Agrobacterium radiobacter strain ASE44 and its use in orchardgrass

Abstract

The application relates to an agrobacterium ASE44 and application thereof. The agrobacterium ASE44 is separated from a desert grassland soil, is preserved in the China Center for Type Culture Collection, and has a preservation number of CCTCC No: M 20251157. The bacteria have a good promoting effect on improving the growth of oat grass seedling stages, can be prepared into a microbial inoculant for the application of the oat grass seedling stages, are applied to the actual production of the oat grass in a dry region, and provide an excellent basic strain for the excavation and application of desert microbial resources.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to an agrobacterium. Agrobacterium sp. ASE44 and its application in promoting seedling growth of oat grass under drought stress. Background Technology

[0002] Drought is a key environmental factor that limits plant growth. It can cause adverse reactions in plants, such as osmotic imbalance, membrane system damage, and reduced respiration and photosynthetic rates, which in turn affect the growth and metabolism of plants at various stages and lead to crop yield reduction.

[0003] Oat grass is characterized by its drought and cold resistance, tolerance to poor soil, high yield, and significant ecological benefits. In recent years, with the adjustment of the national agricultural industrial structure and the development needs of the grass-livestock industry, the planting area and acreage of oat grass have expanded rapidly. However, increasingly severe drought has a significant impact on the growth, development, yield, and quality of oat grass. Currently, most research focuses on its response mechanism to drought stress, molecular mechanisms, and the breeding of drought-resistant varieties, but these studies are often time-consuming. Therefore, it is necessary to develop new methods to improve the yield and quality of oat grass under drought stress, especially to solve the germination problem of oat grass under drought conditions.

[0004] Microorganisms can significantly alleviate the stress effects of drought on plants through multiple mechanisms. They can enhance root water retention and improve soil structure by secreting extracellular polysaccharides to form biofilms, thereby reducing water evaporation; synthesize osmotic regulators such as proline and soluble sugars to help plants maintain osmotic pressure; produce antioxidant enzymes to scavenge reactive oxygen species and mitigate oxidative damage; and degrade ethylene precursors through ACC deaminases, delaying leaf senescence and regulating the balance of auxin and abscisic acid. Furthermore, microorganisms can enhance plant tolerance to drought stress by activating drought-resistance genes and inducing the expression of drought-tolerant proteins.

[0005] Agrobacterium, also known as the genus *Agrobacterium*, is a Gram-negative bacterium. Currently, this genus includes 34 type species, among which *Agrobacterium tumefaciens* is the most studied type species. This genus can transfer exogenous DNA into plants via horizontal transfer, making it an important tool for plant genetic modification. This technology is currently used in crops such as maize, potato, golden rice, soybean, and tomato. Meanwhile, this genus has been reported to have certain growth-promoting effects on plants. *Agrobacterium larrymoorei* (CGMCC No. 24856) exhibits nitrogen-fixing activity, promoting the growth of wheat and rapeseed by increasing nitrogen content (patent number CN115820477A). *Agrobacterium tumefaciens* (CGMCC No. 6289) can enhance oat growth in petroleum-contaminated saline-alkali land by increasing ACC deaminase content (patent number CN102816721A), indicating that this genus, in addition to its ability to perform plant genetic modification, may also possess certain plant growth-promoting capabilities. However, current technology has not reported the application of strains from this genus in drought resistance and growth promotion of oat grass. Therefore, it is necessary to screen strains from the *Agrobacterium* genus that promote oat grass growth and apply them to oat grass cultivation or pasture growth promotion. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to provide a *Agrobacterium* ASE44 isolated from desert grassland soil. This bacterium has a good promoting effect on improving the growth of oat grass seedlings.

[0007] The strain mentioned above is Agrobacterium sp. or Agrobacterium radiobacter, ASE44, deposited at the China Center for Type Culture Collection, accession number CCTCC No: M 20251157.

[0008] The present invention provides Agrobacterium sp. ASE44, which was deposited on May 29, 2025, at the China Center for Type Culture Collection (CCTCC, address: No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China), classified as Agrobacterium sp. ASE44, with accession number CCTCC No: M20251157, and deposit address: Wuhan University, Wuhan, China.

