Pseudomonas LSZ22 and application thereof

Abstract

The invention discloses a pseudomonas LSZ22 and application thereof, the bacterial strain is classified and named as Pseudomonas extrinsic LSZ22, the bacterial strain is preserved in China Center for Type Culture Collection on September 29, 2025, and the preservation number is CCTCC NO: M 20252165. The pseudomonas LSZ22 has the advantages that the bacterial strain is named as Pseudomonas extrinsic LSZ22, the bacterial strain is preserved in China Center for Type Culture Collection on September 29, 2025, and the preservation number is CCTCC NO: M 20252165. The application of the pseudomonas LSZ22 can promote the growth of plants, induce up-regulation expression of drought-resistant related genes of the plants and improve the water absorption capacity of the plants, so that the adaptability of the plants in a drought environment is improved.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, specifically to a Pseudomonas LSZ22 and its applications. Background Technology

[0002] Drought is a major abiotic stress in global agriculture and forestry. The frequency and intensity of drought stress caused by climate change are constantly increasing; it is estimated that by the end of the 21st century, the global dryland area will increase by 11%–23%, leading to serious food security problems. The interaction between crops, soil, and microorganisms jointly maintains the balance of the farmland ecosystem. Faced with drought, plants do not fight alone; they form a complex network of interactions with tens of thousands of microorganisms around their rhizosphere. These microorganisms help plants enhance nutrient absorption, resist pathogen invasion, and alleviate environmental stress. Therefore, this complex plant-associated microbial community is called the plant's "second genome" and is crucial for plant growth and development.

[0003] Rhizosphere microorganisms are an important approach to studying ways to improve plant drought stress resistance. Existing drought-resistant bacteria include *Bacillus belye* (…). bacillus velezensis Aspergillus oryzae ( ) aspergillusoryzae ), Bacillus cereus ( bacillus cereus ), clusters of pantothenic bacteria ( pantoeaagglomerans Pseudomonas is a group of non-spore-forming, capsule-containing, scattered Gram-negative bacilli. They are obligate aerobic, with straight or slightly curved cells, possessing a single or tufted flagella, and are highly motile. Studies have found that some Pseudomonas species exhibit strong resistance to adverse conditions, surviving under extreme conditions such as drought and high temperatures. However, current screening methods for Pseudomonas (…) Pseudomonas Research on Pseudomonas strains as drought-resistant functional bacteria remains limited. Therefore, developing a Pseudomonas strain with a clearly identified origin, strong drought resistance, and stable application in planting areas is of great significance for improving crop drought resistance and ensuring food security. Summary of the Invention

[0004] The purpose of this invention is to provide a Pseudomonas LSZ22 strain and its application, which can grow stably under water stress conditions and exhibits significant drought resistance.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a Pseudomonas aeruginosa ( Pseudomonas extremorientalis LSZ22, this strain has been deposited at the China Center for Type Culture Collection (CCTCC) designated by the State Intellectual Property Office on September 29, 2025. The deposit number is: CCTCC NO: M 20252165.

[0006] Furthermore, the Pseudomonas LSZ22 was isolated from the rhizosphere of wheat.

[0007] Furthermore, the 16S rDNA gene sequence of the Pseudomonas LSZ22 is shown in SEQ ID NO. 1.

[0008] Furthermore, the Pseudomonas LSZ22 colonies on LB medium are round, opaque, smooth, and moist; under a microscope, the Gram-stained strain appears red, indicating it is a Gram-negative bacterium with short, straight rod-shaped cells.

[0009] Furthermore, the optimal growth temperature for the Pseudomonas LSZ22 is 32°C, and the optimal growth pH is 5–9.

[0010] Secondly, the present invention provides a drought-resistant growth promoter, comprising the aforementioned Pseudomonas LSZ22.

[0011] Furthermore, the drought-resistant growth promoter also includes acetic acid, S-inducer and plant growth regulator, wherein the mass ratio of acetic acid, S-inducer and plant growth regulator is 1:(0.1-0.5):(0.5-2).

[0012] Furthermore, the plant growth regulator includes one or more of indoleacetic acid, zeatin, and paclobutrazol.

