Acid-resistant klebsiella pneumoniae and application thereof

Abstract

The invention discloses acid-resistant klebsiella pneumoniae and an application thereof. The strain number of the acid-resistant klebsiella pneumoniae (Klebsiella pneumoniae) is Y41, and the preservation number of the acid-resistant klebsiella pneumoniae (Klebsiella pneumoniae) is CGMCC (China General Microbiological Culture Collection Center) NO.34143. The klebsiella pneumoniae-like Y41 disclosed by the invention can be used for promoting the germination of corn seeds; y41 and metabolites thereof can improve acid soil and promote photosynthesis, oxidation resistance and growth of corn in the acid soil, Y41 and metabolites thereof have a synergistic effect with corn, the pH value of the soil can be better increased, the content of aluminum ions in the soil can be better reduced, and therefore Y41 and metabolites thereof can be combined with corn to be used for improving and treating the acid soil.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to an acid-resistant Klebsiella pneumoniae and an application thereof. Background Art

[0002] Approximately half of the world's arable and potentially arable land is on acidic soils, primarily in tropical and subtropical regions. This has led to soil acidification becoming a global ecological and environmental problem. In China, the total area of ​​acidic soils with a pH below 6.5 is approximately 3.111 million square kilometers, accounting for 32.4% of the land area. In Chongqing, during the Second National Soil Survey (1979-1985), acidic soils with a pH below 5.5 accounted for only 5.2% of the arable land area. However, by 2017-2018, this proportion had increased to 26.7%.

[0003] Soil acidification accelerates the loss of basic cations such as calcium, magnesium, potassium, and sodium, leading to a decrease in soil fertility. Soil pH can affect plant growth and health by limiting nutrient bioavailability. On the other hand, trivalent aluminum (Al 3+ ) and manganese (Mn 2+ ) has a significantly increased solubility in strongly acidic soils. Aluminum toxicity is considered a major factor limiting plant growth by inhibiting root elongation, and the concentration of active aluminum in acidic soils is predicted to increase significantly with decreasing pH. This may hinder and damage plant roots, weakening plant productivity.

[0004] Currently, the main methods for improving acidic soils include chemical, physical, and biological amendments. Chemical amendment is a common soil improvement method that primarily neutralizes soil acidity by applying alkaline substances such as lime, effectively buffering soil acidity. While chemical remediation can quickly and effectively neutralize acidic soil pollutants, it also has some drawbacks, such as excessive application, which can negatively impact the soil environment and damage microbial ecosystems. Physical amendment mitigates the effects of soil acidification by modifying the physical properties of the soil through physical means, such as deep plowing, turning over, and washing. These methods dilute acidic substances in the soil and mix them with the topsoil, thereby reducing soil acidity. These methods are effective for improving small areas but are not suitable for large-scale soil improvement. Microbial amendment promotes the growth and activity of soil microorganisms, increases soil organic matter and nutrient content, improves soil structure, and enhances water retention, thereby improving soil fertility and productivity. Furthermore, microbial remediation can degrade harmful substances, such as pesticides and fertilizers, reducing their impact on the soil and the environment. Compared with traditional chemical remediation technologies, microbial remediation offers advantages such as low cost and minimal environmental impact. The plant rhizosphere is the primary site for microbial growth and interaction. Microorganisms play a key role in underground ecosystems, promoting plant absorption and growth by influencing carbon and nutrient cycles. Plants and microorganisms work synergistically to further increase the pH of acidic soils.

[0005] Therefore, screening rhizosphere growth-promoting bacteria that can improve acidic purple soil, exploring their growth-promoting effects on plants, and constructing an ecological restoration model based on plant-microorganism synergy will not only help improve acidic soil and enhance the quality of cultivated land, but also provide theoretical support and technical paths for achieving a virtuous cycle in soil ecosystems. Summary of the Invention

[0006] In view of this, one of the objects of the present invention is to provide an acid-resistant Klebsiella pneumoniae (Klebsiellaquasipneumoniae), strain numbered Y41, deposited with CGMCC NO.34143, and deposited on April 9, 2025 in the General Microbiology Center of the China Culture Collection Administration Committee, with a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The second object of the present invention is to provide the metabolite of the acid-resistant Klebsiella pneumoniae Y41, that is, the supernatant obtained by centrifuging the Klebsiella pneumoniae in LB liquid culture medium is the metabolite.

