Mn-resistant Klebsiella aerogenes and application thereof

Abstract

The invention discloses Mn-resistant klebsiella aerogenes and an application of the Mn-resistant klebsiella aerogenes. The strain number of the Klebsiella aerogenes is C16, and the preservation number of the Klebsiella aerogenes is CGMCC (China General Microbiological Culture Collection Center) NO.34141. The invention further discloses a preparation method of the Klebsiella aerogenes. The bacterial strain is a manganese-resistant and growth-promoting efficient growth-promoting bacterium, can be colonized in feather cockscomb, promotes growth of feather cockscomb in a manganese-polluted environment, promotes enrichment and transfer of feather cockscomb on manganese in soil, and improves the soil environment. The klebsiella aerogenes C16 and the feather cockscomb are combined, so that the manganese-polluted soil can be effectively repaired, and the klebsiella aerogenes C16 has a wide application prospect in the treatment of the manganese-polluted soil.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a Mn-resistant Klebsiella gas-producing bacterium and an application thereof. Background Art

[0002] Manganese, a vital resource and essential material in the national economy, has significantly driven economic development and modernization through its mining and utilization. However, the increased mining, processing, and smelting of manganese ore has generated significant amounts of mining waste. This mining waste is often directly deposited, leading to the entry of manganese into soil and water bodies through atmospheric deposition, rainfall, and surface runoff, severely harming the local ecological environment and human health.

[0003] Excessive Mn can have multiple toxic effects on plant growth. In leaves, Mn poisoning can disrupt the structure and function of chloroplasts, reduce the content of photosynthetic pigments, interfere with the photosynthetic electron transport process, and thus reduce the photosynthetic rate. Furthermore, excessive Mn can disrupt the balance of the plant's antioxidant system, promote the accumulation of reactive oxygen species, trigger membrane lipid peroxidation, and damage cell membrane integrity, severely affecting plant growth and development, and even leading to plant death. Furthermore, Mn can accumulate in the human body through the food chain, posing a threat to human health, particularly neurological damage such as Parkinson's disease.

[0004] In recent years, a variety of physical, chemical, and biological remediation technologies have been developed to address soil Mn contamination. Physical remediation methods, including soil importation, soil replacement, and separation remediation, can reduce soil Mn concentrations in the short term but cannot fundamentally address the long-term accumulation and migration risks of Mn in soils. They also require significant human, material, and financial resources. Chemical remediation techniques, such as soil leaching and chemical immobilization, can reduce the bioavailability of heavy metals to a certain extent. However, the chemical reagents used may negatively impact soil microbial communities and ecosystem functions, and pose the risk of secondary migration or transformation. Phytoremediation, primarily using hyperaccumulators, has become an effective method for remediating heavy metal-contaminated soils due to its safety, reliability, and environmental friendliness. However, the low efficiency of single-plant remediation and its susceptibility to heavy metal toxicity have limited its widespread application. Microbial remediation utilizes the metabolic activities of microorganisms to reduce heavy metals in soils through mechanisms such as passivation, activation, transformation, and detoxification. However, changes in environmental conditions can cause reversal of heavy metal forms, resulting in unstable remediation results and ecological risks.

[0005] Given the limitations of these single remediation technologies, the combined use of plants and microorganisms for synergistic remediation has become an important research direction for improving the remediation efficiency of heavy metal-contaminated soils. Phytoremediation is a long-term, in-situ process, and the addition of microorganisms can significantly enhance the bioavailability of heavy metals, optimize the soil environment, and further improve phytoremediation efficiency. Summary of the Invention

[0006] In view of this, one of the objects of the present invention is to provide a Mn-resistant gas-producing Klebsiella aerogenes (Klebsiella aerogenes), the strain number is C16, the preservation number is CGMCC NO.34141, and it was deposited in the General Microbiology Center of the China Culture Collection Administration on April 9, 2025, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The second object of the present invention is to provide the use of the above-mentioned Mn-resistant gas-producing Klebsiella C16 in colonizing C. aurantiacum or promoting C. aurantiacum seed germination or C. aurantiacum growth or increasing the chlorophyll a, chlorophyll b and carotenoid contents in C. aurantiacum or promoting oxalic acid secretion in C. aurantiacum roots.

[0008] Preferably, the Celosia chinensis is grown in manganese-contaminated soil.

[0009] Preferably, the promoting the growth of C. acerola is promoting an increase in the fresh weight, dry weight, plant height, stem diameter, maximum leaf area, root surface area, number of root tips, root volume, total root length, and root diameter of C. acerola.

[0010] The third object of the present invention is to provide a use of the above-mentioned Mn-resistant gas-producing Klebsiella in combination with Celosia chinensis in the treatment of manganese-contaminated soil.

[0011] Preferably, the C. chinensis is grown in the manganese-contaminated soil, and the Klebsiella is inoculated into the C. chinensis.

[0012] Preferably, the inoculation into C. aurantifolia is performed by adding a bacterial solution to the rhizosphere soil of C. aurantifolia, or by soaking C. aurantifolia seeds in the bacterial solution.

[0013] Preferably, the treatment of manganese-contaminated soil is to reduce the amount of manganese in the soil and / or improve the soil environment.

[0014] Preferably, the reduction of the amount of manganese in the soil is achieved by the enrichment and transport of manganese in the soil by Celosia chinensis.

[0015] Preferably, the improving the soil environment is to increase the soil nutrient content and reduce the soil pH value.

[0016] The present invention provides a Mn-tolerant Klebsiella aeruginosa C16 strain that can promote the growth of C. acerola. When inoculated with C. acerola, C16 colonizes the plant's roots, promoting its growth in Mn-contaminated soil and enhancing its accumulation, transport, and absorption of Mn in the soil, while also improving the soil environment. The C16 strain, Mn, and C. acerola interact with each other, and the combined use of Klebsiella aeruginosa C16 and C. acerola can effectively remediate manganese-contaminated soil, demonstrating broad application prospects in the remediation of manganese-contaminated soil.