[0009] The nucleotide sequence of the 16S rDNA of Agrobacterium sp. ASE44 is shown in SEQ ID NO:1. The 16S rRNA gene sequence of Agrobacterium sp. ASE44 is most closely related to Agrobacterium radiobacter ATCC 19358 (GenBank: MK424303).

[0010] Agrobacterium sp. ASE44 is a strain isolated from soil. This strain is Gram-negative and grows well on R2A solid medium, with an optimal growth temperature of 28°C and an optimal growth pH of 7.2.

[0011] This invention provides an Agrobacterium, which is cultured on R2A solid medium at a temperature of 25-30°C and a pH of 6.5-7.6; preferably, the culture temperature is 27-29°C and the optimal growth pH is 7.2.

[0012] This invention provides a microbial agent containing Agrobacterium sp. ASE44.

[0013] The above-mentioned inoculants containing Agrobacterium sp. ASE44 can be used as liquid inoculants. They can also be used as any of the following: seed soaking solution, inoculant mud, seed coating, or fertilizer.

[0014] This invention provides a product containing Agrobacterium sp. ASE44, wherein the total viable count of Agrobacterium in the product is not less than 10. 6 CFU / g or 10 6 CFU / mL.

[0015] The above-mentioned Agrobacterium sp. ASE44 bacterial agent can be prepared using conventional techniques and by adding excipients permitted in the field of microbial preparations.

[0016] The present invention also provides the application of Agrobacterium sp. ASE44 or an inoculum containing this strain in the growth of oat grass seedlings.

[0017] This includes the application of the aforementioned *Arbuscular mycorrhizal* or inoculants in improving the growth performance of oat grass. The growth performance refers to improvements in one or more of the following: stem diameter, root length, plant height, stem and leaf weight, and root weight. The oat grass variety mentioned is Zhongcao No. 33.

[0018] The present invention also provides one or more applications of Agrobacterium sp. ASE44 or an agent containing this strain, including: (1) Application in increasing the stem thickness of oat grass; (2) Application in improving the root length of oat grass; (3) Application in improving the height of oat grass plants; (4) Application in increasing the stem and leaf weight and root weight of oat grass.

[0019] The present invention also provides a microbial inoculant for promoting the growth of oat grass seedlings, which contains Agrobacterium sp. ASE44 cells.

[0020] The fermentation product is the bacterial cell obtained by fermenting the Agrobacterium sp. ASE44.

[0021] The above-mentioned Agrobacterium ASE44 can be fermented in R2A medium at pH 7.2 and 28°C with shaking.

[0022] The method specifically involves applying Agrobacterium sp. ASE44 cells to oat grass (Zhongcao No. 33).

[0023] Compared with the prior art, the present invention has the following advantages: (1) This invention provides a soil bacterium ASE44 isolated from desert grassland soil, with accession number CCTCC No: M 20251157.

[0024] (2) The bacterial cells obtained by fermentation of this strain have a significant effect on improving the growth of oat grass seedlings.

[0025] (3) Agrobacterium ASE44 can be used to prepare microbial inoculants for application during the seedling stage of oat grass. It can improve the drought resistance of oat grass and be applied to the actual production of oat grass in arid areas, providing an excellent basic strain for the exploration and application of desert microbial resources. Attached Figure Description

[0026] Figure 1 The image shows the plate morphology of strain Agrobacterium sp. ASE44. The morphology of Agrobacterium sp. ASE44 after 3 days of incubation on R2A plates.

[0027] Figure 2 The growth of oat grass under different treatments.

[0028] Figure 3 The thickness of oat grass stems under different treatments.

[0029] Figure 4 Oat root length and plant height under different treatments.

[0030] Figure 5 The weight of oat stems and leaves and the weight of roots under different treatments.