[0008] The third object of the present invention is to provide the use of the acid-resistant Klebsiella pneumoniae Y41 in promoting corn seed germination.

[0009] Preferably, the corn seeds germinate in an acidic environment; and the promoting of corn seed germination is promoting an increase in the sprout length and / or fresh weight of the corn seeds.

[0010] The fourth object of the present invention is to provide the use of the above-mentioned Klebsiella pneumoniae-like bacteria and / or the above-mentioned metabolites in improving acidic soil.

[0011] Preferably, the acidic soil is improved by increasing the pH value of the soil, reducing the aluminum ion content in the soil and / or increasing the nutrients in the soil, and the nutrients include alkaline nitrogen, available potassium and / or available phosphorus.

[0012] A fifth object of the present invention is to provide a use of the aforementioned Klebsiella pneumoniae-like bacteria and / or metabolites thereof for promoting photosynthesis, antioxidant capacity, and / or growth of corn. The corn is grown in acidic soil, and the Klebsiella pneumoniae-like bacteria are inoculated into the soil and / or metabolites of the Klebsiella pneumoniae-like bacteria are added to the soil.

[0013] Preferably, the promotion of corn growth is to promote an increase in corn plant height, stem diameter, aboveground fresh weight, aboveground dry weight, underground fresh weight, underground dry weight, root length, root surface area, root volume and / or root tip number.

[0014] A sixth object of the present invention is to provide the use of the aforementioned Klebsiella pneumoniae-like bacteria and / or the aforementioned metabolites in combination with corn for the treatment of acidic soil. Corn is grown in acidic soil, and Klebsiella pneumoniae-like bacteria are inoculated into the soil and / or metabolites of Klebsiella pneumoniae-like bacteria are added to the soil.

[0015] Preferably, the treatment of acidic soil is to increase the pH value of the soil and / or reduce the aluminum ion content in the soil.

[0016] The present invention provides an acid-resistant Klebsiella pneumoniae Y41 strain. This strain can promote corn seed germination; this strain and its metabolites can improve acidic soil, including increasing soil pH, reducing soil aluminum ion content, and increasing the content of nutrients such as alkaline-hydrolyzable nitrogen, available potassium, and available phosphorus in the soil. This strain and its metabolites can also promote corn photosynthesis, antioxidant capacity, and growth in acidic soil. Y41 and its metabolites have a synergistic effect with corn. The combination of Y41 and corn can effectively improve the pH value and aluminum ion content of acidic soil, and has broad application prospects in acidic soil management.

[0017] Biological Deposit Description

[0018] Acid-resistant Klebsiella pneumoniae Y41, Latin name Klebsiella quasipneumoniae, is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No.34143, the deposit date is April 9, 2025, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effects of the three strains Y31, Y41 and Y43 of the present invention on the pH value of the culture medium;

[0020] Figure 2 is the phylogenetic tree of the Y41 strain of the present invention;

[0021] Figure 3 is the growth curve of the Y41 strain of the present invention;

[0022] Figure 4 The improvement effect of the Y41 strain of the present invention on acidic purple soil;

[0023] Figure 5 The improvement effect of the metabolites of the Y41 strain of the present invention on acidic purple soil;

[0024] Figure 6 The effects of the Y41 strain and its metabolites on the photosynthetic indexes of corn;

[0025] Figure 7 The effects of the Y41 strain and its metabolites on corn biomass;

[0026] Figure 8 The effects of the Y41 strain and its metabolites on the root development of corn;

[0027] Figure 9 This is a diagram of the corn root system under the action of the Y41 strain and its metabolites of the present invention;

[0028] Figure 10 The effect of the Y41 strain of the present invention on above-ground and underground osmotic regulating substances in corn;