[0017] Biological Deposit Description

[0018] Klebsiella aerogenes C16, Latin name Klebsiella aerogenes, is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC NO.34141, the deposit date is April 9, 2025, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the phylogenetic tree of the C16 strain of the present invention;

[0020] Figure 2 The fluorescently labeled C16 strain of the present invention;

[0021] Figure 3 : The growth curves of the C16 strain of the present invention and the C16 strain labeled with GFP;

[0022] Figure 4 The colonization of the strain C16 of the present invention in the rhizosphere of C.

[0023] Figure 5 The growth of C. amurense treated with 0, 1500, and 3000 mg / kg Mn for 60 days after growth in the control group and the C16 inoculation group is shown in the figure.

[0024] Figure 6 The effect of the strain C16 of the present invention on the root system of C. amurense under different Mn treatment concentrations;

[0025] Figure 7 The effect of the strain C16 of the present invention on the chlorophyll content of Celosia chinensis under different Mn treatment concentrations;

[0026] Figure 8 This is the effect of C16 on the secretion of low molecular weight organic acids by the roots of C. amurense under different Mn treatment concentrations in the present invention;

[0027] Figure 9 The effect of the strain C16 of the present invention on the Mn content in C. amurense under different Mn treatment concentrations;

[0028] Figure 10 The effect of the strain C16 of the present invention on the enrichment coefficient and transport coefficient of C. aurantifolia under different Mn treatment concentrations;

[0029] Figure 11 The effect of the strain C16 of the present invention on the total Mn and available Mn content in the rhizosphere soil of Celosia chinensis under different Mn treatment concentrations;

[0030] Figure 12 The effect of different concentrations of oxalic acid on the chemotaxis of C16 in the present invention;

[0031] Figure 13 The effects of different concentrations of oxalic acid on C16 biofilm formation in the present invention;

[0032] Figure 14 The effect of different concentrations of oxalic acid on the growth of strain C16 of the present invention;

[0033] Figure 15 This is the effect of different concentrations of oxalic acid on the total Mn and available Mn content in soil. DETAILED DESCRIPTION

[0034] The present invention will be described in detail below with reference to the following examples. These examples serve only as illustrations and are not intended to limit the scope of application of the present invention. The present invention is not limited to the following embodiments or examples, and any modifications and variations that do not violate the spirit of the present invention are intended to be included within the scope of the present invention. The experimental materials and reagents used in the following examples are commercially available unless otherwise specified. The manganese used in the following examples for manganese stress, manganese treatment, or manganese pollution is MnSO4·H2O, and the manganese concentration is the concentration of manganese in MnSO4·H2O.

[0035] Example 1. Screening, identification and fluorescent labeling of Mn-resistant strain C16

[0036] 1.1 Screening of manganese-resistant strains

[0037] Rhizosphere soils of plants growing normally in Huangguang Village, Lengshui City, Hunan Province (27°45'56"N, 111°28'47"E) and Yangjiaba, Shifeng District, Zhuzhou City (27°52'32"N, 113°3'58"E) were collected to construct bacterial libraries. 2+ A total of 20 strains were screened from the bacterial library on LB solid medium, and then 2+The minimum inhibitory concentration (MIC) of the bacteria was detected on LB solid culture medium with a concentration gradient of 1000, 2000, 3000, 4000, 5000 and 6000 mg / L, and a qualitative detection experiment (the detection method is a conventional experimental method in the art) of the characteristics of secreting IAA, solubilizing phosphate, siderophore and ACC deaminase was performed to detect its growth-promoting ability. According to the test results, the six strains of C16, M33, Y29, Y30, Y42 and ZH6 have relatively high Mn (6000 mg / L) tolerance, and have the characteristics of secreting IAA, solubilizing phosphate, siderophore and ACC, and then the six strains are further quantitatively determined for their growth-promoting characteristics. The quantitative detection results of the IAA production and phosphate solubilizing ability of the six strains are shown in Table 1, among which the four strains of C16, M33, Y30 and Y42 showed good IAA production and phosphate solubilizing ability. Therefore, these four strains were selected for subsequent germination experiments.

[0038] Table 1 Quantitative test results of growth-promoting characteristics of Mn-resistant strains under different Mn treatment concentrations

[0039]

[0040]

[0041] Note: Data are presented as mean ± standard deviation (n = 3). Different lowercase letters in a column indicate significant differences among bacterial treatments (P < 0.05) according to one-way ANOVA.

[0042] Germination test method: Celosia argentea L. seeds with small differences in size and plumpness (purchased from Hubei Yichang Chengmei Agriculture Co., Ltd.) were selected and rinsed with sterile water, soaked in 10% H2O2 solution for 30 minutes, and then rinsed with distilled water three times; the cleaned seeds were dried with sterile paper and soaked in C16, M33, Y30 or Y42 bacterial suspension containing 10 mmol / L MgSO4·7H2O containing 2% (w / w) glycerol. The OD 600 The value was about 1.5, and the mixture was stirred repeatedly for 2 hours. The seeds of the control group were soaked in sterile water. The seeds after the immersion treatment were placed in a sterilized culture dish, and two layers of sterilized 0, 1500, and 3000 mg / L Mn were placed in each culture dish. 2+ Filter paper moistened with the solution, and sterile water as a control. Place 10 Celosia seeds of the same size in each culture dish, with 3 replicates for each treatment, and culture in a dark incubator at 28°C. During the culture process, sufficient water for germination must be maintained, and the humidity of the filter paper must be closely monitored. To ensure that Mn 2+ The concentration remained constant, and 4 mL of the corresponding concentration of Mn was added every day using a pipette. 2+Solution / sterile water. Record the number of germinations starting on the second day and every 24 hours thereafter. End the germination experiment on the seventh day and measure various indicators, including sprout and root length. Germination index = ∑Gt / Dt, where Gt is the number of germinations on day t and Dt is the corresponding number of days to germination. Vitality index = germination index × plant height.

