Application of a new marine bacterium and its bacterial agent in plant salt tolerance and growth promotion

Abstract

The application provides a new strain of Pontibacter sp. R65 and application thereof in plant salt tolerance and growth promotion. The new strain of Pontibacter sp. R65 is isolated from soil in the Pamirs, and has UVC radiation resistance, IAA activity, tolerance to 5% NaCl, and a microbial agent prepared from fermentation liquor of the new strain of Pontibacter sp. R65 can significantly promote plant growth under salt stress. By treating wheat seeds with the microbial agent of the new strain of Pontibacter sp. R65, the plant height of the wheat is increased by 73%, and the chlorophyll content is also increased, the salt tolerance of the plant is significantly enhanced, and the growth of the plant is promoted, indicating that the new strain of Pontibacter sp. R65 provided by the application not only has certain radiation resistance, but also can promote plant growth, and has wide application value in agriculture.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to the technical field of a new strain of Marinebacterium and its microbial inoculant for plant growth promotion. Background Technology

[0002] Soil salinization is a significant abiotic stress, leading to major ecological and environmental problems for agriculture worldwide. Currently, the area of ​​saline-alkali land in Xinjiang has reached 13.36 million hectares. 2 It accounts for 36.8% of the country's saline-alkali land area. Soil salinity hinders plant growth, development, and productivity by affecting plant morphology, physiological and biochemical characteristics, and metabolic functions, ultimately affecting crop yield and quality and seriously threatening the sustainable development of agriculture.

[0003] Salinity stress manifests in two main phases: the osmotic phase and the ionic phase. Both phases lead to secondary stress, with most phases resulting from oxidative stress and a series of secondary stresses due to the excessive production of reactive oxygen species (ROS). Excessive salt in the soil reduces plant water uptake, a characteristic of the early osmotic stress phase, leading to stomatal closure and reduced aboveground growth. In the ionic phase, the accumulation of several ions, including potassium (K), occurs. + Zn 2+ Mn 2+ Ions cause toxic effects, including damage to membrane structures and organelles, impaired nutrient absorption, altered photosynthetic and transpiration rates, leaf senescence, inhibition of enzyme activity, and reduced chlorophyll content leading to photosynthetic impairment, resulting in a significant decrease in crop yield.

[0004] With the vigorous development of modern agriculture, beneficial microorganisms have attracted much attention due to their significant effects in improving soil quality, enhancing soil fertility, improving crop stress resistance, and increasing yield and quality. For example, under salt stress conditions, plant growth-promoting strains can regulate the levels of endogenous plant hormones such as IAA, ABA, GA, JA, and SA, and promote nutrient absorption through nitrogen fixation, phosphorus solubilization, and potassium solubilization. They also upregulate the activity of key enzymes to activate the plant's antioxidant defense mechanism to prevent membrane deterioration caused by excessive ROS and restore normal photosynthetic pigment synthesis. Related enzymes include superoxide dismutase, peroxidase, catalase, and glutathione reductase. Therefore, beneficial microorganisms, as a new type of biological pesticide, are more environmentally friendly and effective, and are of great significance to the sustainable development of modern agriculture. Summary of the Invention

[0005] Given the advantages of existing microbial agents in promoting salt tolerance in plants, but the limited resources and limited functions of existing growth-promoting strains, there is an urgent need to screen for more high-performance new microbial strains. Furthermore, there is a lack of literature and patent records regarding the application of *Pontibacter* in promoting salt tolerance in plants. This invention provides a novel *Pontibacter* strain R65 and its application in promoting salt tolerance in plants. The invention involves isolating a novel *Pontibacter* strain R65 from Pamir Plateau soil. This strain exhibits UVC radiation resistance, IAA activity, and tolerance to 5% NaCl. Moreover, the microbial agent prepared from the fermentation broth of *Pontibacter* strain R65 significantly promotes plant growth under salt stress. Treating wheat seeds with the novel strain of Pontibacter R65 (sp. R65) provided by this invention resulted in a 73% increase in wheat plant height. Physiological measurements of wheat leaves revealed that salt stress significantly increased the content of antioxidant enzymes in wheat leaves. After using the microbial agent, the content of antioxidant enzymes decreased, while chlorophyll content increased, significantly enhancing the plant's salt tolerance and promoting plant growth. This demonstrates that the novel strain of Pontibacter R65 not only possesses certain radiation resistance but also promotes plant growth, exhibiting broad application value in agriculture, and its potential radiation-related applications remain to be developed.

