Citrobacter freundii and application thereof

Abstract

The present application relates to the field of microbial technology, and more particularly to a citrobacter freundii and application thereof. The present application provides a citrobacter freundii, wherein the citrobacter freundii is citrobacter freundii (C. Citrobacter freundii ) TR37, and the preservation number is CCTCC NO: M2026452. The strain can tolerate 8000 mg / L of sodium selenite, and can efficiently synthesize biological nano-selenium with small particle size and high stability, and can be applied to heavy metal pollution treatment. The strain and nano-selenium can significantly promote the growth and development of coriander, including physiological parameters such as plant height and biomass. The strain and nano-selenium can improve the physiological metabolism of coriander by regulating the content of carotenoids, promoting the accumulation of soluble sugar, reducing the content of soluble protein and malondialdehyde, and enhancing the activity of antioxidant enzyme system, so as to improve the stress resistance of coriander to cadmium.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a type of Citrobacter freundii and its applications. Background Technology

[0002] Cadmium is a toxic heavy metal widely found in nature and is one of the most abundant heavy metals in soil. With the widespread use of cadmium in battery manufacturing, alloy production, and metallurgy, as well as the improper treatment of waste residue and wastewater, the impact of heavy metal pollution on soil and water resources is becoming increasingly serious.

[0003] Leafy vegetables are a significant part of people's daily diet. Studies have shown that leafy vegetables have a strong ability to absorb and accumulate cadmium, with their cadmium content often exceeding that of the soil in which they grow, and generally higher than that of root and fruit vegetables. Ingesting leafy vegetables with excessive heavy metal content poses a threat to human health. Therefore, controlling soil cadmium pollution and reducing cadmium absorption into edible crop tissues is a major challenge for environmental science. Previous research has identified methods for reducing cadmium in soil as physical, chemical, and bioremediation techniques. However, physical and chemical remediation methods are costly and often cause secondary pollution. Phytoremediation methods within bioremediation, such as phytostabilization, phytovolatile matter, and phytoextraction, are safe and reliable. However, these methods are slow, and phytoremediation typically requires prolonged periods of non-use of arable land, which is not practical for societal and agricultural needs. Therefore, there is a need to develop technologies that can directly reduce cadmium accumulation in the edible parts of crops without interrupting soil use. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned shortcomings of the prior art by proposing a Citrobacter freundii strain and its applications.

[0005] The first objective of this invention is to provide a *Citrobacter freundii*, wherein the *Citrobacter freundii* is *Citrobacter freundii* (… Citrobacter freundii TR37, accession number CCTCC NO: M2026452. Deposited on March 16, 2026 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0006] A second objective of the present invention is to provide a microbial inoculum containing Citrobacter freundii TR37 as described in claim 1.

[0007] A third objective of this invention is to provide an application of Citrobacter freundii TR37 as described above in the reduction of Se(IV).

[0008] The fourth objective of this invention is to provide an application of Citrobacter freundii TR37, as described above, in the preparation of bio-nano selenium.

[0009] The fifth objective of this invention is to provide an application of Citrobacter freundii TR37, as described above, in promoting plant growth.

[0010] The sixth objective of this invention is to provide an application of the combined use of Citrobacter freundii TR37 and nano-selenium as described above in cadmium reduction, wherein cadmium reduction refers to reducing the stress of cadmium ions on plant growth or reducing the cadmium content in plants.

[0011] The seventh objective of this invention is to provide an application of the combined use of Citrobacter freundii TR37 and nano-selenium as described above to promote the growth of coriander under cadmium stress.

[0012] The combined use of Citrobacter freundii TR37 and nano-selenium enhances the cadmium resistance of coriander by regulating the content of photosynthetic pigments and nutrients and increasing the activity of the antioxidant enzyme system in coriander. This includes altering the content and / or activity of any one or more of the following physiological indicators: (1) Carotenoids; (2) Soluble sugars; (3) Soluble proteins; (4) Peroxidase (POD); (5) Superoxide dismutase (SOD); (6) Catalase (CAT); (7) Malondialdehyde (MDA); (8) Total antioxidant capacity (T-AOC).

[0013] Furthermore, coriander was sprayed with nano-selenium on the leaves and irrigated with bacterial solution at the roots.

