations lead to structural deterioration, such as swelling, disintegration, dispersion, hardening, and surface crusting, resulting in decreased hydraulic conductivity, water permeability, and porosity. Excessive salinity also causes increased soil pH, organic matter loss, nutrient cycling inhibition, and toxicity of some major anions / cations in the soil. Salinized soils often exhibit nutrient deficiencies (primarily N, P, and K) and reduced availability of micronutrients (such as Al, Fe, Zn, Cu, and Mn), ultimately leading to decreased soil fertility. Soil salinization also creates salt stress for plants, affecting crop growth. Excessive soil salinity causes plants to absorb large amounts of salt, inhibiting physiological metabolic processes and even affecting plant survival. High salt stress also adversely affects plant physiological processes such as ion homeostasis, lipid metabolism, photosynthesis, and protein synthesis. For agricultural production, soil salinization affects crop growth and development, reducing the quality of agricultural products. Therefore, soil salinization is a major factor limiting agricultural production.

[0003] In recent years, some progress has been made in alleviating plant salt stress through the exogenous addition of microorganisms, and this method has gradually developed into a novel approach. Experiments have shown that inoculation with arbuscular mycorrhizal (AM) bacteria can enhance plant resistance to salt stress. AM bacteria improve the salt tolerance of maize, mung beans, and clover by improving osmotic regulation or proline accumulation. Currently, no Pseudomonas bacteria have been identified that can be used to alleviate soil salinization and promote plant growth. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the purpose of this invention is to provide a Pseudomonas bacterium that can improve the salt stress tolerance of crops in saline-alkali land, promote the growth of crops in saline-alkali land, and increase the yield of crops in saline-alkali land.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention provides a Pseudomonas sp. bacterium TJ1-185, with accession number CDMCC No. 64979.

[0007] This invention provides a bacterial agent comprising the Pseudomonas spp. bacteria TJ1-185 described in the above technical solution.

[0008] Preferably, the OD of Pseudomonas spp. TJ1-185 in the bacterial agent is... 600 The value is ≥1.0.

[0009] This invention provides a method for preparing the microbial agent described in the above technical solution, comprising:

[0010] The Pseudomonas bacteria TJ1-185 were cultured in a culture medium to obtain a bacterial agent.

[0011] Preferably, the culture temperature is 30–37°C; the culture time is 18–30 h; and the culture rotation speed is 150–250 rpm.

[0012] This invention provides the application of the Pseudomonas spp. bacteria TJ1-185 described in the above technical solution, the bacterial agent described in the above technical solution, or the bacterial agent prepared by the preparation method described in the above technical solution in promoting plant growth and / or improving plant salt tolerance.

[0013] Preferably, the plant includes corn.

[0014] Preferably, promoting plant growth includes any one or more of the following: increasing plant height, increasing plant root length, increasing the fresh weight of the above-ground parts of the plant, increasing the fresh weight of the plant roots, increasing the dry weight of the above-ground parts of the plant, and increasing the dry weight of the plant roots.

[0015] Preferably, the improvement of plant salt tolerance includes mitigating the damage of salt stress to plants and / or improving the plant's ability to tolerate salt stress.

[0016] This invention provides a method for improving the salt stress tolerance of plants, comprising:

[0017] Apply a bacterial agent containing Pseudomonas bacteria TJ1-185 as described in the above technical solution during plant growth.

[0018] Beneficial effects:

[0019] This invention provides a *Pseudomonas* sp. bacterium, TJ1-185, with accession number CDMCC No. 64979. The *Pseudomonas* sp. bacterium TJ1-185 provided by this invention can improve the salt stress tolerance of plants, alleviate the damage caused by salt stress, promote the growth of crops in saline-alkali land, and increase the yield of crops in saline-alkali land. The results of the embodiments of this invention show that the *Pseudomonas* sp. bacterium TJ1-185 can promote the germination of maize under salt stress, increase the plant height, root length, aboveground fresh weight, root fresh weight, aboveground dry weight, and root dry weight of maize under salt stress conditions, and at the same time, it can reduce malondialdehyde content, thereby promoting the growth of maize under salt stress conditions and alleviating the damage caused by salt stress to maize.