[0013] Further, the concentration of acetic acid in the drought-resistant growth promoter is 0.1–1.0 g / L, the concentration of S-inducer in the drought-resistant growth promoter is 0.01–0.1 g / L, and the concentration of plant growth regulator in the drought-resistant growth promoter is 0.05–0.5 g / L.

[0014] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects: (1) This invention is based on Pseudomonas LSZ22, which was screened from the rhizosphere soil of spring wheat under drought stress and artificial PEG simulated drought conditions. Pseudomonas LSZ22, a strain of Pseudomonas, exhibits remarkable drought tolerance and can grow stably in arid environments. By secreting amino acids (such as arginine) and other bioactive substances (such as extracellular polysaccharides), Pseudomonas LSZ22 helps plants improve water use efficiency and reduce water loss under drought conditions. Application of Pseudomonas LSZ22 can promote plant growth and induce the upregulation of drought-related genes, enhancing the plant's ability to absorb water and thus improving its adaptability to arid environments.

[0015] (2) The present invention uses Pseudomonas LSZ22 to prepare a drought-resistant growth promoter, which can significantly promote the growth of spring wheat, improve its physiological state, and induce the upregulation of drought-resistant genes under drought stress. Its effect is better than using Pseudomonas LSZ22 alone or additives. This confirms the synergistic effect of the components in the drought-resistant growth promoter and provides a basis for the development of highly efficient drought-resistant microbial preparations. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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 the structures shown in these drawings without creative effort.

[0017] Figure 1 This invention describes the colony and cell morphology of the *Pseudomonas* strain. Figure 2 This invention describes the growth of the Pseudomonas LSZ22 strain at different temperatures. Figure 3 This invention describes the growth of Pseudomonas LSZ22 in different pH culture media. Figure 4 This is a phylogenetic tree of the strain LSZ22 of this invention based on the 16S rDNA gene sequence; Figure 5 This invention relates to the effect of PEG stress on the secretion of arginine and extracellular polysaccharides by Pseudomonas LSZ22. Figure 6 This invention describes the effects of exogenous addition of Pseudomonas LSZ22 on plant morphology and root phenotype under drought stress. Figure 7 Adding external sources to this invention Pseudomonas The effect of LSZ22 on the relative expression levels of drought resistance genes in spring wheat; in the figure, A represents... TaLEA2 Relative expression level; B is TaWRKY10 Relative expression level; C is TaERF3 Relative expression level; D is TaBADHb Relative expression level. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0019] Example 1: Isolation and screening of Pseudomonas LSZ22 To obtain drought-resistant bacteria, dilution streak plate method was used to isolate and purify rhizosphere spring wheat soil under drought stress. The purified strains were acclimatized on LB medium with different mass concentrations of PEG6000, and the OD was measured. 600 Drought resistance analysis was conducted under conditions of 60% PEG6000. Pseudomonas The average growth rate (AGR) of LSZ22 was 4.99E. -02 OD max The value is 1.521. Pseudomonas LSZ22 has strong drought resistance (Table 1).

[0020] Table 1. Growth of rhizosphere bacteria under different drought stress conditions

[0021] Example 2: Physiological and biochemical characteristics analysis of Pseudomonas LSZ22 1. Morphology of Pseudomonas LSZ22 Pseudomonas LSZ22 ( Pseudomonas LSZ22) grew well on LB medium, producing round, opaque colonies with smooth, moist surfaces. Under a 10×100 microscope, the Gram-stained strain appeared red, indicating it was a Gram-negative bacterium with short, straight rod-like cells. Figure 1 ).

[0022] 2. Optimal growth temperature of Pseudomonas LSZ22 The growth of *Pseudomonas LSZ22* showed significant differences at temperatures ranging from 28℃ to 49℃ (P<0.05). The optimal growth temperature for *Pseudomonas LSZ22* strain was 32℃, and the OD... 600 The value is 1.15 ( Figure 2 ).

[0023] 3. Optimal pH for the growth of Pseudomonas LSZ22 Pseudomonas LSZ22 exhibits pH preference in its growth. Specifically, its growth is significantly inhibited at pH values ​​between 2 and 4, resulting in lower OD values. 600 The values ​​ranged from 0.042 to 0.057 (P < 0.05). *Pseudomonas* LSZ21 grew well at pH values ​​from 5 to 9. The optimal pH for the growth of *Pseudomonas* LSZ22 was 7, with an OD value of [missing value]. 600 The value is 1.20 ( Figure 3 ).