[0043] The results of the germination experiment are shown in Table 2. Mn treatment concentration and strain type significantly affected root length, shoot length, germination index, and vigor index. C16 was least negatively affected by elevated Mn treatment concentrations, effectively alleviating Mn stress and significantly improving the growth performance of C. aurantium, demonstrating strong Mn resistance. Therefore, C16 was selected as the target strain for further research.

[0044] Table 2 Effects of four strains on C. amurense germination at different Mn concentrations

[0045]

[0046]

[0047] Note: Data are presented as mean ± standard deviation (n = 3). Different lowercase letters in a column indicate significant differences among bacterial treatments (P < 0.05) according to one-way ANOVA.

[0048] 1.2 Identification of strain C16 by 16S rRNA system

[0049] The 16S rRNA of C16 strain was identified, and the homology comparison between the obtained sequencing results and the known 16S rRNA gene sequences in GenBank was performed using BLAST software. The strain species were identified based on the high repetitiveness of the sequence with the target functional strain, such as Figure 1 The phylogenetic tree of strain C16 was used to identify it as Klebsiella aerogenes, which was screened from the rhizosphere soil of Yangjiaba, Shifeng District, Zhuzhou City, and its preservation number is CGMCC NO.34141.

[0050] 1.3 GFP fluorescent labeling and detection of C16 strain

[0051] Prepare C16 strain competent cells and then perform electroporation: Place 100 μL of competent cells in an ice bath (thaw for 1-2 minutes), add 1 μL of pBAV1K-T5-gfp plasmid (purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.), mix gently, and place in an ice bath for 30 minutes. Then, add the mixture to an electroporation cuvette, perform three replicates and a control (the control group does not add plasmid, and all other procedures are the same), electroporate (electroporation conditions are 2.5 kV, 6 seconds), add 1 mL of LB medium preheated at 30°C, transfer to a 1.5 mL centrifuge tube, mix thoroughly, and incubate at 37°C, 180 rpm, and shake on a shaker for 60-90 minutes. Centrifuge the culture at 4°C and 8000 rpm for 4 min, discard 500 μL of supernatant, resuspend the remaining 100 μL, aspirate the transformed competent cells and add them to LB solid agar medium containing kanamycin, spread the cells evenly, invert the plate and culture in a 37°C incubator.

[0052] Fluorescence detection: Pick a single colony from the plate and inoculate it into LB liquid culture medium containing kanamycin. Incubate it in a shaker at 28°C and 180 rpm for 24 hours. Pipette 1 mL of the bacterial solution and centrifuge it. After centrifugation, pour out the supernatant and add 50 μL of sterile water to resuspend it and prepare a slide. Observe the fluorescence of the prepared slide under a fluorescence microscope. Figure 2 As shown, bright green fluorescence was found, indicating that the C16 strain successfully achieved fluorescent labeling expression, and the labeled strain was used in subsequent experiments.

[0053] 1.4 Determination of strain growth curve

[0054] GFP-labeled strain C16 and unlabeled strain C16 were inoculated at 1% in LB liquid medium containing kanamycin and LB liquid medium without kanamycin respectively for activation, and then transferred to 100 mL LB liquid flasks respectively. Uninoculated LB liquid medium was used as blank control. Each treatment was repeated three times and cultured in a shaking incubator at 28°C and 180 rpm. The absorbance of the bacterial suspension at a wavelength of 600 nm (OD) was measured by spectrophotometry at different culture times. 600 ). Draw bacterial growth curve: OD 600 The value is the vertical axis, and the culture time is the horizontal axis. Figure 3 The growth curves before and after C16 labeling were essentially identical, with a delay period of 0-2 hours, rapid logarithmic growth from 2-16 hours, and a stationary phase from 16-48 hours. This indicates that C16 has good adaptability to the transferred plasmid and has no significant effect on its growth, meeting the requirements of subsequent experiments.

[0055] Experimental Example 2: Effect of strain C16 on the growth of C. amurense under Mn stress

[0056] Test strain: C16 strain, preservation number: CGMCC NO.34141.

[0057] Test soil: collected from Xiema Town, Beibei District, Chongqing City. The soil type is purple soil. The physical and chemical properties of the soil were determined to be 0.86 g / kg total nitrogen, 0.73 g / kg total phosphorus, 23.09 g / kg total potassium, 8.45 mg / kg ammonium nitrogen, 3.97 mg / kg nitrate nitrogen, 14.77 mg / kg available phosphorus, 123.34 mg / kg available potassium, 13.07 g / kg organic matter, and pH 7.8.

[0058] Test plants: Celosia argentea L., an annual herb of the genus Celosia in the Amaranthaceae family, seeds were purchased from Hubei Yichang Chengmei Agriculture Co., Ltd.

[0059] 2.1 Preparation of soil and bacterial solution and experimental design

[0060] The soil was collected and naturally dried, sieved, and potted. 2+ Solution (the solution is prepared by quantitatively dissolving MnSO4·H2O in deionized water to prepare 10000 mg / L Mn 2+ storage solution), Mn2 + The soil was mixed at dosages of 0, 1500, and 3000 mg / kg to simulate Mn contamination. The prepared soil was aged for 45 days, air-dried, ground, and repotted into flowerpots (20 cm diameter, 14 cm height; 1 kg / pot). After aging, the total and available Mn contents in the soil were measured.