[0029] Figure 11 The effect of the metabolites of the Y41 strain of the present invention on the above-ground and underground osmotic regulating substances of corn;

[0030] Figure 12 The effect of the Y41 strain of the present invention on MDA and SOD in the aboveground and underground parts of corn;

[0031] Figure 13 The effect of the metabolites of the Y41 strain of the present invention on MDA and SOD in the aboveground and underground parts of corn;

[0032] Figure 14 Effects of Y41 strain and its metabolites of the present application on acidic purple soil before planting corn;

[0033] Figure 15 Effects of Y41 strain and its metabolites of the present application on acidic purple soil after planting corn. DETAILED DESCRIPTION

[0034] The present application will be described in detail below with reference to the examples, which are merely illustrative and not restrictive of the scope of the present application. The present application is not limited to the following embodiments or examples, and any modification and variation made without departing from the spirit of the present application shall be included in the scope of the present application. The experimental materials or reagents used in the following examples are commercially available unless otherwise specified.

[0035] Example 1, screening and identification of acid-tolerant strain Y41

[0036] 1.1 Screening of acid-tolerant strain

[0037] Rhizosphere soil of normally growing plants was collected in Huangguang Village (27°45'56"N, 111°28'47"E) of Lengshui City, Hunan Province, Yangjiaba (27°52'32"N, 113°3'58"E) of Shifeng District, Zhuzhou City, and Xikou Town (28°35'44"N, 108°55'4"E) of Xiushan Tujia and Miao Autonomous County, Chongqing City, and bacterial libraries were constructed, respectively. The strains were activated, and single colonies were picked and cultured in 50 mL LB liquid medium with pH 4 (pH value of the medium was adjusted by 5 mol / L HC1) for 48 h, and then the OD value and pH value of the medium were detected. According to the results, 11 strains that were both acid-tolerant and could increase the pH value of the medium (could increase the pH value of the medium to above 6 (6.41-7.02), and the OD value was between 1.7 and 2.1) were screened out. Then, qualitative detection experiments (detection methods were conventional experimental methods in the art) of phosphorus-dissolving, IAA-secreting, iron carrier-producing, and nitrogen-fixing properties of the 11 strains were performed to detect their growth-promoting ability. The detection results showed that Y31, Y41, and Y43 strains simultaneously had the properties of phosphorus-dissolving, IAA-secreting, iron carrier-producing, and nitrogen-fixing. Therefore, Y31, Y41, and Y43 were subjected to subsequent experiments. 600 600

[0038] The three strains screened were inoculated in LB liquid medium with initial pH 4 (acidic condition, pH value was adjusted by 5 mol / L HC1) and pH 7 (neutral condition, pH value was adjusted by 5 mol / L HC1) (activated strains, single colonies were picked and inoculated in 50 mL LB liquid medium), and cultured at 28°C for 48 h under shaking, and the pH change trend of the medium at different time was recorded, and the results are shown in Table 1. Figure 1 ​​As shown, all three strains were able to increase the pH of the acidic and neutral medium and reach alkalinity after 24 h.

[0039] 1.2 Identification of strain Y41 by 16S rRNA system

[0040] After 16S rRNA identification of the three strains, the 16S rRNA sequences of strains Y31, Y41, and Y43 were compared, and the strains were identified based on the strains with the highest sequence repetition with the target functional strain. Strain Y41 was Klebsiella quasipneumoniae, and the phylogenetic tree was as follows: Figure 2 The results show that Y31 is Serratia nematodiphila, and Y43 is Enterobacter mori. Both strains were isolated from peanut rhizosphere soil in Huangguang Village, Lengshuijiang City (27°45'56"N, 111°28'47"E). Because Enterobacter mori and Serratia nematodiphila have potential pathogenicity in crops such as mulberry, kiwifruit, and strawberry, Y41 (accession number: CGMCC NO. 34143) was selected as the target strain for subsequent experiments.

