Pseudomonas chlororaphis resistant to salt, alkali and drought and application of pseudomonas chlororaphis

Abstract

The invention discloses a salt-alkali resistant and drought resistant pseudomonas chlororaphis and an application thereof. Belongs to the technical field of agricultural microorganism and ecological restoration. The invention overcomes the defect of poor environmental adaptability of the existing potassium bacteria, provides the pseudomonas chlororaphis K5 which still keeps high activity under saline-alkali, drought and barren conditions, and provides application of the pseudomonas chlororaphis K5 in improvement of saline-alkali soil and improvement of crop stress resistance. The problem that the existing potassium bacteria die immediately in the field or do not work in the saline-alkali soil is solved. The pseudomonas chlororaphis K5 can still effectively decompose mineral potassium under the conditions that the pH is 9.0 and the salinity is high, and the blank of potassium bacteria for saline-alkali soil is filled.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology and ecological restoration technology, and more specifically to a salt- and drought-tolerant strain of Pseudomonas aeruginosa and its applications. Background Technology

[0002] Potassium is one of the three essential nutrients for plant growth, participating in key physiological processes such as the activation of more than 60 enzymes, photosynthesis, sugar transport, and protein synthesis. my country's soils are rich in potassium, but over 90% of it exists as insoluble potassium-containing minerals (such as feldspar and mica), which plant roots cannot directly absorb and utilize, resulting in a "potassium-rich but potassium-deficient" soil condition. For a long time, agricultural production has relied mainly on the application of chemical potassium fertilizers to replenish readily available potassium in the soil. However, the production of chemical potassium fertilizers is energy-intensive, and excessive application can easily lead to a series of problems such as soil compaction, salinization, and environmental pollution. To address this contradiction, utilizing potassium-solubilizing microorganisms (KSMs) to convert ineffective mineral potassium in the soil into soluble potassium is considered a highly promising green and sustainable pathway. These microorganisms, by secreting organic acids, enzymes, and capsular polysaccharides, disrupt the crystal structure of potassium-containing minerals, releasing potassium ions and thus improving the bioavailability of potassium in the soil.

[0003] While utilizing potassium-solubilizing microorganisms (KSM) to convert soil potassium is a trend in green agriculture, existing technologies face significant bottlenecks:

[0004] Most of the potassium-solubilizing bacteria reported so far (such as Bacillus mucilaginosus and common Pseudomonas) were screened from fertile farmland soils, and their optimal environment for action is often neutral pH, low salinity, and suitable moisture conditions. However, in the widely distributed saline-alkali lands, arid desert areas, and barren soils in my country, high salt concentrations, high pH values, and water stress can generate enormous osmotic pressure and toxic effects on microorganisms, causing conventional potassium-solubilizing bacteria to fail to colonize or have their metabolic activity inhibited after application, thus failing to perform their potassium-solubilizing function.

[0005] Therefore, screening a "dual-effect" strain that can efficiently solubilize potassium and tolerate extreme environments such as high salt, high alkali, and drought is of great strategic significance for developing microbial agents suitable for saline-alkali land management and marginal land use. Summary of the Invention

[0006] In view of this, the present invention provides a salt- and alkali-tolerant, drought-resistant Pseudomonas aeruginosa and its applications.

[0007] The purpose of this invention is to overcome the shortcomings of existing potassium-solubilizing bacteria in terms of poor environmental adaptability, and to provide a strain of *Pseudomonas aeruginosa* K5 that maintains high activity under saline, arid, and barren conditions.Pseudomonas chlororaphis K5) and its application in improving saline-alkali soil and enhancing crop stress resistance.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A salt- and alkali-tolerant, drought-resistant strain of *Pseudomonas aeruginosa*, named K5, is classified as follows: Pseudomonas chlororaphis It was deposited on October 20, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252259, at Wuhan University, Wuhan, China.

[0009] Furthermore, it can tolerate a maximum NaCl concentration of 6%, a pH value of 9.0, and a PEG-6000 concentration of 20%.

[0010] A biological agent comprising the aforementioned *Pseudomonas aeruginosa*.