[0020] Biological Preservation Instructions

[0021] A bacterium of the genus *Pseudomonas*, TJ1-185, was deposited on May 8, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located on the 5th floor of the Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCCNO.64979. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 A graph showing the statistical results of corn germination rate under different salt concentrations;

[0024] Figure 2 The figure shows the phenotypic results of the whole maize plant of functional bacteria TJ1-185 and blank control group at a salt concentration of 5‰.

[0025] Figure 3 The figure shows the phenotypic results of potted maize with functional bacteria TJ1-185 and the blank control group at a salt concentration of 5‰.

[0026] Figure 4 The figure shows the phenotypic results of the whole maize plant of functional bacteria TJ1-185 and blank control group at a salt concentration of 8‰.

[0027] Figure 5 The figure shows the phenotypic results of potted maize with functional bacteria TJ1-185 and the blank control group at a salt concentration of 8‰.

[0028] Figure 6The graph shows the effects of five functional bacteria on maize plant height.

[0029] Figure 7 The results of the effects of five functional bacteria on maize root length are shown in the figure.

[0030] Figure 8 Figure showing the effects of five functional bacteria on the fresh weight of maize aboveground parts;

[0031] Figure 9 Figure showing the effect of five functional bacteria on the fresh weight of maize roots;

[0032] Figure 10 Figure showing the effects of five functional bacteria on the aboveground dry weight of maize.

[0033] Figure 11 The effect of five functional bacteria on the dry weight of maize roots is shown in the figure.

[0034] Figure 12 The effect of five functional bacteria on malondialdehyde (MDA) content in corn is shown in the figure.

[0035] Figure 13 The figure shows the phylogenetic analysis results of Pseudomonas bacteria TJ1-185. Detailed Implementation

[0036] This invention provides a *Pseudomonas* sp. bacterium, TJ1-185, with accession number CDMCC No. 64979. In this invention, the *Pseudomonas* sp. TJ1-185 is derived from the rhizosphere of the halophyte *Senecio scandens*. In this invention, the nucleotide sequence of the 16S rDNA of the *Pseudomonas* sp. TJ1-185 is shown in SEQ ID NO. 1. In this invention, the *Pseudomonas* sp. TJ1-185 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 8, 2024, with accession number GDMCC NO. 64979.

[0037] This invention provides a bacterial agent comprising *Pseudomonas* bacteria TJ1-185 as described in the above-mentioned technical solution. As an optional embodiment of this invention, the OD of *Pseudomonas* bacteria TJ1-185 in the bacterial agent is... 600 The value can be ≥1.0 or 1.0.

[0038] This invention provides a method for preparing the microbial agent described in the above technical solution, comprising:

[0039] The Pseudomonas bacteria TJ1-185 were cultured in a culture medium to obtain a bacterial agent.

[0040] As an optional embodiment of the present invention, the culture medium includes TSB liquid culture medium; the culture temperature can be 30-37°C, or 30, 31, 32, 33, 34, 35, 36 or 37°C; the culture time can be 18-30h, or 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30h; the rotation speed during the culture process can be 150-250rpm, or 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250rpm.

[0041] After culturing, a culture medium of *Pseudomonas* spp. TJ1-185 is obtained. As an optional embodiment of the present invention, the *Pseudomonas* spp. TJ1-185 culture medium can be used directly as a bacterial agent, or the bacterial cells in the *Pseudomonas* spp. TJ1-185 culture medium can be isolated and resuspended to obtain a bacterial suspension for use as a bacterial agent. The present invention does not specifically limit the isolation method; any conventional method in the art can be used. As an optional embodiment of the present invention, the isolation method can be centrifugation. After the bacterial cells are isolated, they are resuspended in sterile water to obtain a bacterial suspension. As an optional embodiment of the present invention, the OD of *Pseudomonas* spp. TJ1-185 in the bacterial suspension... 600 The value can be ≥1.0 or 1.0.

[0042] This invention provides the application of the *Pseudomonas* bacteria TJ1-185 described in the above-described technical solutions, the bacterial agent described in the above-described technical solutions, or the bacterial agent prepared by the preparation method described in the above-described technical solutions in promoting plant growth and / or improving plant salt tolerance. As an optional embodiment of this invention, the plant includes maize. As an optional embodiment of this invention, promoting plant growth includes increasing plant height, increasing plant root length, increasing aboveground fresh weight, increasing plant root fresh weight, increasing aboveground dry weight, and increasing plant root dry weight, or two or more of these. As an optional embodiment of this invention, improving plant salt tolerance includes alleviating the damage of salt stress to plants and / or improving the plant's ability to tolerate salt stress. The results of the embodiments of this invention show that the *Pseudomonas* bacteria TJ1-185 can promote maize growth under 5‰ and 8‰ salt concentration stress conditions, increasing maize plant height, root length, aboveground fresh weight, root fresh weight, aboveground dry weight, and root fresh weight. Furthermore, the Pseudomonas bacteria TJ1-185 can significantly reduce the malondialdehyde content in maize under salt stress conditions, thereby improving the salt stress tolerance of maize.