[0006] To achieve the above technical effects, the present invention is implemented through the following technical solution.

[0007] Specifically, this invention provides a new strain of marine bacterium R65 (Pontibacter.sp.R65).

[0008] The novel strain of *Pontibacter* R65 provided by this invention was isolated from soil samples from the Pamir Plateau in Xinjiang. Phylogenetic analysis of its 16S rRNA sequence confirmed that strain R65 belongs to the genus *Pontibacter*. Gene sequencing of this strain was performed, and the resulting sequences were cut and assembled using seqman software and uploaded to the NCBI website for BLAST alignment analysis. The 16S rRNA sequence of *Pontibacter* R65 showed the highest similarity (95.23%) to both *Pontibacter qinzhouensis* GY10130 (MZ318048) and *Pontibacter beigongshangeins* T6-1 (NR165736), which is below 97%. A 16S rRNA phylogenetic tree was constructed using sequences with high homology. The results showed that strain R65 (Pontibacter sp. R65) was most closely related to *Pontibacter qinzhouensis* GY10130 (MZ318048) and *Pontibacter beigongshangeins* T6-1 (NR165736) within the same branch. Further polyphasic classification confirmed that strain R65 (Pontibacter sp. R65) is a novel species within the genus *Pontibacter*, exhibiting typical characteristics of a new species.

[0009] This invention provides a new species of *Pontibacter* R65, identified through molecular-level identification using well-known and recognized strain systems, combined with multi-phase classification analysis based on morphological identification, physiological and biochemical characteristics, etc. The invention demonstrates that *Pontibacter* R65 differs from the standard type strain of the same genus *Pontibacter* in several ways, confirming that this strain belongs to the new species of the genus *Pontibacter*. This strain is named *Pontibacter* R65. It has been deposited at the Budapest Treaty International Collection of Microorganisms: China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, 430072, China, on April 12, 2023, with accession number CCTCC No: M2023529.

[0010] The gene sequence of the above-mentioned new strain of the genus *Pontibacter*, *Pontibacter* R65 (*Pontibacter.sp.R65*), is shown in SEQ ID NO:1.

[0011] In this invention, the isolation and purification culture medium for strain Pontibacter sp. R65 is as follows: 0.25 g / L tryptone, 0.5 g / L acid-hydrolyzed casein, 0.5 g / L yeast extract, 0.5 g / L soluble starch, 0.3 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate, 0.3 g / L sodium pyruvate, 12.0 g / L agar, 0.25 g / L peptone, 0.5 g / L glucose, and pH 7.2 ± 0.2.

[0012] In this invention, the culture conditions for strain Pontibacter sp. R65 are: pH 5-8.5, temperature 4-37℃, salinity 0-5%, and the optimal culture conditions are: pH=8, temperature 37℃, salinity 1%.

[0013] In this invention, the strain *Pontibacter* sp. R65 exhibits a certain degree of radiation tolerance. The irradiation conditions and treatment are provided as follows: *Pontibacter* sp. R65 was cultured in NA medium for 2 days, centrifuged at 6000 rpm for 2 min to collect the bacterial cells, washed with sterile 0.05 mol / L potassium phosphate buffer (pH 7.2), and the bacterial solution was diluted to OD0.05. 600 =0.8, take 10 ml of the diluent and irradiate it under a 6W UV lamp for 10 min (distance d = 20 cm between the lamp source and the bacterial suspension), stirring with a magnetic stirrer (30℃, 120 rpm). Every 2 min, aspirate 100 μL of the sample and place it in a sterile centrifuge tube, storing at 4℃. Dilute the sample to 10 with sterile PBS. -5 For each dilution gradient, 10 μL of bacterial culture was spotted onto the corresponding agar medium and incubated at 30°C. The colony count was observed and recorded for 1-3 days. The colony morphology was observed and the survival rate was calculated.

[0014] Meanwhile, this invention provides a method for preparing a microbial agent of Pontibacter R65, specifically including the following steps: inoculating a single colony of Pontibacter R65 into R2A liquid medium, culturing at 30°C for 2 days to obtain a seed culture of Pontibacter R65, inoculating it into R2A liquid medium at an inoculation rate of 2%, fermenting for 4 days to prepare a microbial agent of the new strain of Pontibacter R65.