[0024] 4. Identification of the physiological and biochemical characteristics of the strain The metabolic characteristics of Pseudomonas LSZ22 strain were identified using the BIOLOG GeneⅢ system. The results showed that Pseudomonas LSZ22 had a total of 57 positive reactions ( Figure 4 Among the 26 sugar alcohols, *Pseudomonas LSZ22* can effectively utilize 8 sugar alcohol substrates, including D-trehalose, sucrose, and D-galactose (Table 2). Among the 10 amino acids, *Pseudomonas LSZ22* can effectively utilize 8 amino acid substrates, including L-alanine, L-arginine, and L-glutamic acid (Table 3). Among the 17 antibiotics, *Pseudomonas LSZ22* showed positive reactions with 14 antibiotic substrates, including sodium lactate, tetrazolium violet, lithium chloride, and vancomycin, while being sensitive to antibiotics such as sodium butyrate and dimethylaminotetracycline (Table 4).

[0025] Table 2. Identification of Pseudomonas LSZ22 using sugar alcohol reactions in GenIII identification plates.

[0026] Table 3. Amino acid reaction identification of Pseudomonas LSZ22 in GenIII identification plates.

[0027] Table 4. Antibiotic reaction identification of Pseudomonas LSZ22 in GenIII identification plates

[0028] 5. Molecular biological identification based on 16S rDNA Sequencing of the PCR amplification product of 16S rDNA from strain LSZ22 revealed a sequence length of 1421 bp, as shown in SEQ ID NO. 1. SEQ ID NO. 1:

[0029] In the NCBI database, homology searches were performed using Blester and published 16S rDNA sequences in GenBank, and similarity comparisons revealed that strain LSZ22 was similar to... Pseudomonas The 16S rDNA sequence alignment of *Sp.* showed the highest consistency, with a similarity exceeding 99%. Therefore, the 16S rDNA gene sequences of relevant strains were downloaded from GeneBank, and multiple comparisons were performed using MEGA 11.0 software to construct a phylogenetic tree. The similarity was then calculated based on genetic distance. Figure 6 LSZ22 and Pseudomonas extremorientalis The similarity of strain JM23 (MN758783.1) is 100%. Figure 4 Therefore, LSZ22 was named... Pseudomonas extremorientalis LSZ22.

[0030] Example 3: Drought stress induces Pseudomonas LSZ22 to secrete amino acids and bioactive substances Compared with the control (LB), the arginine and extracellular polysaccharide (EPS) content of *Pseudomonas LSZ22* were significantly increased under 20% and 40% PEG moisture stress (P<0.05). Specifically, the arginine content increased by 20.61% and 44.30%, respectively. Figure 5 A); EPS content increased by 27.17% and 62.35% respectively ( Figure 5 B). Increased arginine levels not only contribute to osmotic regulation but may also enhance the plant's ability to absorb water and nutrients by promoting root growth, while the accumulation of EPS helps improve the water retention capacity of the rhizosphere, thus alleviating water deficit stress to some extent. In summary, these results indicate that... Pseudomonas Under drought stress, LSZ22 can provide drought resistance support to the host plant by accumulating arginine and extracellular polysaccharides.

[0031] Example 4: Pseudomonas LSZ22 promotes crop growth under drought stress In the pot experiment, when the spring wheat reached the three-leaf stage, 10 mL of [amount missing] was added to the flowerpot. Pseudomonas LSZ22 (1×10 8 CFU·mL -1 10 mL of sterile water (WT). After inoculation, a drought treatment period of one week was conducted, and the morphology of spring wheat plants was observed and phenotypic indicators were measured. Figure 6 The results showed that, compared with the control, inoculation under drought (DT) treatment was more effective. Pseudomonas The morphological changes of spring wheat LSZ22 are relatively small. Figure 6 Compared to sterile water (SW) treatment, inoculation... PseudomonasThe aboveground fresh weight, aboveground dry weight, root fresh weight, root dry weight, root length and stem diameter of spring wheat treated with LSZ22 were significantly increased by 39.81%, 37.36%, 22.52%, 28.95%, 19.09% and 47.89% respectively (P<0.05) (Table 5).