[0043] This invention provides a method for improving the salt stress tolerance of plants, comprising:

[0044] Apply a bacterial agent containing Pseudomonas bacteria TJ1-185 as described in the above technical solution during plant growth.

[0045] As an optional embodiment of the present invention, the inoculant can be applied either during seed planting or after the plant has reached the two-leaf-one-heart stage. When applying the inoculant containing the *Pseudomonas* spp. TJ1-185 after the plant has reached the two-leaf-one-heart stage, the application time can be within 1-2 weeks of the plant reaching this stage. As an optional embodiment of the present invention, the application can be performed twice; the interval between the two applications is two weeks; and the amount of *Pseudomonas* spp. TJ1-185 used in each application is 10... 7 CFU / g soil, meaning 10 CFU is applied per gram of soil. 7 CFU is a Pseudomonas bacterium, TJ1-185.

[0046] As an optional embodiment of the present invention, the Pseudomonas bacteria TJ1-185 can also be mixed with solids for fermentation and then mixed with organic fertilizer and applied to the soil.

[0047] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0048] 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 all purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0049] The corn variety used in the following examples is Zhengdan 958.

[0050] Example 1: Construction of a microbial strain resource bank

[0051] Halophytic materials: Suaeda salsa, Sesbania sesquiterpene, and Tamarix chinensis were collected from a farm near the Chinese Milu Deer Park in Dafeng District, Yancheng City (32°59′46″N, 120°49′48″E); the halophyte Spartina alterniflora was collected from the mudflats near the Yellow Sea National Forest Park in Dafeng District, Yancheng City (32°52′25″N, 120°54′24″E).

[0052] Using a macro-omics approach, rhizosphere samples of halophytes were subjected to extreme dilutions with PBS-S, with a dilution gradient of 10-1. -6 10 -7Rhizosphere sample suspensions were cultured in TSB liquid medium in 96-well plates. After 14 days of culture, 96-well plates with a microbial growth probability of 30%-50% were selected. Under aseptic conditions, 80 μL of bacterial culture was aspirated from the wells containing growing microorganisms and added to a PCR tube containing 80 μL of 40% glycerol (a 40% glycerol aqueous solution by volume). The mixture was gently blown and aspirated, labeled, and stored at -80℃ to preserve the microbial strain, thus obtaining a halophyte rhizosphere microbial strain resource bank.

[0053] Experimental results: 3991 effective mycelial pores were isolated from the rhizosphere of four halophytes: Sesbania, Suaeda salsa, Spartina alterniflora and Tamarix chinensis.

[0054] Example 2 Screening of functional strains

[0055] I. Determination of the critical salt concentration for maize seed germination

[0056] 1. Select plump corn seeds of uniform size and disinfect their surface.

[0057] Soak the seeds in a 75% ethanol aqueous solution for 1 minute, then rinse off the ethanol on the seed surface with sterile water. Next, soak the seeds in a 2% sodium hypochlorite solution for 10 minutes, and rinse them 6 times with sterile water.

[0058] 2. After surface disinfection in step 1, the corn seeds were soaked in sterile sodium chloride solutions of different salt concentrations (0mM, 40mM, 80mM, 120mM, 160mM, 200mM, 240mM, 280mM), with three replicates for each treatment and 10 seeds per replicate. The seeds were soaked at 25°C in the dark for 4 hours.

[0059] 3. Place the seeds soaked in step 2 into a petri dish containing sterile moistened filter paper with the appropriate salt concentration, and place it in a 25℃ light incubator for germination, with a day / night ratio of 16h / 8h for 7 days.

[0060] 4. After completing step 3, calculate the germination rate of maize seeds under different treatments (germination rate (%) = (number of normally germinated seeds on day 7 / number of tested seeds) × 100) and find the critical salt concentration for maize seed germination.