[0061] Prepare LB liquid culture medium, sterilize under high pressure, and cool for use. Inoculate the bacterial solution into a 100mL conical flask containing 50mL LB culture medium at a 1% inoculation rate. Place it in a constant temperature shaker and expand the culture at 28°C to the logarithmic growth phase. Collect the cultured bacterial cells by centrifugation at 4°C, 8000r / min, and 10min. Wash with PBS buffer and collect by centrifugation. Repeat the washing twice. Finally, suspend the collected bacteria in PBS and measure the absorbance of the bacterial solution at a wavelength of 600nm (OD 600 ), the prepared bacterial concentration was about 1×10 8 CFU / mL bacterial suspension for later use.

[0062] A. argentea seeds with plump, uniform seeds were selected and disinfected by soaking in a 0.5% NaClO solution for 30 minutes, then rinsed three times with sterile water and soaked in sterile water for 12 hours. They were then soaked in the bacterial suspension for 30 minutes before being evenly spread on a Petri dish lined with double-layer filter paper. A control group was disinfected, rinsed, and then soaked in sterile water for 13 hours before being evenly spread on a Petri dish lined with double-layer filter paper. Germination was performed in an artificial climate chamber at 25±0.5°C, a 14h / 10h light / dark ratio, and 70% relative humidity. After germination, the seeds were transferred to seedling trays filled with nutrient soil and continued to grow. When the seedlings developed 6-8 true leaves, healthy and uniformly growing A. argentea seedlings were selected and transplanted, one per pot, into pots containing different Mn treatments (Mn concentrations of 0, 1500, and 3000 mg / kg). For each treatment, a control group (uninoculated) and an inoculated group (inoculated with C16 seeds soaked in bacterial solution) were set up, and 7 replicates were performed. All plants were cultured in a constant temperature tissue culture room. To avoid Mn loss, a tray pot was placed under each flower pot to collect seepage water. All plants maintained the same lighting conditions (14h light / 10h dark) during the culture process, and water was added to the pot every 5 days to keep the soil at 50%-70% of the field water capacity. The culture cycle was 60 days, during which soil moisture was monitored regularly to ensure consistency of experimental conditions. At the end of the experiment, samples were collected uniformly on the 60th day, and subsequent testing and analysis of the effects of different Mn treatment concentrations on the growth of Celosia chinensis will be carried out.

[0063] 2.2 Colonization of strain C16 in the rhizosphere of C.

[0064] After the experiment, select C. aurantifolia with the same growth from each treatment group, cut the roots and rinse them with sterile water, and dry the surface moisture with sterile filter paper. Then, the root samples were solidified with embedding agent complex, cut into 10-20 μm slices by freezing microtome, and observed using laser confocal microscope. Figure 4 It can be seen that GFP-labeled C16 successfully colonized into the root tissues of C. chinensis under various Mn treatment concentrations.

[0065] Effects of 2.3C16 strain on the growth of C. amurense under Mn stress

[0066] 2.3.1 Appearance of Atractylodes lancea plants

[0067] After 60 days of growth, Figure 5 It is obvious that C16 treatment promoted the growth of C. aurantifolia plants in each group shown.

[0068] The growth parameters of C. aurantifolia were measured: the plant height and maximum leaf area of C. aurantifolia in each treatment group were measured with a ruler, and the stem diameter of C. aurantifolia in each treatment group was measured with a vernier caliper and recorded, with each measurement repeated three times. After harvest, C. aurantifolia was divided into three parts: roots, stems, and leaves. The roots were gently rinsed with deionized water to remove the attached soil, and the fresh weight of each part was measured. The samples were then placed in an oven at 105°C for 30 minutes, and then dried at 70°C to constant weight, and then the dry weight was measured and recorded. To ensure the accuracy of the data, the growth parameters (fresh weight, dry weight, plant height, stem diameter, and maximum leaf area) of the plants in each group were measured three times. The mean ± standard deviation was used as the final calculation result.

[0069] The results of growth parameters are shown in Table 3. The Mn treatment concentration had a significant inhibitory effect on the growth of C. amurense, and the degree of inhibition increased with the increase of Mn treatment concentration. Under the 1500 mg / kg Mn treatment, the fresh weight, dry weight, plant height, stem diameter, and maximum leaf area decreased by 37.47%, 56.52%, 19.60%, 38.89%, and 34.49%, respectively, while under the 3000 mg / kg Mn treatment, they decreased by 59.37%, 79.71%, 29.05%, 55.56%, and 53.98%, respectively. Inoculation with C16 significantly improved plant growth. Fresh weight, dry weight, plant height, stem diameter, and maximum leaf area increased by 143.4%, 179.71%, 56.86%, 14.81%, and 70.61%, respectively, at 0 mg / kg Mn; by 164.59%, 150.00%, 84.62%, 51.52%, and 31.13%, respectively, at 1500 mg / kg Mn; and by 29.94%, 64.29%, 21.50%, 29.17%, and 30.58%, respectively, at 3000 mg / kg Mn. In summary, C16 demonstrated its ability to promote C. aestivum growth at different Mn concentrations and effectively alleviated the inhibitory effects of Mn stress on C. aestivum growth, demonstrating its strong adaptability and functionality.

[0070] Table 3 Effects of C16 on growth parameters of Celosia chinensis under different Mn treatment concentrations

[0071]

[0072] Note: Data are presented as mean ± standard deviation (n = 3). Different lowercase letters within a column indicate significant differences between treatments (P < 0.05) according to one-way ANOVA. To determine the effects of Mn stress in the substrate and strain C16, a two-way ANOVA was performed. The test results are expressed as test statistics and are shown as follows: ***, significant effect at the P < 0.001 level; **, significant effect at the P < 0.01 level; *, significant effect at the P < 0.05 level.