[0041] 1.3 Determination of the growth curve of Y41 strain

[0042] The growth curves of strain Y41 in LB medium at pH 4 and pH 7 are shown in Figure 2. Figure 3 As shown in the figure, the growth curves of strain Y41 showed significant differences under different pH conditions. Under neutral conditions of pH 7, the strain grew faster, entered the exponential growth phase after 4 hours, and reached the stable phase after 24 hours. 600 Finally, it stabilized at around 2.25. Under acidic conditions of pH 4, the growth of the strain was inhibited to a certain extent, the exponential growth phase was delayed, and the growth rate was slow, but it could eventually reach an OD close to 2.1 after 48 hours. 600 In contrast, neutral conditions were more conducive to its growth, indicating that strain Y41 had higher metabolic efficiency in a neutral environment.

[0043] Example 2: Effects of Y41 strain and its metabolites on soil and corn growth

[0044] Test soil: Acidic purple soil collected from Nanchuan District, Chongqing (29°07'57.0"N, 107°01'37.0"E). Large stones, residual plastic film, and plant roots were removed from the soil samples. The soil samples were passed through a 2 mm sieve and air-dried for later use. The physical and chemical properties of the test soils are shown in Table 1.

[0045] Table 1 Basic physical and chemical properties of the tested soil

[0046]

[0047] Note: Values ​​represent mean ± standard deviation.

[0048] The test corn seeds were selected from Zhengdan 985 and National Approved Yu 20000009 bred by the Institute of Grain Crops, Henan Academy of Agricultural Sciences.

[0049] Test strain: Y41 strain (deposit number: CGMCC NO.34143).

[0050] 3.1 Germination experiment and soil culture experiment

[0051] 3.1.1 Germination experiment

[0052] The strain Y41 was cultured in a constant temperature shaker at 28°C for about 12 hours, centrifuged at 10000 rpm for 10 minutes, the supernatant was removed, the cells were shaken and then prepared with sterile water to a volume of 1×10 8 A bacterial suspension with a CFU / mL concentration was prepared. Select corn seeds with minimal variability and rinse them with sterile water. Then, soak them in a 10% hydrogen peroxide solution for 30 minutes. The rinsed seeds were blotted dry with sterile paper towels and soaked in the bacterial suspension separately, repeatedly shaking for 2 hours. A control group of seeds was soaked in sterile water. The soaked seeds were placed in sterilized Petri dishes. Each dish contained two layers of filter paper moistened with sterile water at pH 4.3 or pH 7 (adjusted with HCl or NaOH). Sterile water served as the control. Fifteen uniformly sized corn seeds were placed in each dish, with three replicates per treatment. The seeds were incubated in a dark incubator at 28°C. The seeds were kept adequately hydrated, and the filter paper was monitored for moisture. Water was replenished daily with a fixed amount (4 mL) of sterile water (pH 4.3 and pH 7). The number of germinations was recorded starting on the second day, and the germination rate was recorded every 24 hours. The germination experiment was concluded on the sixth day, and various indicators such as sprout length and fresh quality were measured.

[0053] As shown in Table 3, under acidic stress and neutral environments, the sprout length and fresh weight of corn seeds in the Y41 treatment group were higher than those in the control, indicating that Y41 can promote corn germination under both acidic and neutral conditions.

[0054] Table 3 Effect of Y41 on corn germination

[0055]

[0056] Note: Values ​​represent mean ± standard deviation.

[0057] 3.1.2 Soil culture experiment

[0058] Preparation of bacterial suspension: After the strain stored at -80℃ was inoculated into solid culture medium for activation, a single colony was inoculated into 500mL culture medium and cultured for 8h. The OD value was measured. 600 After centrifugation, discard the supernatant and add sterile water to prepare OD 600 =1 concentration gradient bacterial suspension;

[0059] Preparation of supernatant: After the strain stored at -80°C is inoculated into solid culture medium for activation, a single colony is inoculated into 500 mL of culture medium and cultured for 8 hours. The supernatant is retained after centrifugation.