[0015] Furthermore, this invention provides the application of Pontibacter sp. R65 microbial inoculant in promoting plant growth under salt stress. It can regulate the plant's ability to scavenge free radicals and significantly promote plant elongation, showing broad development and application prospects in plant salt tolerance and growth promotion.

[0016] Through the above technical solutions, the present invention achieves the following technical effects:

[0017] (1) This invention provides a new species of the genus *Pontibacter* (sp. R65). After systematic molecular identification, combined with multi-phase classification and identification analysis based on morphological identification, physiological and biochemical characteristics and chemical characteristics, *Pontibacter* (sp. R65) has several differences from the closest homologous strains of the genus *Pontibacter* (sp.) *Pontibacter qinzhouensis* GY10130 (MZ318048) and *Pontibacter beigongshangeins* T6-1 (NR165736). It is determined to be a new species of the genus *Pontibacter* (sp.). It is confirmed that the strain *Pontibacter* (sp. R65) with the species number M2023529 in the Pontibacter category is a typical new species, and therefore it is necessary to preserve it in accordance with legal requirements.

[0018] (2) The novel strain of *Pontibacter* sp. R65 provided by this invention exhibits a certain degree of UVC tolerance. Under the same UVC stress conditions, its survival rate is 600 times that of the negative control strain *Escherichia coli* No. CMCC(B)44102, but lower than that of the positive strain *Deinococcus radiodurans* R1, indicating a certain degree of UVC radiation resistance. After 3 minutes of UVC radiation treatment, the survival rate of the novel strain *Pontibacter* sp. R65 is approximately 10%.

[0019] (3) The application of a novel strain of marine bacillus R65 (Pontibacter.sp.R65) microbial agent provided by this invention in promoting plant growth under salt stress. Compared with salt stress, the wheat seedlings treated with the microbial agent prepared by this invention increased in length by 73%, while reducing the accumulation of cellular antioxidant enzymes, scavenging free radicals, increasing the chlorophyll content of plants, significantly improving plant salt resistance and promoting plant growth. It has broad development and application prospects in plant salt tolerance and growth promotion, and can be widely used in the agricultural industry. Attached Figure Description

[0020] Figure 1 The diagram shows a phylogenetic tree of a new strain of the genus *Pontibacter*, R65, based on the 16S rRNA gene sequence.

[0021] Figure 2 This image shows the colony morphology of Pontibacter sp. R65 on a culture medium.

[0022] Figure 3 The image shown is a scanning electron microscope image of the novel strain Pontibacter sp. R65.

[0023] Figure 4 The graph shows the survival curves of strains Escherichia coli No. CMCC(B)44102, (Pontibacter.sp) R65, and Deinococcus radiodurans R1 under UVC stress.

[0024] Figure 5 The image shows the results of the IAA production capacity assay for the new strain of Marine Bacteria R65 (Pontibacter.sp.R65).

[0025] Figure 6 The image shows the growth of wheat under different treatments.

[0026] Figure 7 The graph shows the superoxide dismutase activity in wheat leaves under different treatments.

[0027] Figure 8 The graph shows the peroxidase activity of wheat leaves under different treatments.

[0028] Figure 9 The graph shows the glutathione peroxidase activity in wheat leaves under different treatments.

[0029] Figure 10 The graph shows the proline content in wheat leaves under different treatments.

[0030] Figure 11 The graph shows the chlorophyll content of wheat leaves under different treatments. Detailed Implementation

[0031] To better explain the present invention, the main contents of the invention are further illustrated below in conjunction with specific embodiments, but the contents of the present invention are not limited to the following embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0032] Example 1: Isolation, screening and identification of Pontibacter sp. R65

[0033] (I) Separation and Screening

[0034] Soil samples were collected from the Pamir region, and microorganisms were isolated and purified using the plate dilution method. 5g of soil sample was weighed and added to 45ml of sterile physiological saline (0.9%, w / v). The mixture was shaken at 150rpm and 30℃ for 30min, then serially diluted to 10⁻⁴. 100μL of the diluted solution was plated onto R2A solid medium and incubated at 30℃ for 3 days. Single colonies were picked for isolation and purification. The purified strains were then transferred to R2A medium slant for storage.