[0011] The aforementioned *Pseudomonas aeruginosa* and the aforementioned biological agent possess at least one of the following properties: 1) Potassium solubilization; 2) Alleviating salt and alkali stress in plants; 3) Improve plant stress resistance; 4) Promotes plant growth; 5) Promotes an increase in the above-ground fresh weight of plants; 6) Promotes increased plant height.

[0012] The application of the aforementioned *Pseudomonas aeruginosa* and the aforementioned biological agent, wherein the application is any one of the following: 1) Applications in potassium solubilization or in the preparation of products for increasing the available potassium content in soil; 2) Applications in alleviating salt and alkali stress in plants or in the preparation of products for alleviating salt and alkali stress in plants; 3) Applications in promoting plant growth or in the preparation of products for promoting plant growth; 4) Applications in promoting the increase of aboveground fresh weight of plants or in the preparation of products for promoting the increase of aboveground fresh weight of plants; 5) Applications in promoting plant height increase or in the preparation of products for promoting plant height increase; 6) Applications in improving plant stress resistance or in the preparation of products for improving plant stress resistance.

[0013] Furthermore, the plants mentioned are tobacco and alfalfa.

[0014] This invention provides the application of Pseudomonas aeruginosa K5 or an inoculum containing this strain in agricultural production, particularly in the preparation of products for increasing the available potassium content in soil.

[0015] Application methods: It can be applied to the soil or crop roots through seed dressing, root dipping, trenching, hole application, or irrigation.

[0016] Application rate: Depending on soil type, crop type, and application method, the recommended application rate is 10. 7 ~10 8 CFU / g soil. Can be used in combination with organic fertilizer or bio-compound fertilizer; shows good growth-promoting effect on various plants such as tobacco and alfalfa.

[0017] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) Outstanding potassium solubilization ability: The *Pseudomonas aeruginosa* K5 (CCTCC NO: M 20252259) screened and identified in this invention has a very strong potassium solubilization ability. The potassium-solubilizing zone formed on Aleksandrov medium can reach a diameter of 3.5 cm, which can more efficiently activate the unavailable potassium in the soil.

[0018] (2) Superior strain performance: Pseudomonas aeruginosa K5 not only has strong potassium solubilization ability, but also has the characteristics of fast growth rate, good environmental adaptability and easy industrial fermentation culture, which lays a solid foundation for its large-scale production and application as an active ingredient of microbial agents.

[0019] (3) Overcoming environmental limitations: It solves the problem that existing potassium-solubilizing bacteria "die immediately upon planting" or "do not work in saline-alkali land". Pseudomonas aeruginosa K5 can still effectively decompose mineral potassium under high salinity conditions at pH 9.0, filling the gap in potassium-solubilizing bacteria for saline-alkali land.

[0020] (4) Green and environmentally friendly, reducing the application of chemical fertilizers: This invention supplements the soil with readily available potassium through biological means, which can partially or completely replace the use of chemical potassium fertilizers, reducing soil degradation and environmental pollution caused by excessive fertilization, and conforming to the direction of green agriculture and sustainable development. Attached Figure Description

[0021] 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 embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 This refers to the potassium solubilization ability of strain K5 in Example 1 of the present invention on Aleksandrov medium.

[0023] Figure 2 This is a Gram staining image of strain K5 in Example 1 of the present invention.

[0024] Figure 3 This is a colony diagram of strain K5 in Example 1 of the present invention on a TSA plate.

[0025] Figure 4 This is the phylogenetic tree of strain K5 in Example 1 of the present invention. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0027] Example 1 Isolation and identification of strains Soils from secondary salinized tobacco fields were collected from the tobacco-growing area of ​​Guiyang County, Chenzhou City, Hunan Province. 3% NaCl was added to the screening medium, and the pH was adjusted to 9.0 as the selection pressure; only colonies exhibiting a potassium-solubilizing zone under these conditions were selected. The soil suspension was spread onto Aleksandrov inorganic potassium solid medium (potassium feldspar powder was the sole potassium source, from CHINOOK, Aleksandrov Biotechnology Co., Ltd.; the specific formula was: glucose 5 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferric chloride 0.005 g / L, calcium carbonate 2 g / L, potassium feldspar powder 2 g / L, agar 15 g / L) using a dilution plating method. After incubation at 30℃ for 5 days, single colonies with a clearly visible transparent potassium-solubilizing zone were selected, purified, and named strain K5. Figure 1 Measurements showed that the ratio (D / d) of the potassium-solubilizing zone diameter to the colony diameter was consistently greater than 2, and the potassium-solubilizing zone diameter could reach 3.5 cm.