[0060] Bacterial suspension test group: 10 g of sterilized soil was weighed and placed in a 100 mL Erlenmeyer flask for sterilization. 30 mL of the bacterial suspension was added to each flask under a sterile operating table. An equal amount of sterile water was added to the flask as a control. Two treatments were conducted, with three replicates per treatment. The flask and the soil-bacterial suspension mixture were shaken at 28°C (200 rpm) for 2 h to reach equilibrium. After equilibrium, the mixture was transferred to a centrifuge tube and centrifuged at 11,000 g for 5 min. The supernatant (unattached to bacteria) was discarded. Then, 20 mL of sterile water was added to the centrifuge tube under a sterile operating table (the control group was treated similarly). The suspension was shaken at 28°C (200 rpm) for 2 h and then stored for 1, 3, and 7 days. The pH of the suspension was measured using a PHS-3C pH meter. The soil was dried and stored for other physical and chemical property measurements.

[0061] Supernatant test group: 10g of sterilized soil was weighed and placed in a 100mL Erlenmeyer flask for sterilization. 5mL (low concentration) and 15mL (high concentration) of the supernatant metabolic solution were added to the flask under a sterile operating table. Sterilized LB was then added to the flask to make up to 30mL (the control group received 30mL of sterilized LB). Three treatments were performed, with three replicates per treatment. The flasks were shaken at 28°C (200 rpm) for 2h and then allowed to stand for 1, 3, and 7 days. The pH of the suspension was measured using a PHS-3C pH meter. The soil was dried and stored for determination of other physical and chemical properties.

[0062] Methods for determining soil physical and chemical properties: exchangeable aluminum ions were extracted using KCl; available phosphorus was extracted using sodium bicarbonate-molybdenum cake colorimetric method; available potassium was extracted using ammonium acetate-flame photometer method; and alkaline nitrogen was extracted using alkaline diffusion method.

[0063] The soil improvement effects of strain Y41 and its metabolites are as follows: Figure 4 and 5As shown in the results, strain Y41 and its metabolites can significantly improve acidic purple soil and increase the pH value of acidic soil; reduce the concentration of aluminum ions in the soil, reduce the toxic effects of acidic soil on plants; promote the increase of alkaline nitrogen in the soil, improve soil fertility, increase the content of available potassium, and further optimize the nutrient content of the soil. Phosphorus is an important element for plant growth, especially in acidic soil. It promotes the availability of phosphorus in the soil, enables plants to better absorb it, and improves plant growth conditions. There was no significant difference in the physical and chemical properties of the soil measured at different time points (day 1, day 3, and day 7), indicating that the effects of strain Y41 and its metabolites are relatively stable in the short term. In summary, strain Y41 and its metabolites have a good improvement effect on acidic soil.

[0064] 3.2 Potted plant experiment

[0065] The potted experiments included four treatments, one containing a Y41 bacterial suspension (with a control group of distilled water) and one containing a Y41 metabolic solution (with a control group of sterilized LB), with seven replicates per treatment. Each pot was filled with 1 kg of soil and seeded with five germinated corn seeds. After three days of incubation, the remaining seedlings were removed, leaving only three seedlings in each pot.

[0066] Seed preparation: After washing and selecting the seeds, disinfect them with hydrogen peroxide for 20 minutes, soak them in water at room temperature for 6 hours to germinate, and transplant them into treated soil after 3 days of germination.

[0067] Bacterial suspension treatment (Y41): The target bacteria in the liquid culture medium were placed in a constant temperature shaker at 28°C for about 12 hours, centrifuged at 10,000 rpm for 10 minutes, the supernatant was removed, the bacteria were shaken and then prepared with sterile water to a concentration of 1×10 8 A bacterial suspension with a concentration of 100 CFU / mL was added to each pot of soil. A total of 10 pots of soil were prepared. After 5 days of stagnation, the soil from three of the pots was dried and used to measure soil physical and chemical properties before planting corn. Germinated corn was then transplanted into the remaining seven pots. A control group (CK) was treated with sterile water.