[0035] (II) Classification and Identification

[0036] Colony PCR was used to identify strains that grew well on agar plates. The PCR reaction conditions were: 95℃ for 5 min, 94℃ for 30 s, 54℃ for 30 s, 72℃ for 30 s (30x cycles), 72℃ for 7 min, and storage at 4℃. The sequences were analyzed by agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) for sequencing. The returned sequences were trimmed and assembled using seqman software, and the processed sequences were uploaded to the NCBI database for sequence alignment. The new strain, *Pontibacter* sp. R65, was identified as having a sequence length of 1379 bp (SEQ ID No: 1). It belongs to the *Pontibacter* genus and showed the highest similarity (95.23%) to *Pontibacter qinzhouensis* GY10130 (MZ318048) and *Pontibacter beigongshangeins* T6-1 (NR165736), both below 97%. A phylogenetic tree was constructed using MEGA 11.0 software via the Neighbor-joining method (with 1000 repeated samplings). The results are attached. Figure 1 As shown, through a comprehensive assessment of the similarity and homology of the bacterial species, it was confirmed that the bacterial species with the number M2023529 within the Pontibacter category belongs to a typical new bacterial species, R65 of the genus *Pontibacter* (*Pontibacter.sp.R65*).

[0037] Example 2: Polyphasic taxonomic identification of Pontibacter sp. R65

[0038] (I) Colony morphology and physiological and biochemical characteristics

[0039] Experiments have confirmed that Pontibacter sp. R65 is a Gram-negative, aerobic, non-motile, rod-shaped, pale pink bacterium. See the appendix for colony morphology. Figure 2 The strain *Pontibacter* sp. R65 can grow on 1 / 3 MA, R2A, NA, and TSA media (optimal medium NA). Culture conditions are: pH 5-8.5, temperature 4-37℃, salinity 0-5%, with optimal growth conditions of pH 8, temperature 37℃, and salinity 1%. After 3 days of cultivation on NA medium, colony characteristics were observed, recorded, and photographed. Scanning electron micrographs of the colonies were also recorded. See the attached image for the electron micrograph morphology of the strain. Figure 3 .

[0040] The novel strain Pontibacter R65 was inoculated into NA liquid medium. Physiological and biochemical tests were performed using API20E and API20NE. Carbon source utilization and chemosensitivity of the strain were detected using Biolog GenIII microplates. The results showed that strain Pontibacter R65 was positive for nitrate reduction, and also positive for arginine hydrolysis, urease, aescin, gelatin, galactosidase, β-galactosidase, and glucose fermentation / oxidation reactions. In the GenIII experiment, strain Pontibacter R65 could utilize dextrin, maltose, trehalose, cellobiose, gentiobiose, sucrose, D-minobiose, stachyose, raffinose, α-D-lactose, melibiose, β-formyl-D-glucosidase, D-salicylic acid, N-acetyl-D-glucosamine, N-acetyl-β-D-mannosamine, α-D-glucose, D-mannose, D-fructose, D-galactose, L-fructose, D-sorbitol, and D-glucose. Mannitol, D-arabinol, inositol, glycerol, D-glucose-6-phosphate, D-fructose-6-phosphate, gelatin, aminoacetyl-L-proline, L-alanine, L-arginine, L-glutamic acid, L-histamine, L-pyroglutamic acid, pectin, D-galacturonic acid, D-glucuronic acid, D-glucuronic acid, glycosylate, D-methyl lactate, L-lactic acid, citric acid, α-ketoglutarate, L-malic acid, bromosuccinic acid, γ-aminobutyric acid, β-hydroxy-D,L-butyric acid, acetoacetic acid, acetic acid, formic acid. The strain showed positive results for 1% sodium lactate, rifamycin SV, nalidixinone acid, lithium chloride, potassium tellurite, and aztreonam.

[0041] (II) Chemical characteristics of new strains of Marinebacterium

[0042] Whole-cell hydrolysates, cellular fatty acids, polar lipids, and major quinones of Pontibacter sp. R65 were detected, and the specific results are shown in Table 1.

[0043] The whole-cell hydrolysate of *Pontibacter* sp. R65 is primarily mannose, with small amounts of rhamnose, glucose, galactose, and xylose. The only respiratory quinone is MK-7, and the main cellular fatty acids are iso-C15:0 and Sumin feature 4 (anteiso-C17:1B / iso-C17:1I). Polar lipids include PE and two unidentified lipids.