[0061] Experimental results: Based on the statistical results of corn germination rate under different salt concentrations ( Figure 1 ), and determined that 240 mM is the critical salt stress concentration for corn germination.

[0062] II. Screening of Mycelial Wells

[0063] Test strains: 3991 effective wells isolated in Example 1.

[0064] 1. After surface disinfection, corn seeds were soaked in a 240mM sodium chloride solution at 25°C in the dark for 4 hours. The control group was soaked in sterile water.

[0065] 2. Place the seeds soaked in step 1 in a petri dish containing sterile water and 240mM sodium chloride solution and lined with filter paper, with 3 seeds per petri dish and three replicates.

[0066] 3. Take 10 μL of bacterial culture from each PCR tube of the halophyte strain resource bank constructed in Example 1 and transfer it to a 2 mL centrifuge tube containing 1 mL of TSB liquid medium. Gently mix and incubate at 30°C and 170 rpm in a shaker until OD reaches the target value. 600 =1.0.

[0067] 4. In step 2, drop 100 μL of the corresponding bacterial solution from step 3 directly above each corn seed. That is, add the bacterial solution obtained from step 3 to one effective well directly above each corn seed. Number the wells and place them in a 25℃ light incubator for germination. The day / night ratio is 16h / 8h for 4 days.

[0068] 5. Observe the germination of different treatments in step 4, and preliminarily screen out mycelial pores that can promote corn germination under salt stress conditions.

[0069] A positive control was obtained by treating with Bacillus amyloliquefaciens SQR9 (accession number CGMCC NO.5808, disclosed in ZL201710797445.0), a negative control was obtained by adding only salt and no bacteria, and a control was obtained by neither adding salt nor bacteria.

[0070] Experimental results: Based on the statistical results of the germination rate of corn after inoculation with different mycelium wells at a salt concentration of 240mM, 33 mycelium wells that can promote the germination of corn under salt stress were screened out.

[0071] III. Purification of the bacterial strain

[0072] Based on the initial screening results of the germination test under salt stress, the mycelial wells that can promote the germination of maize under 240mM salt stress were identified and isolated and purified as single bacteria.

[0073] 1. Spread the bacterial culture from the wells obtained from the initial screening onto a TSB plate and incubate upside down at 30°C for 24 hours.

[0074] 2. Select individual colonies that grow rapidly and have different morphologies from step 1, and continue to streak the remaining colonies that have not yet appeared on TSB plates until individual colonies appear.

[0075] 3. Then, purify each single colony from step 2 by streaking it onto a TSB plate three times.

[0076] 4. Pick a single colony from step 3 into a test tube containing 3 mL of TSB liquid culture medium, and shake at 30°C and 170 rpm until the culture becomes turbid.

[0077] 5. Take 80 μL of the bacterial culture from step 4 into a 40% glycerol tube and store it in a refrigerator at -80°C.

[0078] Based on the preliminary screening results of crop germination experiments under salt stress, a single-strain resource bank was constructed.

[0079] Experimental results: Five strains that can promote the germination of maize under salt stress were purified, namely TJ1-160, TJ1-229, TJ1-238, TJ1-185 and TJ1-179.

[0080] Example 3: Pot culture verification of functional strains

[0081] Test strains: 5 strains screened in Example 2

[0082] Soil materials used in the test: saline-alkali soil from Cangdong Farm, Yancheng City, Jiangsu Province, divided into two salinity gradients (5‰ and 8‰). The pH of the 5‰ saline-alkali soil was 7.84, and the pH of the 8‰ saline-alkali soil was 7.80.

[0083] I. Preparation of bacterial suspension

[0084] 1. The five functional strains and the control strain SQR9 obtained from the screening were inoculated into TSB liquid medium and cultured at 30°C and 170 rpm.

[0085] 2. After 24 hours, centrifuge the bacterial suspension, then resuspend the bacterial suspension in sterile water to adjust the OD of the suspension. 600 The value is 1.0.

[0086] II. Germination and Seedling Raising of Corn Seeds

[0087] 1. First, the surface of the corn seeds needs to be sterilized. This step is carried out in a clean bench. Select plump Zhengdan 958 corn seeds that are basically the same size, disinfect the corn seeds with an alcohol solution with a volume fraction of 75% for 1 minute, then soak the corn seeds in a sodium hypochlorite solution with a mass concentration of 2% for 10 minutes, and rinse them 6 times with sterile ultrapure water to wash away the sodium hypochlorite solution adhering to the seed surface.