[0068] Metabolomics treatment (SQY): After activating the strain stored at -80°C by inoculating it onto solid culture medium, a single colony was inoculated into 500 mL of liquid LB medium (six bottles, 3 L total). The culture was placed in a 28°C constant-temperature shaker for approximately 48 hours. The supernatant, representing the strain's metabolites, was obtained by centrifugation at 10,000 rpm for 10 minutes. 300 mL of the supernatant was added to each pot of soil, for a total of 10 pots. After 5 days of stagnation, soil from three of the pots was dried and used for soil physicochemical property analysis before planting corn. Germinated corn was then transplanted into the remaining seven pots of soil. A control group was treated with sterilized LB (LB).

[0069] Determination of the effect of strains on corn growth: One month after planting, plants with the same growth in each treatment group were taken to measure their plant height, fresh weight, etc. After the photosynthetic index was measured, corn samples were collected and soil was collected for subsequent measurements.

[0070] 3.2.1 Sample collection and preservation

[0071] Plant Sampling: Fresh samples were collected after photosynthetic index determination. A portion of the plants (both aboveground and underground) were immediately wrapped in tin foil, labeled, and quickly frozen in liquid nitrogen. They were then stored at -80°C for subsequent osmotic pressure and enzyme activity analysis. The remaining portion was weighed, and data such as plant height, root length, and fresh weight were recorded. Root scans were then performed to determine corn root system indicators.

[0072] Soil sampling: The first soil sampling was conducted 5 days after soil mixing (to measure the effect of the strain and its supernatant on acidic soil before corn planting); the second soil sampling was conducted 30 days after corn plants were harvested (to measure the effect of the strain and its supernatant on acidic soil after corn planting).

[0073] 3.2.2 Determination of photosynthetic indicators

[0074] The transpiration rate (Tr), photosynthetic rate (Pn), and stomatal conductance (Cleaf) of the leaves were measured using a portable photosynthetic meter in clear weather. Five pots were selected for each treatment, and two well-growing corn plants were selected from each pot. Healthy leaves at the same position of each plant were selected for measurement and these data points were recorded. Figure 6 As shown in the results, the Y41 group showed significantly improved net photosynthetic rate, stomatal conductance and transpiration rate, and the indicators of the SQY group were also higher than those of the CK group and LB group, indicating that Y41 and its metabolites can promote corn photosynthesis.

[0075] 3.2.3 Determination of growth parameters

[0076] After sampling one month after the plants were cultured, plants with the same growth in each treatment group were taken to measure their plant height, fresh weight, dry weight, and stem diameter. Each corn plant was measured three times, and the average ± standard deviation was used as the final calculation result.

[0077] a. Plant height: Use a ruler to measure the length from the point where the rhizomes separate to the highest point of the plant and record it.

[0078] b. Fresh weight: Rinse the corn root repeatedly with deionized water, wipe dry the surface moisture, and measure the weight of the whole corn and record it.

[0079] c. Stem diameter: Use a vernier caliper to measure the diameter of the corn.

[0080] The results are as follows Figure 7As shown in the results, among all treatment groups, the Y41 group performed best, significantly outperforming the control group (CK) and other treatments in terms of plant height, stem diameter, fresh weight and dry weight of above-ground and underground parts. The plant height of the Y41 group was 55.80 cm, which was significantly higher than the CK group (44.80 cm), the SQY group (37.00 cm) and the LB group (25.40 cm). Figure 7 A). The dry weight and fresh weight of the Y41 group were nearly twice that of the CK group. The aboveground dry weight data of Y41 was the most different from that of CK ( Figure 7 CF), the stem diameter of group Y41 was also the highest ( Figure 7 B). LB had the worst performance of all treatments, with significantly lower plant height (25.40 cm) and aboveground fresh weight (0.35 g) than SQY, and also exhibited a trend of underperformance in other parameters. In summary, Y41 and its metabolites can promote maize growth.