[0044] Table 1: Fatty acid composition of Pontibacter sp. R65

[0045] Saturated <![CDATA[C 16:0 ]]> - <![CDATA[C 17:0 ]]> <![CDATA[C 18:0 ]]> - <![CDATA[iso-C 15:0 ]]> 34.2 <![CDATA[iso-C 17:0 ]]> 2.2 <![CDATA[anteiso-C 15:0 ]]> 1.0 <![CDATA[iso-C 15:0 3OH]]> 5.1 <![CDATA[iso-C 17:0 3OH]]> 10.7 Unsaturated <![CDATA[C 16:1 ω5c]]> 8.4 <![CDATA[C 17:1 ω6c]]> 1.9 <![CDATA[C 18:1 ω7c11-methyl]]> - Others <![CDATA[Suminfeature1(C 13:0 3OH / C 15:1 iH)]]> 1.2 <![CDATA[Suminfeature2(C 14:0 3OH / iso-C 16:1 I)]]> - <![CDATA[Suminfeature3(C 16:1 ω6c / C 16:1 ω7c)]]> 6.4 <![CDATA[Suminfeature4(anteiso-C 17:1 B / iso-C 17:1 I)]]> 22.6 <![CDATA[Suminfeature8(C 18:1 ω6c / C 18:1 ω7c)]]> -

[0046] Example 3: UVC radiation tolerance characteristics of a new strain of Pontibacter R65

[0047] The positive strain used in the experiment was Deinococcus Radiodurans R1, and the negative strain was Escherichiacoli No. CMCC(B)44102.

[0048] The novel strain of *Pontibacter* sp. R65 was cultured in NA medium for 2 days. The bacterial cells were collected by centrifugation at 6000 rpm for 2 min, washed with sterile 0.05 mol / L potassium phosphate buffer (pH 7.2), and the bacterial solution was diluted to OD0.05. 600 =0.8, take 10 ml of the diluent and irradiate it under a 6W UV lamp for 10 min (distance d = 20 cm between the lamp source and the bacterial suspension), stirring with a magnetic stirrer (30℃, 120 rpm). Every 2 min, aspirate 100 μL of the sample and place it in a sterile centrifuge tube, storing at 4℃. Dilute the sample to 10 with sterile PBS. -5 For each dilution gradient, 10 μL of bacterial culture was spotted onto the corresponding agar medium and incubated at 30°C. Colony counts were observed and recorded for 1-3 days. Colony morphology was observed, and survival rate was calculated. See attached table for results. Figure 4 .

[0049] The results showed that under the same UVC stress conditions, the survival rate of the negative control strain *Escherichia coli* No. CMCC(B) 44102 was 600 times higher, but lower than that of the positive strain *Deinococcus radiodurans* R1, indicating a certain degree of UVC radiation resistance. After 3 minutes of UVC radiation treatment, the survival rate of the new strain *Pontibacter* sp. R65 was approximately 10%.

[0050] Example 4: Growth-promoting function of a new strain of Pontibacter R65

[0051] (I) Determination of IAA production capacity of the new strain Pontibacter R65

[0052] The new strain of *Pontibacter* sp. R65 was inoculated into R2A liquid medium containing L-tryptophan (100 mg / L), which was dissolved in NaOH. After culturing on a shaker (30℃, 180 r / min) for 3 days, 100 μL of the bacterial suspension was added to a white ceramic plate, along with an equal volume of Salkowski colorimetric solution (50 mL 35% HClO4 + 1 mL 0.5 mol / L FeCl3). A control was prepared by adding 100 μL of uninoculated R2A and NA liquid medium to an equal volume of colorimetric solution. After culturing in the dark for 30 min, the absorbance at 530 nm was measured using a microplate reader. The white ceramic plate was then placed at room temperature in the dark for 30 min, and the color change to red was observed.

[0053] From the appendix Figure 5 The results showed that after 30 minutes of dark treatment, the positive control bacteria PVC-14 and the test bacteria, the new strain of Pontibacter spp. R65, turned red, while other treatments showed no color development, proving that strain Pontibacter spp. R65 has the ability to produce IAA.