[0014] This invention provides a *Citrobacter freundii* TR37 strain that is tolerant to selenium and multiple heavy metals, and possesses growth-promoting properties such as inorganic phosphorus solubilization, nitrogen fixation, siderophore production, and IAA production. This strain can tolerate 8000 mg / L sodium selenite and can efficiently synthesize small-particle, highly stable bio-nano-selenium, which can be applied to the remediation of heavy metal pollution. The combined use of this strain and nano-selenium significantly promotes the growth and development of coriander, including physiological parameters such as plant height and biomass. Furthermore, it can improve the physiological metabolism of coriander by regulating carotenoid content, promoting soluble sugar accumulation, reducing soluble protein and malondialdehyde content, and enhancing the activity of antioxidant enzyme systems, thereby improving coriander's resistance to cadmium. The strain of this invention can serve as a functional microbial resource, providing a scientific basis for developing efficient microbial remediation technologies in selenium-rich areas contaminated with cadmium. Attached Figure Description

[0015] Figure 1 The growth of Citrobacter freundii TR37 proposed in this invention on 100 mg / L cadmium chloride (a) and 8640 mg / L sodium selenite (b) plates; Figure 2 This is the phylogenetic tree of Citrobacter freundii TR37 of the present invention; Figure 3 The growth-promoting effect of Citrobacter freundii TR37 in this invention is shown in the following diagrams: a) shows the effect of inorganic phosphorus solubilization, b) shows the effect of nitrogen fixation, d) shows the effect of iron carrier production, and d) shows the effect of IAA production colorimetric reaction. Figure 4 The results of sodium selenite reduction rate determination of Citrobacter freundii TR37 in this invention; Figure 5 This is a scanning electron microscope image of the selenium nanoparticles synthesized by *Citrobacter freundii* TR37 according to the present invention. Figure 6 The image shows the FTIR spectrum of the selenium nanoparticles synthesized by *Citrobacter freundii* TR37 in this invention. Figure 7 The effect of the combined use of Citrobacter freundii TR37 and nano-selenium on the plant height and biomass of Coriander is presented in this invention, where a is the plant height and b is the biomass. Figure 8 The effect of the combined use of Citrobacter freundii TR37 and nano-selenium on the carotenoid content of coriander in this invention; Figure 9 The present invention describes the effect of the combined use of Citrobacter freundii TR37 and nano-selenium on the nutrient content of coriander, wherein a is soluble sugar and b is soluble protein; Figure 10 The effect of the combined use of Citrobacter freundii TR37 and nano-selenium on cadmium content in the aboveground and underground parts of coriander is presented in this invention, where a represents the aboveground parts and b represents the underground parts; Figure 11 The effect of the combined use of Citrobacter freundii TR37 and nano-selenium on the antioxidant enzyme activity and membrane lipid peroxidation of coriander, wherein a is SOD, b is POD, c is CAT, and d is MDA; Figure 12 This invention investigates the effect of the combined use of Citrobacter freundii TR37 and nano-selenium on the total antioxidant capacity of coriander. Detailed Implementation

[0016] The following are specific embodiments of the present invention, which are described in conjunction with the accompanying drawings to further illustrate the technical solutions of the present invention. However, the present invention is not limited to these embodiments.

[0017] It should be emphasized that any equivalent modifications, substitutions, or variations made by those skilled in the art within the scope of the technology disclosed in this invention, based on the core ideas of this invention, should be included within the protection scope of this invention. Furthermore, unless otherwise expressly stated, the experimental procedures used in the various embodiments of this invention are all conventional standard methods in the art, and the reagents and instruments used can be purchased through publicly available commercial channels.

[0018] Example 1: Screening of strains Cadmium- and selenium-tolerant bacterial strains were isolated from soil collected in Xiangyang, Hubei Province. 10 g of soil was added to 50 mL of sterile water and incubated at 37°C and 180 rpm for 30 min with shaking. The culture was then diluted and spread onto LB agar plates. After multiple subculturings and acclimatization, single colonies were picked and cultured in a solution containing 100 mg / L Cd. 2+ In LB solid medium, strains with good growth status were isolated and then inoculated into LB solid medium containing 8640 mg / L sodium selenite. Based on their ability to convert sodium selenite into red nano-selenium, strains were screened to select the best strain. Figure 1 Single colonies of the strain were inoculated into LB liquid medium and cultured until OD. 600 When the concentration is 1, mix it evenly with an equal volume of sterilized 50% glycerol and store it in a refrigerator at -80°C until use.