[0081] 3.2.4 Scanning analysis of root morphological characteristics

[0082] Clean the plant roots, place the plant roots under a root scanner, soak the roots with sterile water, and disperse the plant roots as much as possible. Analyze and record the root length, root number, and root surface area, and analyze them using winRHIZIO root analysis software. Figure 8 As shown in the figure, the total root length of the Y41 treatment was 377.16 cm, which was much longer than that of the other treatment groups (CK: 248.75 cm, LB: 103.16 cm and SQY: 143.04 cm); the root surface area of ​​the Y41 group (44.78 cm 2 ) and root tip number (758.20) were also significantly higher than those of the other three treatment groups. The root system of the Y41 treatment group showed significant improvement, with a more developed root system, more root hairs, and a wide root distribution area. The root system data of the LB group was the worst, with a root surface area (15.17cm 2 ) was significantly lower than that of the SQY group (23.23cm 2 ) and the other two groups; the root length of the SQY group was nearly 40% longer than that of the LB group. Figure 15 As shown, the root system of the Y41-treated group showed significant improvement, with a more developed root system, more root hairs, and a wide root distribution. The root system of the CK group was relatively short and dense, with a more restricted root distribution and fewer root hairs. Compared with the CK group, Y41 significantly enhanced root growth, demonstrating a stronger expansion capacity. Compared with LB, SQY had a more developed root system and an increased number of root hairs. This indicates that Y41 and its metabolites can promote maize root development.

[0083] 3.2.5 Determination of osmotic regulating substance content

[0084] a. Determination of soluble sugar content. Take 0.1g of leaf tissue ground with liquid nitrogen and follow the reagent instructions and test procedures in the manual. Mix the sample and reagent in a 95°C water bath for 10 minutes. After cooling to room temperature, read the absorbance at 620nm and record it as A1. A blank control is required for soluble sugar determination, and the absorbance value is recorded as A0, where A = A1 - A0. The calculation formula is as follows:

[0085] Soluble sugar content (mg / 8) = 0.718*(A+0.0103) / (W*D)

[0086] In the formula: W is the sample mass, unit: g; D is the dilution factor.

[0087] b. Determination of soluble protein content. Weigh 0.1 g of ground plant material stored at -80°C, add 1 mL of distilled water, mix thoroughly, and then fully extract at room temperature for 1 hour. Then centrifuge at 4000 rpm for 20 minutes. Aspirate 0.01 mL of the supernatant, add 0.5 mL of Coomassie Brilliant Blue, mix thoroughly for 2 minutes, and measure the OD. 595 , using the blank as the control, substitute into the standard curve and calculate the protein content in the sample according to the following formula:

[0088] Protein content in sample (mg / g) = (m*Vt*N) / (m0*Vs*1000)

[0089] In the formula: m is the amount of protein obtained from the standard curve (μg); Vt is the total volume of the extract (mL); m0 is the sample mass (g); Vs is the volume during measurement (mL); N is the dilution factor; 1000 means 1 μg = 1*10-3 mg.

[0090] The above-ground parts are as follows Figure 10 The soluble sugar content of the Y41 treatment group in the aboveground part of the corn was significantly higher than that of the other treatment groups. In contrast, the soluble sugar content of the SQY group was significantly higher than that of its control group, the LB group, which had the lowest soluble sugar content. The measured soluble protein content showed that the soluble protein content of the Y41 group was also significantly higher than that of the other three groups. Figure 11As shown, the Y41-treated group had significantly higher soluble sugars in the underground parts of corn than the other three groups. In contrast, the SQY group had significantly higher soluble sugars than its control group, the LB group, which had the lowest soluble sugar content. Soluble protein content also showed that the Y41 group had significantly higher soluble protein than the other three groups. These results indicate that strain Y41 significantly promoted the accumulation of soluble sugars and soluble proteins in both the aboveground and underground parts of corn, potentially enhancing the plant's osmotic regulation. Compared to the CK, LB, and SQY groups, the Y41 group exhibited a significant advantage in increasing the content of osmotic regulators, suggesting that Y41 may contribute to improving corn's stress resistance. The LB group had the least effect, with the SQY group exceeding the LB group in both soluble sugar and soluble protein. The supernatant contained both enrichment medium (LB) and bacterial metabolites. LB exhibited a significant inhibitory effect on corn, suggesting that bacterial metabolites may, to some extent, promote the production of osmotic regulators.