[0088] 2. Place the soaked corn seeds in a petri dish containing sterile water and lined with sterile filter paper, and germinate them in the dark at 25°C for 4 days.

[0089] 3. Select corn seedlings with uniform growth from step 2 and transplant them into a seedling substrate for seedling cultivation. When the corn grows to the two-leaf-one-heart stage, select corn seedlings with uniform growth and transplant them into designated soil for subsequent experiments.

[0090] III. Pot Experiment Design

[0091] This experiment was conducted in the greenhouse of Baima Research Base of Nanjing Agricultural University in Nanjing, Jiangsu Province, from November 2023 to January 2024, with an experimental period of 28 days.

[0092] Two salinity gradients were set up in this experiment: 5‰ and 8‰. Each pot was loaded with 500g of soil. The experiment set up a control group and a treatment group. The control group pots were either uninoculated (blank control group) or inoculated with SQR9 (positive control group). The treatment groups were inoculated with 5 functional strains.

[0093] 1. Select maize seedlings with uniform growth from step two and transplant them into saline soils with two salinity gradients (5‰ and 8‰). Each pot contains 5 maize seedlings, and each treatment has 3 control pots. Incubate at room temperature and water appropriately to maintain soil moisture content.

[0094] 2. One week after corn transplanting, irrigate the area around the roots of the seedlings with bacterial suspensions of five functional bacteria and SQR9, maintaining an inoculation concentration of 10 for each bacteria. 7 CFU / g soil, meaning 10 CFU is applied per gram of soil. 7 CFU bacteria. The specific steps are as follows: one type of bacteria is inoculated into one corn treatment group, the blank control group is not inoculated, and an equal volume of sterile water as the bacterial suspension is added. The positive control group is inoculated with Bacillus amyloliquefaciens SQR9.

[0095] 3. Two weeks later, the bacterial suspension was added again in the same manner, while the control group was watered with the same volume of sterile water per pot. The plants were then cultured at 30℃ under 16 / 8h light conditions for another two weeks.

[0096] 4. After completing step 3, measure various physiological and biochemical indicators of the corn plant.

[0097] The malondialdehyde (MDA) content was detected using a commercially available reagent kit (purchased from Nanjing Jiancheng Biotechnology Co., Ltd.).

[0098] The results of various physiological and biochemical indicators of maize plants are shown in Tables 1-2 and 2. Figures 2-12 As shown.

[0099] Figure 2 The results show the phenotypic observation of whole maize plants under a 5‰ salt concentration, with the three plants on the left representing the blank control group and the three plants on the right representing the functional bacteria TJ1-185 treatment group. Figure 3 The results show the phenotypic observations of potted maize plants with functional bacteria TJ1-185 and the blank control group at a salt concentration of 5‰. The two pots on the left are the blank control group, and the two pots on the right are the functional bacteria TJ1-185 treatment group. Figure 4The results show the phenotypic observation of whole maize plants under 8‰ salt concentration, with the two plants on the left representing the blank control group and the two plants on the right representing the functional bacteria TJ1-185 treatment group. Figure 5 The results show the phenotypic observations of potted maize plants with functional bacteria TJ1-185 and the blank control group at a salt concentration of 8‰. The two pots on the left are the blank control group, and the two pots on the right are the functional bacteria TJ3-57 treatment group.

[0100] Figure 6 The graph shows the effects of five functional bacteria on maize plant height. In the graph, A shows the effects of the five functional bacteria on maize plant height at a salt concentration of 5‰, and B shows the effects of the five functional bacteria on maize plant height at a salt concentration of 8‰.

[0101] Figure 7 The graph shows the effects of five functional bacteria on maize root length. In the graph, A shows the effects of the five functional bacteria on maize root length at a salt concentration of 5‰; B shows the effects of the five functional bacteria on maize root length at a salt concentration of 8‰.

[0102] Figure 8 The figures show the effects of five functional bacteria on the fresh weight of maize aboveground parts; where A represents the effect of the five functional bacteria on the fresh weight of maize aboveground parts at a salt concentration of 5‰; and B represents the effect of the five functional bacteria on the fresh weight of maize aboveground parts at a salt concentration of 8‰.