[0019] LB medium formula (g / L): 10 g peptone, 5 g yeast extract, 10 g sodium chloride, and distilled water to a final volume of 1000 mL.

[0020] Example 2: Identification of the strain Physiological and biochemical tests of strain TR37 were performed according to Bergey's Manual of Bacteriological Identification.

[0021] Physiological and biochemical results of strain TR37

[0022] Genomic DNA was extracted from the strain using the CTAB method. Universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3') were used to amplify 16S rRNA. The PCR amplification reaction system was as follows: ddH2O 8 μL, 2×M5 HiPer plus Taq HiFi PCR mix 10 μL, primer 27F 0.5 μL, primer 1492R 0.5 μL, and template 1 μL. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After comparing the sequencing results with the NCBI database, sequences of strains with high similarity were downloaded, and a phylogenetic tree was constructed using the neighbor-joining method in MEGA 7.0 software to determine the evolutionary relationship of the strains. Figure 2 It can be seen that strain TR37 is related to Citrobacter freundii ( Citrobacter freundii The 16S rDNA of strain TR37 (LMG3246) showed the highest homology, reaching 99.50%. Therefore, based on the physiological and biochemical results of the strain, strain TR37 was identified as *Citrobacter freundii*. The accession number is CCTCC NO: M2026452, and the accession date is March 16, 2026.

[0023] Example 3: Evaluation of the ability of Citrobacter freundii TR37 to decompose inorganic phosphorus, fix nitrogen, produce iron carriers, and produce IAA. 1. Take 1 μL of Citrobacter freundii seed culture and inoculate it with... Pikovskaya If the strain can grow and produce a clear zone on a solid culture medium at 37°C for 48 h, it indicates that the strain has the ability to solubilize inorganic phosphorus.

[0024] Pikovskaya Solid culture medium formula (g / L): 0.5 g yeast extract, 10 g glucose, 0.3 g NaCl, 0.3 g KCl, 0.3 g MgSO4·7H2O, 0.03 g MnSO4·H2O, 0.03 g FeSO4·7H2O, 0.5 g (NH4)2SO4, 5.0 g Ca3(PO4)2, distilled water to a final volume of 1 L, pH adjusted to 7.0~7.5, and 20 g agar powder.

[0025] 2. Streak the seed culture of Citrobacter freundii on Ashby nitrogen-free solid medium and incubate at 37°C for 48 h. If the strain can grow on the medium and produce a clear zone, it indicates that the strain has nitrogen-fixing ability.

[0026] Ashby Nitrogen-Free Solid Culture Medium Formula (g / L): Mannitol 10 g, KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g, CaSO4·2H2O 0.1 g, CaCO3 5.0 g, distilled water to a final volume of 1 L, pH adjusted to 7.0, agar powder 20 g.

[0027] 3. Take 1 μL of seed culture of Citrobacter freundii and inoculate it into the siderophore primary screening solid medium. Incubate at 37℃ for 48 h. If the strain can grow on the medium and produce an orange-yellow transparent zone, it indicates that the strain has the ability to produce siderophores.

[0028] The formulation of the primary screening medium for siderogenetic aspirant is as follows: Reagent a is prepared by dissolving 0.121 g of chromaffin (CAS) in 50 mL of 0.04 mM ferric chloride solution. Reagent b is prepared by dissolving 0.03 g of hexadecyltrimethylammonium bromide in 20 mL of distilled water. Slowly pour reagent a into reagent b and mix thoroughly. Add 10 mL of the colorimetric solution to 90 mL of LB medium to obtain the primary screening medium for siderogenetic aspirant.

[0029] 4. Inoculate *Citrobacter freundii* at a 1% inoculum into LB liquid medium (containing 100 mg / L L-tryptophan) and shake at 37℃ and 180 rpm for 48 h. After the culture is completed, centrifuge the fermentation broth at 8000 rpm for 10 min, take the supernatant and mix it with Salkowski colorimetric solution at a volume ratio of 1:1, react in the dark for 30 min, and observe the color of the reaction solution. If pink appears, it indicates that the strain has the ability to produce IAA.