[0054] (II) Verification of the growth-promoting ability of the new strain of Pontibacter R65

[0055] Preparation of microbial inoculum: A single colony of the novel strain *Pontibacter* sp. R65 was inoculated into R2A liquid medium and cultured at 30°C for 2 days to obtain a seed culture of the novel strain *Pontibacter* sp. R65. This seed culture was then inoculated into R2A liquid medium at a 2% inoculum size and fermented for 4 days to prepare the microbial inoculum of the novel strain *Pontibacter* sp. R65. The absorbance at 600 nm was measured and diluted to OD200. 600 =0.02. Wheat seeds were cleaned, soaked overnight in purified water for 12 hours, rinsed with sterile water, and then soaked in the prepared inoculant for 4 hours. After soaking, the wheat seeds were spread evenly in a petri dish containing 10 mL of 1.2% NaCl (the dish contained double-layered filter paper) and grown at 25℃. The control group consisted of an equal volume of sterile water (CK H2O) and 1.2% NaCl (CK NaCl). The number of germinations was counted daily. After 7 days of growth, the germination rate was calculated, and the root length and seedling length were measured. (See attached...) Figure 6Data shows that after 10 days of growth, there was no significant difference in the germination rate of wheat among different treatments, all being 95%. After 20 days of growth, there were significant differences in wheat seedling length among different treatments. Salt stress significantly inhibited the normal growth of wheat, reducing plant height by 59.3%. However, the new strain of Pontibacter R65 promoted the growth of wheat plants under salt stress, increasing the plant height by 73% compared to the salt-stressed wheat. This indicates that the new strain of Pontibacter R65 has a significant effect on plant growth under salt stress.

[0056] (III) Effects of the new strain (Pontibacter.sp.R65) of Marine Bacteria R65 on the content of antioxidant enzymes in wheat leaves

[0057] After culturing the newly strain of Pontibacter spp. R65 for 10 days, various salt stress-related indicators in wheat leaves were measured, including superoxide dismutase (SOD), peroxidase (POD), glutathione peroxidase (GPX), proline (PRO), and chlorophyll content. The results are shown in the appendix. Figure 7 -Appendix Figure 11 The results showed that salt stress significantly increased the content of antioxidant enzymes in wheat leaves. Wheat leaves treated with the new strain of *Pontibacter* R65 (*Pontibacter* sp. R65) showed a decrease in antioxidant enzyme content, indicating that *Pontibacter* sp. R65 can alleviate the oxidation level of wheat plants, remove excess reactive oxygen species, and thus reduce the damage of salt stress to wheat plants. Simultaneously, the application of the new strain of *Pontibacter* sp. R65 significantly increased the chlorophyll content of wheat leaves, enhanced photosynthesis, improved the synthesis capacity of various substances, accelerated nutrient absorption, and promoted rapid crop growth.

[0058] The above data analysis shows that the new strain of *Pontibacter* R65 possesses IAA activity. Under salt stress conditions, wheat treated with the *Pontibacter* R65 strain provided by this invention showed a significant increase in plant height. The contents of SOD, POD, GPX, and PRO were similar to or lower than those of the control group, while the chlorophyll content increased significantly. This indicates that the *Pontibacter* R65 microbial agent provided by this invention can promote plant growth and can be used as a bio-fertilizer to improve the growth of crops in saline-alkali land to a certain extent, showing broad development and application prospects in agricultural economics.

[0059] In summary, the above embodiments are merely descriptions of preferred embodiments of this experiment and are not intended to limit the scope of this experiment. Without departing from the spirit of this experiment design, all modifications and improvements made by those skilled in the art to the technical solutions of this experiment should fall within the protection scope determined by this experiment.

Claims

1. A novel strain of Pontibacter sp. R65, characterized in that, Marinebacterium R65 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number M2023529.

2. The application of Marine Bacterium R65 as described in claim 1 in promoting wheat growth.

3. A Marine Bacillus R65 microbial inoculant as described in claim 1, characterized in that, This microbial agent was prepared by fermentation of Marine Bacillus R65.

4. The Marine Bacillus R65 microbial inoculant as described in claim 1, characterized in that, The preparation method of this microbial agent specifically includes the following steps: a single colony of Marinebacterium R65 is inoculated into R2A liquid medium and cultured at 30℃ for 2 days to obtain a seed liquid of Marinebacterium R65 strain. The seed liquid is then inoculated into R2A liquid medium at an inoculation rate of 2% and fermented for 4 days to prepare the microbial agent of Marinebacterium R65.

5. The application of the Marine Bacillus R65 microbial agent as described in claim 4 in promoting plant growth under salt stress.

Images