[0073] 2.3.2 Root morphology and vitality index determination

[0074] Root morphology measurement: C. aurantifolia roots were immersed in a small amount of sterile water to allow them to fully stretch and disperse, avoiding root entanglement to ensure accurate scanning results. A root scanner was used to perform high-precision morphological scans of the C. aurantifolia roots under different treatments, obtaining three-dimensional root morphological data, including total root length, root surface area, number of root tips, root volume, and root diameter.

[0075] Root Vitality Index Determination: Accurately weigh 0.5g of C. aurantifolia root tip sample and place it in a small Petri dish. Mix equal amounts of phosphate buffer and 0.4% TTC solution, and submerge the sample in 10mL of the mixture. The Petri dish is then placed in a 37°C incubator in the dark for 1 hour. The reaction is terminated by adding 2mL of 1mol / L H₂SO₄ (for a blank test, H₂SO₄ is added first, followed by the root sample; the remaining steps remain the same). Gently dry the roots that have developed color with filter paper, add 3-4mL of ethyl acetate and an appropriate amount of quartz sand, and grind. Wash the residue two or three times with a small amount of ethyl acetate, transfer it to a volumetric flask, and dilute to 10mL with ethyl acetate. Compare the color at 485nm, using the blank as a control to measure absorbance. The tetrazolium reduction strength is calculated using a standard curve. Tetrazolium reduction strength = tetrazolium reduction strength (mg) / root weight (g) × time (h).

[0076] The results are as follows Figure 6 As shown in the data, Mn treatment significantly inhibited the root activity and morphology of C. aurantiacum, while inoculation with C16 strain could significantly alleviate the inhibitory effect of Mn stress on the root system. The root surface area, number of root tips, root volume, total root length, root diameter and root activity were all increased. Although the promoting effect of C16 was weakened under high Mn treatment concentration, it was still able to alleviate the inhibitory effect of Mn stress on the root activity and morphology of C. aurantiacum, showing the potential to enhance C. aurantiacum's resistance to Mn stress.

[0077] 2.4 Effect of strain C16 on chlorophyll content in Celosia chinensis

[0078] Select the third, fully expanded, healthy leaf from the middle and upper part of a Celosia argentea plant, clean it, remove the veins, and then cut it into small pieces. Weigh 0.1 g of the chopped leaf and extract it with 5 mL of 95% ethanol for 24-36 hours. The absorbance of the extract is measured at 665, 649, and 470 nm, and the photosynthetic pigment content is calculated according to the following formula.

[0079] C a =13.95OD 665 -6.88OD 649

[0080] C b =24.96OD 649 -7.32OD 665

[0081] C x =(1000OD 470 -2.05C a -114.8C b ) / 245

[0082] Pigment content in leaves (mg / g) = pigment concentration (mg / L) × total volume of extract (mL) × dilution factor / sample mass (g).

[0083] result Figure 7 As shown in the results, the increase of Mn treatment concentration significantly inhibited the chlorophyll synthesis of Celosia argentea. After inoculation with C16, the chlorophyll a, chlorophyll b, carotenoids, total chlorophyll content and the chlorophyll a / b ratio were significantly increased, indicating that C16 can effectively improve the chlorophyll-related indicators of Celosia argentea, optimize the chlorophyll composition ratio, and alleviate the negative impact of Mn stress.

[0084] 2.5 Analysis of low molecular weight organic acids secreted by strain C16 from the roots of C.

[0085] Place the roots of Cinnamomum aviculare in deionized water containing 3 drops of thymol aqueous solution (0.05%, v / v) and soak for 5 minutes. Rinse the roots 3 times with deionized water, place them in a light-proof culture bottle containing sterilized deionized water, and culture them in an incubator for 4 hours. The collected culture solution is filtered through a 0.45μm filter membrane and dried at low temperature using a freeze dryer to obtain root exudate solids. Take an appropriate amount of Cinnamomum aviculare root exudate solids, add them to PBS buffer to dissolve, and prepare a root exudate concentrate. After filtering the concentrate through a 0.45μm filter membrane, use a Shimadzu LC-20AD high performance liquid chromatograph to determine the organic acid components. The low molecular weight organic acids present in the root exudates of Cinnamomum aviculare were analyzed by HPLC. The column was an AQ-C18 (4.6 × 250 mm, Ultimate, USA), and the mobile phase consisted of 20 mmol / L NaH2PO4 (pH 2.60 adjusted with phosphoric acid): methanol (A) at a ratio of 99:1 to methanol (B). The gradient composition was as follows: 0 min, 100% A + 0% B at a flow rate of 0.7 mL / min → 9 min, 100% A + 0% B at a flow rate of 0.7 mL / min → 20 min, 50% A + 50% B at a flow rate of 0.7 mL / min → 23 min, 20% A + 80% B at a flow rate of 0.7 mL / min → 23.1 min, 100% A + 0% B at a flow rate of 0.7 mL / min → stop. The UV detector wavelength was set at 210 nm. Standard organic acids (oxalic acid, malic acid, citric acid, succinic acid, and fumaric acid) were run under the same conditions as the control. Peaks obtained from the root exudate samples were compared to standard controls; major peaks were identified by comparing their retention time and peak area to the retention time of the matched standards.