[0103] Figure 9 The figure shows the effect of five functional bacteria on the fresh weight of maize roots. In the figure, A shows the effect of the five functional bacteria on the fresh weight of maize roots at a salt concentration of 5‰; B shows the effect of the five functional bacteria on the fresh weight of maize roots at a salt concentration of 8‰.

[0104] Figure 10 The figures show the effects of five functional bacteria on the aboveground dry weight of maize. In Figure A, the effect of the five functional bacteria on the aboveground dry weight of maize at a salt concentration of 5‰ is shown; and in Figure B, the effect of the five functional bacteria on the aboveground dry weight of maize at a salt concentration of 8‰ is shown.

[0105] Figure 11 The graph shows the effects of five functional bacteria on the dry weight of maize roots. In the graph, A shows the effects of the five functional bacteria on the dry weight of maize roots at a salt concentration of 5‰; B shows the effects of the five functional bacteria on the dry weight of maize roots at a salt concentration of 8‰.

[0106] Figure 12 The graph shows the effects of five functional bacteria on malondialdehyde (MDA) content in corn. In the graph, A shows the effects of the five functional bacteria on MDA content in corn at a salt concentration of 5‰; B shows the effects of the five functional bacteria on MDA content in corn at a salt concentration of 8‰.

[0107] Table 1. Results of various physiological and biochemical indicators of maize plants in each treatment group at a salt concentration of 15‰ (mean values).

[0108]

[0109]

[0110] Table 2. Results of various physiological and biochemical indicators of maize plants in each treatment group at a salt concentration of 25‰ (mean values).

[0111]

[0112] From Tables 1-2 and Figures 2-12 The experimental results are as follows:

[0113] I. For example Figure 2 As shown, under salt stress, the growth and development of maize are severely inhibited. At a salt concentration of 5‰, maize growth is essentially halted, but inoculation with functional bacteria TJ1-185 can help maize recover its growth. At a salt concentration of 8‰, maize growth is severely inhibited, with leaves turning yellow and curling; inoculation with functional bacteria TJ1-185 can help maize survive.

[0114] II. Figure 6 As shown, with increasing salt concentration, the maize plant height under high salt stress (8‰) was significantly lower than that under low salt stress (5‰). At both salt concentrations, the maize seedling height under the inoculated treatment was significantly higher than that under the blank control (CK) and SQR9 treatments.

[0115] 1. At a salt concentration of 5‰, compared with the control group, the maize plant height inoculated with functional bacteria TJ1-185 increased by 65.5% compared with the blank control.

[0116] 2. At a salt concentration of 8‰, compared with the control group, the maize plant height inoculated with functional bacteria TJ1-185 increased by 75.0%.

[0117] III. Figure 7 As shown, with increasing salt concentration, the root length of maize under high salt stress (8‰) was significantly lower than that under low salt stress (5‰). At both salt concentrations, the root length of maize seedlings treated with the inoculum was significantly higher than that of the control (CK) and SQR9 treatments.

[0118] 1. At a salt concentration of 5‰, compared with the control group, the root length of maize inoculated with functional bacteria TJ1-185 increased by 124.2%.

[0119] 2. At a salt concentration of 8‰, compared with the control group, the root length of maize inoculated with functional bacteria TJ1-185 increased by 200.9%.

[0120] IV. Figure 8 As shown, with increasing salt concentration, the aboveground fresh weight of maize under high salt stress (8‰) was significantly lower than that under low salt stress (5‰). At both salt concentrations, the aboveground fresh weight of maize seedlings treated with the inoculated fungus was significantly higher than that of maize seedlings treated with the blank control (CK) and SQR9.

[0121] 1. At a salt concentration of 5‰, compared with the control group, the fresh weight of the aboveground parts of maize inoculated with functional bacteria TJ1-185 increased by 314.0%.

[0122] 2. At a salt concentration of 8‰, compared with the control group, the fresh weight of the aboveground parts of maize inoculated with functional bacteria TJ1-185 increased by 266.7% compared with the blank control.

[0123] V. For example Figure 9 As shown, with increasing salt concentration, the fresh weight of maize roots under high salt stress (8‰) was significantly lower than that under low salt stress (5‰). At both salt concentrations, the fresh weight of maize seedling roots treated with the inoculum was significantly higher than that of maize roots treated with the blank control (CK) and SQR9.

[0124] 1. At a salt concentration of 5‰, compared with the control group, the fresh weight of maize roots inoculated with functional bacteria TJ1-185 increased by 110.1% compared with the blank control.