[0028] Morphological identification: Strain K5 is a Gram-negative bacillus with rod-shaped cells and motility. Figure 2 After culturing on TSA medium for 24 hours, strain K5 formed round, well-defined, smooth, moist, and slightly translucent white colonies. Figure 3 ).

[0029] Physiological and biochemical identification: Physiological and biochemical identification of strain K5 was performed. The results showed that strain K5 is strictly aerobic, and both oxidase and catalase reactions were positive. This strain can utilize multiple carbon sources such as glucose, glycerol, and citrate. The gelatin liquefaction test was positive, and the starch hydrolysis test was negative (see Table 1 for details).

[0030] Table 1. Physiological and Biochemical Identification Results

[0031] Molecular biological identification: Genomic DNA was extracted from strain K5, and the 16S rRNA gene was amplified using universal primers 27F and 1492R. A valid sequence of 1227 bp (as shown in SEQ ID NO:1) was obtained by bidirectional sequencing. This sequence was then BLAST-aligned in the NCBI database, and the results showed that it was consistent with... Pseudomonas chlororaphis The homology reached over 99%. A phylogenetic tree was constructed using MEGA software. Figure 4 ).

[0032]

[0033] Based on morphological, physiological and biochemical characteristics and molecular biological identification results, strain K5 was identified as *Pseudomonas aeruginosa*. Pseudomonas chlororaphis ).

[0034] Example 2 Preservation of Pseudomonas aeruginosa K5 Pseudomonas aeruginosa K5, its classification name is Pseudomonas chlororaphis It was deposited on October 20, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252259, at Wuhan University, Wuhan, China.

[0035] Example 3 Salt tolerance and potassium solubilization ability test of Pseudomonas aeruginosa K5 *Pseudomonas aeruginosa* K5 was inoculated onto Aleksandrov plates (pH 7.0–7.2) containing 0%, 2%, 4%, 6%, and 8% NaCl. The relative humidity in the incubator was controlled at 60%–70%, and the plates were inverted and incubated at 30°C for 10 days. The results showed that *Pseudomonas aeruginosa* K5 still had a clear potassium-solubilizing zone at a 6% NaCl concentration, and the ratio of the potassium-solubilizing zone to the colony diameter was greater than 2, indicating its strong potassium-solubilizing ability (Table 2).

[0036] Table 2. Potassium solubilization effect of *Pseudomonas aeruginosa* K5 on Aleksandrov inorganic potassium solid medium under different salinity conditions.

[0037] Example 4 Alkali-tolerant potassium-solubilizing ability test of Pseudomonas aeruginosa K5 The pH of Aleksandrov medium was adjusted to 7.0, 8.0, 9.0, and 10.0. *Pseudomonas aeruginosa* K5 was inoculated, and the relative humidity in the incubator was controlled at 60%–70%. The plates were inverted and incubated at 30°C for 10 days. The results showed that the potassium-soluble zone / colony diameter ratio of *Pseudomonas aeruginosa* K5 was still greater than 2 at pH 9.0 (Table 3).

[0038] Table 3. Potassium solubilization effect of Pseudomonas aeruginosa K5 on Aleksandrov inorganic potassium solid medium under different pH conditions.

[0039] Example 5 Drought resistance (PEG simulation) test of Pseudomonas aeruginosa K5 PEG-6000 (polyethylene glycol) was used to simulate drought stress. 0%, 10%, 20%, and 30% (w / v) PEG-6000 were added to LB liquid medium, respectively. *Pseudomonas aeruginosa* K5 (initial inoculum 1%) was inoculated and cultured at 30°C and 180 rpm with shaking for 24 hours. The absorbance (OD600) of the bacterial culture was measured to assess growth. Medium without PEG was used as a control (CK).

[0040] The results are shown in Table 4. With increasing PEG concentration, the environmental water potential decreased, which somewhat affected the growth of *Pseudomonas aeruginosa* K5. However, under the simulated moderate drought conditions of 20% PEG-6000, the OD600 value of *P. aeruginosa* K5 still reached more than 65% of the control group, and the growth status remained turbid; even under the severe drought conditions of 30% PEG, significant growth was still observed. This indicates that *P. aeruginosa* K5 possesses extremely strong water use capacity and drought tolerance.