[0086] The results are as follows Figure 8As shown in Figure 1 (A: standard chromatogram; B: chromatogram of C. amurense sample; C: oxalic acid secretion), C. amurense roots primarily secrete oxalic acid. With increasing Mn concentration, oxalic acid secretion by C. amurense roots also showed an upward trend, increasing from 0.32 mg / L at 0 mg / kg Mn to 0.53 mg / L at 3000 mg / kg Mn, suggesting that C. amurense may alleviate Mn stress by increasing oxalic acid secretion. Inoculation with C16 significantly increased oxalic acid secretion by C. amurense roots. Oxalic acid secretion increased from 0.32 mg / L to 0.40 mg / L, from 0.35 mg / L to 0.47 mg / L, and from 0.53 mg / L to 0.66 mg / L at 0, 1500, and 3000 mg / kg Mn treatments, respectively, representing increases of approximately 25.00%, 34.29%, and 24.53%, respectively. In summary, under different Mn treatment concentrations, C16 can further promote the secretion of oxalic acid from the roots of C. aurantifolia, indicating that C16, Mn, and C. aurantifolia have an interactive effect.

[0087] In summary, C16 can colonize in the rhizosphere of C. aurantifolia. C16 significantly enhanced the tolerance and repair efficiency of C. aurantifolia to Mn stress by promoting root secretion of oxalic acid, improving root morphology and vitality, and increasing chlorophyll content.

[0088] Example 3: Effect of strain C16 on the promotion of Mn absorption by C. amurense under Mn stress

[0089] The test soil, strains, plants, treatments, and sample collection methods were the same as in Example 2.

[0090] 3.1 Effect of C16 on Mn Accumulation in C. amurense

[0091] Select Celosia chinensis with the same growth in each treatment, divide the plant samples into three parts: roots, stems and leaves, grind them separately and pass them through a 0.25mm sieve to obtain a uniform powder. Weigh 0.1g of sample and place it in a 50mL conical flask, add HNO3 and HClO4 in a volume ratio of 5:2, put a small funnel on it, and place the conical flask in a fume hood and let it stand overnight. The next day, place the conical flask on a hot plate and boil it at 180℃ for 30min, then raise the temperature to 250℃ and boil it until the digestion liquid is clear. After the digestion is completed, cool it and transfer the solution to a 50mL volumetric flask. Wash the conical flask 3 times with 1% HNO3 solution, combine the washings and filter it through medium-speed quantitative filter paper, and adjust the volume to 50mL. Use a flame atomic absorption spectrophotometer to determine the Mn content in the supernatant, and calculate its enrichment coefficient (BCF) and transport coefficient (BTF) according to the formula:

[0092] Concentration factor (BCF) = heavy metal content in plants (roots, stems and leaves) (mg / kg) / heavy metal content in soil (mg / kg)

[0093] BTF = heavy metal content in plant aboveground parts (mg / kg) / heavy metal content in plant roots (mg / kg)

[0094] Effects of C16 on Mn content in C. amurense under different Mn treatment concentrations Figure 9 As shown, Mn treatment significantly affected the Mn content in both the aboveground and underground parts of C. aurantifolia. With increasing Mn concentration, both aboveground and underground parts showed a significant increasing trend. After inoculation with C16, the Mn content in the aboveground parts of C. aurantifolia increased by approximately 6.95%, 15.19%, and 17.74% to 115.16, 2627.82, and 6035.06 mg / kg, respectively, at 0, 1500, and 3000 mg / kg Mn treatments. The Mn content in the underground parts of C. aurantifolia increased by approximately 6.67%, 14.62%, and 11.14%, respectively, to 52.18, 1104.89, and 2335.05 mg / kg, respectively. Therefore, C16 inoculation promoted Mn accumulation in C. aurantifolia at all Mn concentrations, and this promoting effect became more pronounced with increasing Mn concentration, indicating an interactive effect among C16, Mn, and C. aurantifolia.

[0095] Effects of C16 on the enrichment coefficient and transport coefficient of C. aurantifolia under different Mn treatment concentrations Figure 10 As shown in the results, after inoculation with C16, the enrichment coefficient of C. aurantifolia reached 1.37, 1.97, and 2.26 at 0, 1500, and 3000 mg / kg Mn treatments, representing increases of approximately 17.09%, 23.12%, and 22.82%, respectively. The transport coefficient of C. aurantifolia increased from 2.20 to 2.21 and from 2.37 to 2.39 at 0 and 1500 mg / kg Mn treatments, respectively. However, at 3000 mg / kg Mn, C16 significantly increased the transport coefficient of C. aurantifolia from 2.44 to 2.56, an increase of approximately 5.70%. This indicates that C16 enhances the Mn accumulation and transport capacity of C. aurantifolia, with the promoting effect significantly enhanced under Mn conditions. This also suggests an interactive effect among C16, Mn, and C. aurantifolia.

[0096] Effects of 3.2C16 on manganese content in rhizosphere soil of Celosia chinensis

[0097] Determination of total manganese content in soil: Weigh 0.25g of sample and place it in a 100mL Erlenmeyer flask. In a fume hood, add 6mL of HNO3, 3mL of HCl, and 2mL of HF in sequence. Mix the sample and digestion solution thoroughly. Place a funnel in the flask and let it sit overnight in the fume hood. The next day, place the flask on a hot plate and boil at 180°C for 30 minutes. Then, raise the temperature to 230-260°C and boil until the solution turns off-white. During the heating process, regularly monitor the solution to ensure complete evaporation. After digestion, cool the flask and rinse it with a small amount of 1% HNO3 using a pipette. Use the residual heat to dissolve any residue adhering to the flask walls. Then, transfer the rinse solution to a 25mL volumetric flask and repeat the above steps with a small amount of 1% HNO3 using a pipette. Finally, dilute to the mark with 1% HNO3, mix thoroughly, and let it sit for 60 minutes. The supernatant solution is then analyzed for Mn content using a flame atomic absorption spectrophotometer.