[0125] 2. At a salt concentration of 8‰, compared with the control group, the fresh weight of maize roots inoculated with functional bacteria TJ1-185 increased by 50.5% compared with the blank control.

[0126] VI. For example Figure 10 As shown, with increasing salt concentration, the aboveground dry weight of maize under high salt stress (8‰) was significantly lower than that under low salt stress (5‰). At both salt concentrations, the aboveground dry weight of maize seedlings treated with the inoculum was significantly higher than that of the control (CK) and SQR9 treatments.

[0127] 1. At a salt concentration of 5‰, compared with the control group, the aboveground dry weight of maize inoculated with functional bacteria TJ1-185 increased by 168.5%.

[0128] 2. At a salt concentration of 8‰, compared with the control group, the aboveground dry weight of maize inoculated with functional bacteria TJ1-185 increased by 84.7%.

[0129] VII. For example Figure 11 As shown, with increasing salt concentration, the dry weight of maize roots under high salt stress (8‰) was significantly lower than that under low salt stress (5‰). At both salt concentrations, the dry weight of maize seedling roots treated with the inoculum was significantly higher than that of maize roots treated with the blank control (CK) and SQR9.

[0130] 1. At a salt concentration of 5‰, compared with the control group, the dry weight of maize roots inoculated with functional bacteria TJ1-185 increased by 58.7% compared with the blank control.

[0131] 2. At a salt concentration of 8‰, compared with the control group, the dry weight of maize roots inoculated with functional bacteria TJ1-185 increased by 84.7%.

[0132] When plants are stimulated, they produce a large number of superoxide free radicals, leading to membrane lipid peroxidation. Therefore, membrane lipid peroxidation is an important indicator of plant cell membrane damage. The process of membrane lipid peroxidation produces malondialdehyde (MDA), the content of which can directly reflect the peroxidation level of the cell membrane.

[0133] 8. For example Figure 12 As shown, under salt stress, the malondialdehyde (MDA) content in plant leaves increased significantly. At both salt concentrations, the MDA content in maize leaves treated with the inoculated fungus was significantly lower than that in the control group (CK) and the SQR9 treatment.

[0134] 1. At a salt concentration of 5‰, compared with the control group, the malondialdehyde (MDA) content in maize inoculated with functional bacteria TJ1-185 was reduced by 28.1%.

[0135] 2. At a salt concentration of 8‰, compared with the control group, the malondialdehyde (MDA) content in maize inoculated with functional bacteria TJ1-185 was reduced by 32.6%.

[0136] Example 4: 16S phylogenetic identification of strain TJ1-185

[0137] 1. The 16S rDNA sequence of strain TJ1-185 was amplified and sequenced. The sequencing results are shown in SEQ ID NO.1.

[0138] GATGGGGCGGCCTACACATGCAGTCGAGCGGATGAAGGGAGCTTGCTCCC

[0139] TGATTTAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGG

[0140] GGGATAACGTTCCGAAAGGAACGCTAATACCGCGTACGTCCTACGGGAGA

[0141] AAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTA

[0142] GCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCT

[0143] GAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACG

[0144] GGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCC

[0145] ATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGG

[0146] AGGAAGGGCTGCTGGTTAATACCCTGCAGTTTTGACGTTACCAACAGAATA

[0147] AGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAG

[0148] CGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTT

[0149] GGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGA

[0150] GCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGC

[0151] GTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATA

[0152] CTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTG

[0153] GTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGATTT

[0154] TAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCA

[0155] AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCAT

[0156] GTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATGCTG

[0157] AGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACACAGGTGC

[0158] TGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAA

[0159] CGAGCGCAACCCTTGTCCTTAGTTACCAGCACCTCGGGTGGGCACTCTAA

[0160] GGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCA

[0161] TCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAAA

[0162] GGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTC

[0163] CGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCG

[0164] TGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCC

[0165] GTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCTAACCTTCGGGGGGACGGTACCACGGAGATCCTGG。

[0166] II. The sequencing results were compared and analyzed using BLAST to construct a phylogenetic tree. Figure 13 Based on the name with high homology to the test strain, the species of the strain was preliminarily identified. Strain TJ1-185 belongs to the Pseudomonas bacterium, therefore it was renamed Pseudomonas TJ1-185.