[0041] Table 4. Growth of Pseudomonas aeruginosa K5 under simulated drought conditions with different concentrations of PEG-6000.

[0042] Example 6 Preparation of potassium-solubilizing bacteria (1) Activation and seed liquid preparation Pseudomonas aeruginosa K5 was activated by streaking in LB or beef extract peptone medium and cultured at 30°C for 24 hours. Single colonies were picked and inoculated into seed culture medium (10 g / L glucose, 1 g / L (NH4)2SO4, 1 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, 0.5 g / L NaCl) and cultured with shaking at 30°C and 180 rpm for 18–24 hours until the bacterial concentration (OD600) reached 1.0–1.2, thus obtaining the seed culture.

[0043] (2) Fermentation culture The seed culture was inoculated into the fermentation medium (20 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 1 g / L K₂HPO₄, 0.5 g / L MgSO₄·7H₂O, 2 g / L betaine, 1 L water, pH 7.0) at a 5% (v / v) inoculation rate. It was cultured at 30℃ and 180 rpm for 48 hours at pH 7.2–7.5 and dissolved oxygen >30% to obtain the fermentation broth. The viable cell count was measured to be 2.0 × 10⁻⁶. 9 cfu / mL, pH decreased to 5.2.

[0044] (3) Preparation of microbial agents ① Liquid inoculant: When the viable cell count in the fermentation broth reaches 2.0 × 10⁻⁶9 Immediately stop aeration and stirring when the concentration of cfu / mL is reached, and rapidly lower the fermenter temperature to 10-15°C to induce dormancy in the bacterial cells. Pump the cooled fermentation broth into a double-layer stainless steel vibrating screen (80-100 mesh) for filtration to remove undissolved particles, large protein precipitates, and potential clumping impurities from the culture medium, preventing subsequent clogging of the irrigation equipment. Simultaneously, retain all bacterial monomers and add a compound protective agent consisting of 1.5% trehalose, 1.0% betaine, 0.5% xanthan gum, and 2.0% glycerol.

[0045] ② Solid inoculants: Bentonite and humic acid powder were mixed at a mass ratio of 4:1, sterilized at 121℃ for 30 minutes, and dried to obtain a composite carrier. 100 mL of fermentation broth treated with the composite protectant was evenly sprayed onto 300 g of the composite carrier while stirring to ensure uniform adsorption. The mixture was then placed in a 35-40℃ oven for low-temperature variable-temperature drying (first at 35℃ for 4 hours, then at 40℃ to constant weight), controlling the moisture content to below 10%, to obtain a solid inoculum. Plate counting showed that the effective viable count of the solid inoculum was approximately 5 × 10⁻⁶. 8 cfu / g.

[0046] Example 7 Pot experiment on the effects of potassium-solubilizing bacteria on potassium-solubilizing and growth-promoting effects The test soil was collected from tobacco fields in Xiangxi Tujia and Miao Autonomous Prefecture, Hunan Province. The soil was air-dried and passed through a 20-mesh sieve before use. To verify the ability of *Pseudomonas aeruginosa* K5 to decompose mineral potassium, 2% (w / w) potassium feldspar was added to the soil as the sole supplementary potassium source. The initial physicochemical properties of the mixed soil were: pH 6.5, organic matter 12.5 g / kg, and initial available potassium content 295.6 mg / kg.

[0047] Select "Yunyan 87" tobacco seedlings with uniform growth for transplanting. Fill each pot with 3kg of soil and plant one seedling.

[0048] The experiment included two treatment groups: CK group (control): 50 mL of sterile water was applied as a root drenching at transplanting; subsequent management was normal, with no chemical potassium fertilizer applied. K5 group (treatment): The liquid inoculant prepared in Example 5 was applied as a root drenching at transplanting. Dosage: The liquid inoculant was diluted 1:100, and 50 mL was applied per pot for root drenching (equivalent to approximately 10 mL of inoculant per gram of soil). 6 ~10 7 cfu).

[0049] Plants were harvested after 45 days of cultivation, and plant height, maximum leaf length, and maximum leaf width were measured. Available potassium in the soil was determined using ammonium acetate extraction-flame photometry.