[0098] Determination of Available Manganese Content in Soil: Weigh 1.0 g of sample and place it in a 100 mL Erlenmeyer flask. Add 2.0 mL of DTPA extract and seal the flask tightly. Shake in a thermostatic shaker at 22°C and 200 rpm for 2 hours. Slowly pour the extract into a 50 mL centrifuge tube and centrifuge at 4°C and 5000 rpm for 10 minutes. Filter the supernatant through medium-speed quantitative filter paper and determine the Mn content in the supernatant within 48 hours using a flame atomic absorption spectrophotometer.

[0099] The results are as follows Figure 11 As shown in the data, after inoculation with C16, the total Mn content and the available Mn content in the soil decreased, indicating that C16 played an important role in enhancing the absorption of Mn by plants and soil remediation by regulating the bioavailability of Mn in the soil, especially showing stronger remediation potential in high-concentration Mn pollution environments.

[0100] 3.3 Determination of soil nutrient content

[0101] Rhizosphere soil samples were collected from C. aurantifolia plants of similar growth in each treatment group and their physical and chemical properties were determined. The samples were air-dried, passed through a 100-mesh nylon sieve, and stored for later use.

[0102] pH value: measured by potentiometric method;

[0103] Soil organic matter (SOM): potassium dichromate volumetric method-external heating method

[0104] Soil total nitrogen (TN): determined by semi-micro Kelvin method;

[0105] Soil total phosphorus (TP): NaOH fusion-molybdenum antimony countercolorimetry method;

[0106] Soil total potassium (TK): NaOH fusion-flame photometry;

[0107] Soil ammonium nitrogen (NH4-N): 0.01 mol / L CaCl2 extraction-indophenol blue colorimetric method;

[0108] Soil nitrate nitrogen (NO3-N): 0.01 mol / L CaCl2 extraction-UV spectrophotometer method;

[0109] Soil available phosphorus (AP): 0.5 mol / L NaHCO3 extraction-molybdenum antimony colorimetric method;

[0110] Soil available potassium (AK): NH4OAc extraction-flame photometry.

[0111] As shown in Table 4, no significant differences were observed in soil nutrient content between C16 inoculation and total nitrogen, total phosphorus, and total potassium (P>0.05). Regarding available nutrient content, C16 inoculation significantly increased ammonium nitrogen and decreased nitrate nitrogen at both 1500 and 3000 mg / kg Mn treatments. Available phosphorus was positively correlated with Mn concentration and increased with increasing Mn concentration. At 3000 mg / kg Mn, C16 inoculation increased available phosphorus from 18.38 mg / kg to 19.33 mg / kg, an increase of approximately 5.16%. Available potassium slowly increased with increasing Mn concentration, and Mn concentration significantly affected it (P<0.01). Regarding other soil parameters, C16 inoculation significantly decreased pH, with extremely significant effects of Mn concentration, C16, and their interaction on pH (P<0.001). However, C16 inoculation had no significant effect on soil organic matter (P>0.05), while Mn treatment concentration had a significant effect on organic matter content (P<0.01), and the interaction between the two was also insignificant (P>0.05). In summary, C16 plays an important role in improving the soil environment and enhancing plant tolerance to Mn stress by regulating soil available nutrient content and pH.

[0112] Table 4 Effects of C16 on the physical and chemical properties of rhizosphere soil of Celosia chinensis under Mn stress

[0113]

[0114] Example 4: Oxalic acid-mediated C16 ecological behavior and soil manganese form regulation mechanism

[0115] The test soil and strain were the same as those in Example 2. It was determined that the main organic acid present in the root exudates of C. aurantifolia was oxalic acid, and that C16 promoted the secretion of oxalic acid from the roots of C. aurantifolia. Therefore, the mechanism by which C16 and C. aurantifolia enhance soil remediation was studied. This included experiments on the effects of different concentrations of oxalic acid on C16 chemotaxis, biofilm formation, and growth, as well as on the effects of different concentrations of oxalic acid on total and available manganese content in soil. The experimental methods are not described in detail here. The results are as follows:

[0116] 4.1 Effects of different oxalic acid concentrations on C16 chemotaxis

[0117] Effects of different concentrations of oxalic acid on C16 chemotaxis Figure 12 As shown in the figure, oxalic acid and Mn treatment concentrations have a significant effect on the chemotaxis of C16. As the oxalic acid concentration increases, the chemotaxis of C16 is significantly enhanced, and the increase is more prominent under the Mn treatment concentration. When treated with 3000 mg / kg Mn, oxalic acid increases the number of C16 chemotaxis from 0.84×10 6 The CFU / mL increased to 1.94×10 6 The CFU / mL increased by approximately 2.31 times. This indicates that the effect of oxalic acid on enhancing C16 chemotaxis was more significant under higher Mn treatment concentrations. The interactive effect between oxalic acid and Mn treatment concentration was extremely significant (P < 0.001). Oxalic acid's activation of C16 chemotaxis was more significant in a high Mn environment, possibly driving the chemotactic behavior of the strain by regulating the rhizosphere microenvironment.

[0118] 4.2 Regulatory effects of different concentrations of oxalic acid on C16 biofilm formation

[0119] Effects of different concentrations of oxalic acid on C16 biofilm formation Figure 13 As shown in the figure, oxalic acid and Mn treatment concentrations had a significant effect on the amount of biofilm formed by strain C16. Under different Mn treatment concentrations, as the oxalic acid concentration increased from 0 mg / L to 0.7 mg / L, the amount of biofilm formed gradually increased and reached a maximum at an oxalic acid concentration of 0.7 mg / L. Under 1500 mg / kg Mn treatment, the biofilm formation amount was 1.77 (OD 595 ), increased by about 19.47%, and was 2.24 (OD 595 ), an increase of approximately 51.14%. The interaction effect between oxalic acid and Mn treatment concentration was extremely significant (P < 0.001), indicating that increasing Mn treatment concentration enhanced the effect of oxalic acid on C16 biofilm formation, especially at higher Mn treatment concentrations, where the promoting effect of oxalic acid on biofilm formation was more pronounced.