[0031] in, Figures 2-5 The study included four treatments: a control group treated with sterile water; a treatment with ATCC 19358 treated with ATCC 19358; a treatment with sterilized ASE44 treated by soaking the ASE44 strain in water; and a treatment with ASE44 treated with ASE44. Detailed Implementation

[0032] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0033] The R2A medium used in the following examples has the following formulation: starch 0.5 g / L, glucose 0.5 g / L, acid-hydrolyzed casein 0.5 g / L, yeast extract 0.5 g / L, bacteriological peptone 0.5 g / L, sodium pyruvate 0.3 g / L, potassium dihydrogen phosphate 0.05 g / L, magnesium sulfate 0.025 g / L, TES 1.0 g / L, pH 7.2.

[0034] Example 1: Isolation and screening of Agrobacterium sp. ASE44 1. Material collection: Sand samples were collected from desert grassland in Siziwang Banner, Inner Mongolia Autonomous Region, China. They were mixed and placed in sterile sampling bags, placed in a vehicle refrigerator (4℃) and sent to the laboratory for storage at 4℃ for later use.

[0035] 2. Sample pretreatment: Collect soil samples, mix them evenly, sieve to remove litter, weigh 5 g of sample, add the soil sample to 45 mL of sterile distilled water under sterile conditions, shake and incubate for 30 min to prepare soil suspension for bacterial isolation.

[0036] 3. Isolation and purification of the bacterial strain: The treated soil suspension was serially diluted, and the original solution and 10... -1 Dilute the solution onto the surface of the solid culture medium (set up 3 replicates and 1 blank control), spread the suspension evenly with a sterile spreader, wait for the liquid to be absorbed, incubate upside down at 28°C, and purify the strain by streak plating.

[0037] Using the above method, Agrobacterium sp. strain number ASE44 was obtained through screening.

[0038] Example 2: Identification of Agrobacterium sp. ASE44 After growing the Agrobacterium sp. ASE44 strain obtained in Example 1 on R2A agar medium for 3 days, the strain was pale yellow and secreted polysaccharides. Colony morphology is shown in [insert image here]. Figure 1 .

[0039] Total DNA from *Agrobacterium sp.* ASE44 was extracted using resin extraction. A suitable amount of bacterial cells was picked from the solid culture medium using a sterile inoculation loop and placed into a 1.5 mL sterile centrifuge tube. 50 μL of 5% Chelex 100 solution was added, and the tube was incubated in a boiling water bath for 30 min. After cooling to room temperature, the tube was centrifuged at 12000 rpm for 5 min. The supernatant was used as a DNA template and stored at -20 ℃ for later use. Primers used for PCR amplification of the 16S rDNA gene were synthesized by Shanghai Sangon Biotech Co., Ltd., and their sequences are: 27F (SEQ ID NO:2): 5′-AGAGTTTGATCCTGGCTCAG-3′; 1492R (SEQ ID NO:3): 5′-TACGGCTACCTTG TTACGACTT-3′. The PCR reaction system consisted of 20 μL of DNA template, 10 μL of 1×EasyTap PCR SuperMix enzyme, 1.0 μL each of 27F and 1492R primers, and 7.0 μL of sterile, enzyme-free water. The reaction program was as follows: pre-denaturation at 95°C for 5 min; amplification included denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 2 min, and final extension at 72°C for 10 min, with multiple PCR cycles. The PCR products were analyzed using 1.0% agarose gel electrophoresis to ensure unique and bright bands. The products were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, as shown in SEQ ID NO:1. The obtained gene sequence was entered into the EZBioCloud database (http: / / www.ezbiocloud.net / ) for 16S rRNA sequence alignment, and the sequence similarity comparison results were obtained.