[0091] 3.2.6 Determination of malondialdehyde (MDA) and superoxide dismutase (SOD) content

[0092] The measurement methods of MDA and SOD were determined according to the methods in the manual and the actual situation. Figure 12 As shown, strain Y41 significantly improved the antioxidant capacity of the aerial part of corn, and enhanced the resistance of corn to oxidative stress by reducing MDA content and increasing SOD activity. Compared with the control group and LB group, the Y41 group had obvious advantages in reducing oxidative damage and increasing antioxidant enzyme activity. Although the SQY group also had a certain antioxidant effect, its effect was not as good as that of the Y41 group. The LB group performed poorly in both MDA content and SOD activity, indicating that its defense against oxidative damage to corn was weak. Figure 13 The results of the four treatments on MDA and SOD in the underground parts of maize show similar increasing and decreasing trends, indicating that Y41 and its metabolites can enhance the antioxidant capacity of plants.

[0093] 3.2.7 Determination of soil pH and aluminum ion content

[0094] The soil pH was measured after mixing soil with water in a ratio of 1:2.5; exchangeable aluminum ions were extracted using KCl. The results of soil pH and aluminum ion values ​​before planting are as follows: Figure 14 As shown, after planting Figure 15 As shown in the data, the pH values ​​of the Y41 group and the SQY group were higher than those of the control, and the aluminum ion content was lower than that of the control; in particular, the pH value of the soil after planting was higher and the aluminum ion content was lower than that before planting, indicating that Y41 and its metabolites have a synergistic effect with corn and can be better used in the improvement of acidic soil.

[0095] In summary, Klebsiella pneumoniae Y41 can promote corn seed germination, Y41 and its metabolites can improve acidic soil, and promote the photosynthesis, antioxidant capacity and growth of corn in acidic soil. Y41 and its metabolites have a synergistic effect with corn, which can better increase soil pH and reduce the aluminum ion content in the soil. Therefore, Y41 and its metabolites can be combined with corn to improve and control acidic soil.

[0096] The conventional techniques and schemes not described in detail in the above embodiments are well known in the art and will not be described in detail here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple variations of the technical scheme of the present invention can be made, and these simple variations all fall within the scope of protection of the present invention.

Claims

1. An acid-resistant Klebsiella pneumoniae (Klebsiella quasipneumoniae), characterized in that The strain number is Y41, the preservation number is CGMCC NO.34143, and it was deposited in the General Microbiology Center of China Culture Collection Administration on April 9, 2025. The preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The metabolite of Klebsiella pneumoniae-like bacteria according to claim 1, characterized in that The metabolite is obtained by the following method: the Klebsiella pneumoniae-like bacteria are cultured in LB liquid culture medium and then centrifuged to obtain the supernatant.

3. Use of the Klebsiella pneumoniae-like bacteria according to claim 1 in promoting corn seed germination.

4. The use according to claim 3, characterized in that The corn seeds germinate in an acidic environment; and the promoting of corn seed germination is promoting an increase in the sprout length and / or fresh weight of the corn seeds.

5. Use of the Klebsiella pneumoniae-like bacteria according to claim 1 and / or the metabolites according to claim 2 in improving acidic soil.

6. The use according to claim 5, characterized in that The acidic soil is improved by increasing the pH value of the soil, reducing the aluminum ion content in the soil and / or increasing the nutrients in the soil, and the nutrients include alkaline-hydrolyzable nitrogen, available potassium and / or available phosphorus.

7. Use of the Klebsiella pneumoniae-like bacteria according to claim 1 and / or the metabolites according to claim 2 in promoting photosynthesis, antioxidant capacity and / or growth of corn.

8. The use according to claim 7, characterized in that The promoting of corn growth is to promote the increase of corn plant height, stem diameter, aboveground fresh weight, aboveground dry weight, underground fresh weight, underground dry weight, root length, root surface area, root volume and / or root tip number.

9. Use of the Klebsiella pneumoniae-like bacteria according to claim 1 and / or the metabolites according to claim 2 in combination with corn in treating acidic soil.

10. The use according to claim 9, characterized in that The acidic soil treatment is to increase the soil pH value and / or reduce the aluminum ion content in the soil.

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