[0120] 4.3 Effects of different oxalic acid concentrations on C16 growth

[0121] The growth curves of C16 at different oxalic acid concentrations are shown in Figure 2. Figure 14 As shown. Under 0 mg / kg Mn treatment ( Figure 14 A), under the conditions of oxalic acid concentration of 0.3 mg / L and 0.5 mg / L, C16 reached 1.17 (OD 600 ), close to the growth level of the control group, indicating no significant inhibitory effect. When the oxalic acid concentration was 0.7 mg / L, the growth of C16 showed a more obvious improvement. At 24 h, it reached 1.23 (OD 600 ), which was significantly higher than that of other experimental groups. This shows that 0.7 mg / L oxalic acid concentration has a promoting effect on the growth of C16. Under 1500 mg / kg Mn treatment ( Figure 14 B), oxalic acid has a certain promoting effect on the growth of C16, but compared with the 0 mg / L Mn treatment, the overall growth is slightly slowed down. Under the condition of oxalic acid concentration of 0.7 mg / L, the growth of C16 is significantly improved, and the OD value is 1.20 at 24 h. 600 ), which is the highest value among all oxalic acid concentration groups. When the oxalic acid concentration is 0.3mg / L and 0.5mg / L, the growth rate of strain C16 is 1.11 (OD 600 ) and 1.18(OD 600 ), with little difference from the 0mg / L oxalic acid group. Under 3000mg / kg Mn treatment ( Figure 14 C), the growth of C16 was significantly inhibited, but with the increase of oxalic acid concentration, the inhibitory effect on growth decreased. When the oxalic acid concentration was 0.3 mg / L and 0.5 mg / L, the growth of C16 was 0.88 (OD 600 ) and 0.93(OD 600 ), which were higher than 0.83 (OD 600 When the oxalic acid concentration was 0.7 mg / L, the OD 600 ) was significantly higher than that of the other oxalic acid concentration groups. It can be seen that the promoting effect of oxalic acid on C16 growth increased with the increase of Mn treatment concentration, especially under the treatment of 3000 mg / kg Mn, the effect was most significant when the oxalic acid concentration was 0.7 mg / L.

[0122] 4.4 Regulation of soil available manganese content by different concentrations of oxalic acid

[0123] The results of the effects of different concentrations of oxalic acid on the available Mn content in soil are as follows: Figure 15As shown in the data (A: 0 mg / LMn; B: 1500 mg / L Mn; C: 3000 mg / L Mn), different concentrations of oxalic acid under Mn stress significantly affected the available Mn content in the soil. As the oxalic acid concentration increased from 0 mg / L to 0.7 mg / L, the available Mn content showed an upward trend. This indicates that increasing oxalic acid concentration can increase the available Mn content. This suggests that oxalic acid may alleviate Mn stress by complexing with Mn ions and can promote the release of available Mn under different Mn stress levels, further confirming its positive role in the remediation of Mn-contaminated soils.

[0124] The above results indicate that the synergistic mechanism of the combined effect of C16 and C. aurantium on the remediation of Mn-contaminated soil includes that Mn promotes the secretion of oxalic acid by the C. aurantium roots, and oxalic acid enhances the chemotaxis, biofilm formation and growth of C16, thereby significantly improving the colonization and function of C16 in the C. aurantium rhizosphere (C16 also promotes the secretion of oxalic acid by the C. aurantium roots). At the same time, oxalic acid promotes the transformation of Mn into an exchangeable state, thereby promoting the morphological transformation and bioavailability of soil Mn. Therefore, the interaction among C16, Mn and C. aurantium promotes the combined effect of C16 and C. aurantium to effectively enhance the remediation of manganese-contaminated soil.

[0125] 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. A Mn-resistant Klebsiella aerogenes, characterized in that The strain number is C16, the preservation number is CGMCC NO.34141, 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. Use of the Mn-resistant gas-producing Klebsiella according to claim 1 in colonizing C. acerola, promoting C. acerola seed germination, promoting C. acerola growth, increasing the content of chlorophyll a, chlorophyll b and carotenoids in C. acerola, or promoting oxalic acid secretion in C. acerola roots.

3. The use according to claim 2, characterized in that The Celosia chinensis grows in manganese-contaminated soil.

4. The use according to claim 2, characterized in that The method for promoting the growth of C. amurense is to promote the increase of the fresh weight, dry weight, plant height, stem diameter, maximum leaf area, root surface area, number of root tips, root volume, total root length and root diameter of C. amurense.

5. Use of the Mn-resistant Klebsiella aeruginosa according to claim 1 in combination with Celosia chinensis in the treatment of manganese-contaminated soil.

6. The use according to claim 5, characterized in that The Cinnamomum aviculare is grown in the manganese-contaminated soil, and the Klebsiella is inoculated into the Cinnamomum aviculare.

7. The use according to claim 6, characterized in that The inoculation into C. amurense is performed by adding bacterial solution to the rhizosphere soil of C. amurense, or by soaking C. amurense seeds in bacterial solution.

8. The use according to claim 5, characterized in that The treatment of manganese-contaminated soil is to reduce the amount of manganese in the soil and / or improve the soil environment.

9. The use according to claim 8, characterized in that The reduction of the amount of manganese in the soil is achieved by the enrichment and transportation of manganese in the soil by Celosia chinensis.

10. The use according to claim 8, characterized in that The improving of soil environment is to increase soil nutrients and reduce soil pH.

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