[0032] Table 5. Effects of exogenous Pseudomonas LSZ22 on plant morphology and root system under drought stress.

[0033] Example 5: Pseudomonas LSZ22 induces upregulation of drought-related genes in plants Will Pseudomonas Culture medium of LSZ22 strain (concentration of 1×10⁻⁶) 8 CFU·mL -1 The gene was added to the soil of spring wheat plants that had reached the three-leaf stage, and root samples were collected at 0h, 12h, 24h, and 48h after the addition for relative gene expression analysis. qRT-PCR results showed that, compared with the control group (0h), Pseudomonas LSZ22 bacterial inoculation significantly upregulated genes related to drought response in spring wheat. TaLEA2 , TaWRKY10 , TaERF3 and TaBADHb The relative expression level (P<0.05) Figure 7 Among them, compared with the control group (0h), Pseudomonas LSZ22 bacterial culture inoculation for 12 hours resulted in drought resistance genes in the roots of spring wheat. TaLEA2 , TaWRKY10 , TaERF3 , TaBADHb The relative expression levels were significantly higher at 24h and 48h. Pseudomonas LSZ22 bacterial culture inoculated for 12 hours resulted in drought resistance genes in the roots of spring wheat. TaLEA2 The expression level increased by up to 22.05 times, while TaERF3 The lowest increase was 32.32 times.

[0034] Example 6: Determination of the effect of drought-resistant and growth-promoting agents To verify the effectiveness of the drought-resistant growth promoter, the following pot experiment was designed. The experiment referenced Examples 4 and 5 in the document, but was optimized for the drought-resistant growth promoter to evaluate its synergistic effect.

[0035] 1. Experimental Materials Spring wheat (variety: "Xinchun No. 6"); Pseudomonas LSZ22 bacterial suspension: prepared at a concentration of 1×10⁻⁶. 8 CFU·mL -1 (The method is the same as in Example 1); Drought-resistant growth promoter: 1×10⁻⁶ Pseudomonas LSZ22 bacterial suspension 8 CFU·mL -1 Acetic acid: 0.5 g / L, S-inducer: 0.05 g / L, plant growth regulator: mixture of indoleacetic acid and zeatin (mass ratio 1:1), total concentration 0.2 g / L; Control treatments: sterile water (SW), Pseudomonas LSZ22 bacterial culture alone (LSZ22), drought-resistant growth promoter (LSZ22 + additives); Potting soil: Use sandy loam soil with a pH of 6.5-7.0; Drought treatment: Simulate drought stress by stopping irrigation (soil moisture content drops to about 30%).

[0036] 2. Experimental Methods Four treatment groups were set up, with 10 pots of spring wheat in each group (5 plants per pot): T1: Sterile water (SW) treatment (control); T2: Treatment with Pseudomonas LSZ22 bacterial culture alone (1×10) 8 CFU·mL -1 ); T3: Drought-resistant and growth-promoting agent treatment (including bacterial solution and the above-mentioned additives). T4: Additive treatment only (without bacterial solution, used to evaluate the effect of the additive alone); When spring wheat reaches the three-leaf stage, apply 10 mL of the corresponding treatment solution, followed by drought treatment (lasting 7 days).

[0037] 3. Measurement indicators: 3.1 Growth indicators: Measurements were taken after the drought treatment ended, including aboveground fresh and dry weight, root fresh and dry weight, root length, and stem diameter of spring wheat. Data are expressed as mean ± standard deviation, and different letters indicate significant differences between treatments at the P < 0.05 level (Table 6).

[0038] Table 6 Effects of drought-resistant growth promoters on growth indicators of spring wheat

[0039] Compared to T1 (sterile water control), the T2 treatment (LSZ22 bacterial solution alone) significantly improved all growth indicators of spring wheat (above-ground fresh weight, dry weight, root fresh weight, root dry weight, root length, and stem diameter), with increases ranging from 22.52% to 47.89%, consistent with the results of Example 4. The T3 treatment (drought-resistant growth promoter) further significantly improved all growth indicators, with even higher increases than the T2 treatment (e.g., above-ground fresh weight increased by 77.14% compared to T1 and by 26.53% compared to T2), indicating that the additives in the drought-resistant growth promoter (acetic acid, S-inducer, and plant growth regulators) have a synergistic growth-promoting effect with Pseudomonas LSZ22. The T4 treatment (additives only) showed some improvement in growth indicators, but the effect was far lower than that of T2 and T3, indicating that the additives alone have limited effect and need to be combined with the bacterial strain to achieve the best effect.