[0050] The results showed that after 45 days, compared with the CK group, the plant height of the K5 group increased by 26.82%, and the available potassium content in the soil increased by 34.29% (Table 5). This proves that the potassium-releasing bacteria agent of the present invention can effectively promote crop growth and significantly improve the availability of potassium in the soil.

[0051] Table 5. Application effects of potassium-solubilizing bacteria in pot experiments

[0052] Example 8 Effects of potassium-solubilizing bacteria on potted plants under salt-alkali stress Poor sandy loam soil was collected, air-dried, and sieved. To verify the ability of *Pseudomonas aeruginosa* K5 to decompose insoluble potassium, 2% (w / w) potassium feldspar powder was added to the soil. The total potassium content of the mixed soil was approximately 18.2 g / kg, but the initial available potassium content was only 98.5 mg / kg (indicating severe potassium deficiency). 3 g of NaCl was added per kilogram of soil, and the pH was adjusted to 8.5 to artificially simulate saline-alkali soil.

[0053] Alfalfa was selected as the observation plant, with 5 seedlings per pot.

[0054] The experiment included two treatment groups: CK group (salt-alkali control): After transplanting, 50 mL of sterile water was applied to the roots of each pot. K5 group (treatment group): After transplanting, the liquid inoculant prepared in Example 5 of this invention was applied. The inoculant was applied by root irrigation, with the liquid inoculant diluted at a ratio of 1:100, and 50 mL was applied to the roots of each pot (to ensure root colonization).

[0055] After 45 days of cultivation, relevant indicators were measured to observe its salt tolerance. Available potassium in the soil was determined by ammonium acetate extraction-flame photometry; proline in the leaves was determined by acidic ninhydrin colorimetric method (the higher the proline accumulation, the stronger the plant's osmotic regulation ability).

[0056] Under salt-alkali stress, alfalfa in the CK group was stunted and its leaves turned yellow (potassium deficiency and salt damage); alfalfa in the K5 group grew at a level close to that of normal soil. Data comparison: the available potassium in the soil of the K5 group was 55.9% higher than that of the CK group, and the proline content in the plant leaves (a stress resistance indicator) was significantly increased. Therefore, K5 not only releases potassium but also helps plants resist salt-alkali stress.

[0057] Table 6. Effects of K5 inoculant on alfalfa growth under salt-alkali stress.

[0058] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0059] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A salt- and alkali-tolerant, drought-resistant strain of *Pseudomonas aeruginosa*, characterized in that... The *Pseudomonas aeruginosa* strain described was named K5, and its classification was named... Pseudomonas chlororaphis It was deposited on October 20, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252259, at Wuhan University, Wuhan, China.

2. The *Pseudomonas aeruginosa* as described in claim 1, characterized in that, It can tolerate a maximum NaCl concentration of 6%, a pH value of 9.0, and a PEG-6000 concentration of 20%.

3. A biological agent, characterized in that, Includes *Pseudomonas aeruginosa* as described in claim 1.

4. The *Pseudomonas aeruginosa* as described in claim 1 or 2, and the biological agent as described in claim 3, characterized in that, It has at least one of the following properties: 1) Potassium solubilization; 2) Alleviating salt and alkali stress in plants; 3) Improve plant stress resistance; 4) Promotes plant growth; 5) Promotes an increase in the above-ground fresh weight of plants; 6) Promotes increased plant height.

5. The application of *Pseudomonas aeruginosa* as described in claim 1 or 2, or the biological agent as described in claim 3, characterized in that... The application is any one of the following: 1) Applications in potassium solubilization or in the preparation of products for increasing the available potassium content in soil; 2) Applications in alleviating salt and alkali stress in plants or in the preparation of products for alleviating salt and alkali stress in plants; 3) Applications in promoting plant growth or in the preparation of products for promoting plant growth; 4) Applications in promoting the increase of aboveground fresh weight of plants or in the preparation of products for promoting the increase of aboveground fresh weight of plants; 5) Applications in promoting plant height increase or in the preparation of products for promoting plant height increase; 6) Applications in improving plant stress resistance or in the preparation of products for improving plant stress resistance.

6. The application as described in claim 5, characterized in that, The plants mentioned are tobacco and alfalfa.

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