[0167] III. Preservation of Pseudomonas TJ1-185

[0168] The Pseudomonas sp. TJ1-185 provided by this invention was deposited on May 8, 2024 at the Guangdong Provincial Center for Microbial Culture Collection (CDMCC No. 64979; address: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province), with accession number CDMCC No. 64979.

[0169] Example 5

[0170] 1. Inoculate Pseudomonas TJ1-185 and Pseudomonas putida PLW1 (preservation number CCTCC NO: M 20241352) into TSB liquid medium and incubate at 30°C and 170 rpm.

[0171] 2. After 24 hours, centrifuge the bacterial suspension, then resuspend the bacterial suspension in sterile water to adjust the OD of the suspension. 600 The value was 1.0, and Pseudomonas TJ1-185 bacterial suspension and Pseudomonas PLW1 bacterial suspension were obtained respectively.

[0172] 3. Place the surface-sterilized corn seeds in a 280mM sodium chloride solution and soak them at 25°C in the dark for 4 hours.

[0173] 4. Place the seeds soaked in step 3 in petri dishes containing sterile water and 280mM sodium chloride solution and lined with filter paper, with 3 seeds per petri dish and 6 replicates.

[0174] 5. Add 100 μL of the *Pseudomonas* TJ1-185 suspension prepared in step 2 directly above the corn seeds in step 4 as the *Pseudomonas* TJ1-185 treatment group; add 100 μL of the *Pseudomonas putida* PLW1 suspension prepared in step 2 directly above the corn seeds in step 4 as the *Pseudomonas putida* PLW1 treatment group. Simultaneously, set up a negative control group without added bacterial agent (adding an equal volume of sterile water as the *Pseudomonas putida* PLW1 treatment group). Germinate in a 25℃ light incubator with a day / night ratio of 16h / 8h for 4 days.

[0175] 6. The germination status of different treatment groups in step 5 is shown in Table 3.

[0176] Table 3 Germination rates (mean) of different treatment groups

[0177] negative control group 0 Pseudomonas TJ1-185 treatment group 78.6％ Pseudomonas putida PLW1 treatment group 37.3％

[0178] Table 3 shows that at a salt concentration of 280 mM, the germination rate of maize treated with strain TJ1-185 suspension was 78.6%, while the germination rate of maize treated with *Pseudomonas putida* PLW1 suspension was 37.3%. This indicates that strain TJ1-185 can significantly improve the germination rate and salt tolerance of maize compared to *Pseudomonas putida* PLW1.

[0179] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type of Pseudomonas bacterium ( Pseudomonas sp.)TJ1-185, characterized in that, The preservation number is CDMCCNo: 64979; the strain of Pseudomonas spp. TJ1-185 promotes crop seed germination and / or growth under salt stress.

2. A microbial agent, characterized in that, Includes the Pseudomonas bacteria TJ1-185 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The OD600 value of Pseudomonas bacteria TJ1-185 in the bacterial agent is ≥1.

0.

4. The method for preparing the microbial agent according to claim 2 or 3, characterized in that, include: The Pseudomonas bacteria TJ1-185 were cultured in a culture medium to obtain a bacterial agent.

5. The preparation method according to claim 4, characterized in that, The culture temperature is 30–37°C; the culture time is 18–30 h; and the culture rotation speed is 150–250 rpm.

6. The application of the Pseudomonas spp. bacteria TJ1-185 of claim 1, the bacterial agent of claim 2 or 3, or the bacterial agent prepared by the preparation method of claim 4 or 5 in promoting plant germination, growth, and / or improving plant salt tolerance; wherein the plant includes corn; and the salt tolerance includes the ability to tolerate a salinity of 5‰ to 8‰.

7. The application according to claim 6, characterized in that, The promotion of plant growth includes any one or more of the following: increasing plant height, increasing plant root length, increasing the fresh weight of the above-ground parts of the plant, increasing the fresh weight of the roots of the plant, increasing the dry weight of the above-ground parts of the plant, and increasing the dry weight of the roots of the plant.

8. The application according to claim 6, characterized in that, Improving plant salt tolerance includes mitigating the damage caused by salt stress to plants and / or enhancing their ability to tolerate salt stress.

9. A method for improving the salt stress tolerance of plants, characterized in that, include: Apply a bacterial agent containing the Pseudomonas bacteria TJ1-185 as described in claim 1 during plant growth.

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