[0040] 3.2 Physiological indicators: The relative water content (RWC) and chlorophyll content (SPAD value) of leaves were measured, as shown in Table 7.

[0041] Table 7 Effects of drought-resistant growth promoters on physiological indicators of spring wheat

[0042] Relative water content (RWC) and chlorophyll content (SPAD value) are important indicators reflecting plant water status and photosynthetic capacity. Treatment T3 significantly increased RWC and SPAD values, by 24.53% and 35.94% respectively compared to T1, and by 10.50% and 15.10% respectively compared to T2, indicating that drought-resistant growth promoters can effectively alleviate drought stress and enhance plant water retention capacity and photosynthetic efficiency. Treatment T2 also significantly improved physiological indicators, but not as much as treatment T3, further confirming the synergistic effect.

[0043] 3.3 Molecular Indicators: Root samples were collected (0h, 12h, 24h, and 48h after treatment), and the relative expression levels of drought-resistant genes were analyzed by qRT-PCR (Table 8), using the same method as in Example 5.

[0044] Table 8 Effects of drought-resistant growth promoters on the relative expression levels of drought-resistant genes in spring wheat (12 h after treatment)

[0045] At the gene expression level, the T3 treatment significantly upregulated the expression of all four drought-resistance genes (TaLEA2, TaWRKY10, TaERF3, and TabADHb) 12 hours after inoculation, with increases greater than those in the T2 treatment. For example, the expression level of TaLEA2 in T3 was 35.8 times that in T1, while it was 22.5 times that in T2. ​​The T4 treatment only slightly induced gene expression, indicating that the additive itself had limited upregulation effect on genes, but could synergistically enhance the plant's drought resistance response with the strain.

[0046] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A Pseudomonas LSZ22 strain, characterized in that, Pseudomonas ( Pseudomonas extremorientalis LSZ22, this strain has been deposited at the China Center for Type Culture Collection (CCTCC) designated by the State Intellectual Property Office on September 29, 2025. The deposit number is: CCTCC NO: M 20252165.

2. The *Pseudomonas* LSZ22 according to claim 1, characterized in that, The pseudomonad LSZ22 was isolated from the rhizosphere of wheat.

3. The *Pseudomonas* LSZ22 according to claim 1, characterized in that, The 16S rDNA gene sequence of the pseudomonad LSZ22 is shown in SEQ ID NO.

1.

4. The *Pseudomonas* LSZ22 according to claim 1, characterized in that, The Pseudomonas LSZ22 colonies on LB medium are round, opaque, smooth, and moist. Under a microscope, the Gram-stained strain appears red, indicating it is a Gram-negative bacterium with short, straight rod-shaped cells.

5. The *Pseudomonas* LSZ22 according to claim 1, characterized in that, The optimal growth temperature for the Pseudomonas LSZ22 is 32℃, and the optimal pH for growth is 5–9.

6. A drought-resistant and growth-promoting agent, characterized in that, Includes Pseudomonas LSZ22 as described in any one of claims 1 to 5.

7. The drought-resistant and growth-promoting agent according to claim 6, characterized in that, The drought-resistant growth promoter also includes acetic acid, S-inducer and plant growth regulator, wherein the mass ratio of acetic acid, S-inducer and plant growth regulator is 1:(0.1-0.5):(0.5-2).

8. The drought-resistant and growth-promoting agent according to claim 7, characterized in that, The plant growth regulators include one or more of the following: indoleacetic acid, zeatin, and paclobutrazol.

9. The drought-resistant and growth-promoting agent according to claim 8, characterized in that, The concentration of acetic acid in the drought-resistant growth promoter is 0.1–1.0 g / L, the concentration of S-inducer in the drought-resistant growth promoter is 0.01–0.1 g / L, and the concentration of plant growth regulator in the drought-resistant growth promoter is 0.05–0.5 g / L.

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