[0040] The sequence of SEQ ID NO:1 is shown below: GGCAGACGGG TGAGTAACGC GTGGGAACAT ACCCTTTCCT GCGGAATAGC TCCGGGAAAC TGGAATTAAT ACCGCATACG CCCTACGGGG GAAGAATTAAT TCGGGGAAGG ATTGGCCCGC GTTGGATTAG CTAGTTGGTG GGGTAAAGGC CTACCAAGGC GACGATCCAT AGCTGGTCTG ACTIVITY CAGCCACATT GGGACTGAGA CACGGGCCAA ACTCCTACGG GAGGCAGCAG TGGGGAATAT TGGACAATGG GCGCAAGCCT GATCCAGCCA TGCCGCGTGA GTGATGAAGG CCTTAGGGTT GTAAAGCTCT TTCACCGGAG AAGATAATGA CGGTATCCGG AGAAAAGCC CCGGCTAACT TCGTGCCAGC AGCCGCGGTA ATACGAAGGG GGCTAGCGTT GTTCGGAATT ACTGGGCGTA AAGGCACGT AGGCGGATT TTAAGTCAGG GGTGAAATCC CAGAGCTCAA CTCTGGAACT GCCTTTGATA CTGGGTATCT GGAATTCGA GTGTAGAGGT GAAATTCGTA GATATTCGGA GGAACACCAG TGGCGAAGGC GGCTTACTGG TCCATTACTG ACGCTGAGGT GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG TAAACG Compound strain, ASE44-based strain (Agrobacterium sp.), and strain ASE44 (Agrobacterium radiobacter ATCC). 19358) (GenBank: MK424303) is an invaluable manufacturer.

[0041] This strain was deposited on May 29, 2025, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, China Center for Type Culture Collection, Wuhan University. Its classification name is Agrobacterium sp. ASE44, and its accession number is CCTCC No: M20251157.

[0042] Example 3: Analysis of the growth-promoting effect of Agrobacterium sp. ASE44 on oat grass 1. Experimental Methods Prepare bacterial suspension. Inoculate purified single colonies into R2A liquid medium and incubate at 28°C and 180 rpm for 48 h using a constant temperature shaker. When collecting the bacterial cells, discard the supernatant using a high-speed refrigerated centrifuge (4°C, 8000 rpm, 10 min), resuspend in an appropriate amount of sterile water, and dilute to a final concentration of 1×10⁻⁶. 6 CFU / mL available for use.

[0043] Seed selection and sowing. Plump seeds of *Oatella esculenta* variety 33 were selected. The seed surface was disinfected with 5% sodium hypochlorite for 10 min and rinsed three times with sterile water. Thirty seeds were randomly selected and placed in 10 mL sterile centrifuge tubes, and 2 mL of bacterial suspension was added for soaking for 2 h (the control group received the same volume of sterile water). After air-drying, the seeds were sown in sterilized seedling substrate (seed soil:quartz sand = 2:1, autoclaved at 121℃ for 30 min), with 30 seeds per pot. Each treatment was replicated in triplicate, including a control treatment, a treatment with strain ATCC 19358, a treatment with sterilized strain ASE44, and a treatment with strain ASE44 (four treatments in total).

[0044] Cultivation and growth index determination. The culture pots were placed on a light-controlled cultivation rack for cultivation (25±1℃, photoperiod 16 h / d). 35 mL of sterile water was applied daily. After 28 days, complete plants were collected and relevant indicators were measured, including: stem diameter (vernier caliper), taproot length (mm), plant height (mm), and aboveground and underground dry weights of stems, leaves and roots after drying in a 70℃ oven (analytical balance ±0.1 mg).

[0045] 2. Experimental Results 2.1 Overall growth effect of oat grass under different treatments The oat grass growth promotion experiment included four treatments: a control group treated with sterile water; a treatment with strain ATCC19358 treated with strain 19358; a treatment with sterilized strain ASE44 treated by soaking seeds after sterilization; and a treatment with strain ASE44 treated with ASE44. After 28 days of cultivation, the oat grass growth was observed, revealing differences among the four treatments, particularly in root length and plant height, where the strain ASE44 treatment showed significantly better results than the other treatments. Figure 2 ).

[0046] 2.2 Coarse analysis of oat grass stems under different treatments Five oat grass plants from four different treatments were taken, and the stem diameter of the five oat grass plants was measured and analyzed using vernier calipers. The results are shown in Table 1.

[0047] Table 1. Measurement results of oat grass stem diameter (mm) Both microbial treatments (live and dead bacteria) significantly increased the stem diameter of oat grass, with live bacteria treatments (strains ASE44 and ATCC19358) showing the most significant effect. Specifically, the stem diameter of oat grass treated with strain ASE44 was 1.35 mm, significantly higher than other treatments. Compared to the control group, strains ATCC19358, sterilized strains ASE44, and ASE44 treatments increased stem diameter by 3.968%, 2.381%, and 7.142%, respectively. Figure 3 ).

[0048] 2.3 Analysis of root length and plant height of oat grass under different treatments The two treatments with added live bacteria (strain ASE44 and ATCC19358) did not show a significant difference in oat grass plant height, but compared with the sterilized ASE44 and sterile water control treatments, they significantly increased the root length and plant height of oat grass (see Tables 2 and 3).

[0049] Table 2. Measurement results of taproot length of oat grass (cm) Table 3. Measurement results of oat grass plant height (cm) The root length of oat grass treated with strain ASE44 was 19.46 cm, significantly higher than that of sterilized ASE44 and the control group, increasing by 5.78% and 6.107%, respectively. Regarding plant height, all three treatments with added microorganisms showed significant increases compared to the sterile water control. The ASE44 strain treatment resulted in a plant height of 49.40 cm, the largest increase of 10.812% compared to the control treatment of 44.58 cm. Figure 4 ).

[0050] 2.4 Analysis of stem, leaf, and root weights of oat grass under different treatments All three treatments involving the addition of microorganisms significantly increased stem and leaf weight and root weight, with the treatment using live bacterial strain ASE44 showing the most significant effect. The results are shown in Tables 4 and 5.

[0051] Table 4. Results of oat grass stem and leaf weight measurement (g) Table 5. Results of oat grass root weight measurement (g) Regarding stem and leaf weight, the stem and leaf weight of the ASE44 treatment was 1.46, 1.12, and 1.42 times that of the ATCC19358 treatment, the sterilized ASE treatment, and the control treatment, respectively (compared to the control group, ATCC19358, sterilized ASE44, and ASE44 increased by -3.921%, 25.490%, and 41.176%, respectively); regarding root weight, the root weight of the ASE44 treatment was 1.64, 1.32, and 1.76 times that of the ATCC19358 treatment, the sterilized ASE treatment, and the control treatment, respectively. Figure 5 (Compared to the control group, ATCC19358, sterilization ASE44 and ASE44 increased by 9.375%, 34.375% and 78.125%, respectively).

[0052] The results of this embodiment demonstrate that live Agrobacterium sp. ASE44 bacteria can significantly promote stem diameter, root length, plant height, stem and leaf weight, and root weight in oat grass seedlings, especially at a concentration of 1×10⁻⁶. 6 The presence of CFU / mL indicates that live Agrobacterium sp. ASE44 bacteria can significantly promote the growth of oat grass seedlings.

[0053] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. An Agrobacterium ASE44, characterized in that, It is Agrobacterium sp. The accession number is ASE44, and it is deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 20251157.

2. A microbial agent, characterized in that, Containing the contents of claim 1 Agrobacterium sp. ASE44.

3. A product comprising the *Agrobacterium* or its fermentation product as described in claim 1, characterized in that, The product is any one of the following: seed soaking solution, bacterial mud, seed coating, or fertilizer.

4. The product as described in claim 3, characterized in that, The total viable count of Agrobacterium in the product is not less than 10. 6 CFU / g or 10 6 CFU / mL.

5. The application of the *Agrobacterium* as described in claim 1 or the microbial agent as described in claim 2 in improving the growth performance of oat grass.

6. The application as described in claim 5, characterized in that, The application described is for improving the growth of oat grass seedlings.

7. The application as described in claim 5, characterized in that, The fermentation culture conditions for Agrobacterium ASE44 were as follows: cultured on R2A medium at a temperature of 25-30℃ and a pH of 6.5-7.

6.

8. The application as described in claim 5, characterized in that, The application is one or more of the following: Application in increasing the stem thickness of oat grass; Application in improving the root length of oat grass; Applications to improve the high efficiency of oat grass plants; Application in increasing the stem, leaf, and root weight of oat grass.

9. The application as described in claim 8, characterized in that, The total viable count of Agrobacterium is not less than 10. 6 CFU / g or 10 6 CFU / mL.

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