[0030] Salkowski colorimetric solution formulation: 35% HClO4 and 0.5 M FeCl3 mixed at a volume ratio of 50:1.

[0031] Depend on Figure 3 It is known that Citrobacter freundii TR37 has growth-promoting properties such as inorganic phosphorus decomposition, nitrogen fixation, iron carrier production, and IAA production, which can improve the available nutrients in the soil and promote plant growth.

[0032] Example 4: Evaluation of the tolerance of Citrobacter freundii TR37 to multiple heavy metals and sodium selenite The tolerance of strain TR37 to heavy metals and sodium selenite was assessed using the minimum inhibitory concentration (MIC). Citrobacter freundii TR37 was inoculated onto substrates containing Cd. 2+ (25, 50, 100, 200, 400, 500 mg / L), Pb 2+ (25, 50, 100, 200, 400, 500 mg / L), Zn 2+(25, 50, 100, 200 mg / L), Fe 2+ (25, 50, 100, 200, 400, 500 mg / L), Cu 2+ (25, 50, 100, 200 mg / L), Mn 2+ (25, 50, 100, 200, 400, 500 mg / L), Se 4+ The OD was measured in LB liquid medium at concentrations of 2000, 4000, 8000, 16000, and 32000 mg / L. The medium was incubated at 37 °C with shaking at 180 rpm for 24 h. 600 To assess the growth status of the strain.

[0033] Results of the tolerance of Citrobacter freundii TR37 to heavy metals and sodium selenite

[0034] Example 5: Determination of sodium selenite reduction rate of Citrobacter freundii TR37 Establishment of the sodium selenite standard curve The specific steps are as follows: Accurately weigh 17.29 mg of sodium selenite and dilute to a 100 mL volumetric flask, then shake well. Pipette 0, 0.5, 1, 1.5, 2, and 2.5 mL of the sodium selenite standard solution into test tubes, and then add deionized water to a final volume of 5 mL. Transfer 1 mL of the standard solution from each test tube to a new test tube, add 0.5 mL of 4 M hydrochloric acid and 1 mL of 1 M ascorbic acid, shake well, and let stand at room temperature for 10 min. Measure the absorbance at 500 nm. Plot a standard curve with the concentration of sodium selenite on the x-axis and absorbance on the y-axis. The standard curve for sodium selenite is y = 0.5158x + 0.0446, R0 2 =0.9992.

[0035] Citrobacter freundii TR37 was inoculated at a 1% inoculum in LB liquid medium (containing 0.864 g / L sodium selenite). The medium was incubated at 37°C and 180 rpm for 84 h with shaking. Samples were taken every 12 h, centrifuged at 12000 rpm for 10 min, and 1 mL of the supernatant was accurately aspirated. 0.5 mL of 4 M hydrochloric acid and 1 mL of 1 M ascorbic acid were added, and the mixture was shaken to mix. After standing at room temperature for 10 min, the absorbance was measured at 500 nm. The reduction rate of sodium selenite was calculated using the formula: A = [(C0 - C...]]. t [A / C0]×100%, where A is the sodium selenite reduction rate, C0 (g / L) is the initial concentration of added sodium selenite, and C... t (g / L) represents the concentration of sodium selenite in the supernatant.

[0036] The results of the strain's ability to reduce sodium selenite are shown in Figure 4. Within the culture time range of 0–48 h, the reduction rate of sodium selenite by TR37 increased with increasing culture time. The reduction rate of sodium selenite by strain TR37 showed the greatest change within the 0–12 h range, reaching 81.15 ± 2.57% at 12 h. After 12 h, the reduction rate tended to stabilize, reaching a maximum of 88.65 ± 2.23% at 48 h. This strain exhibits a strong reducing ability for sodium selenite and can be applied to the efficient synthesis of bio-nano selenium.

[0037] Example 6: Scanning electron microscopy analysis of nanometer selenium Citrobacter freundii TR37 was inoculated at a 1% inoculum in LB liquid medium (containing 0.864 g / L sodium selenite) and cultured at 37°C and 180 rpm with shaking for 24 hours. After culture, the precipitate was collected by centrifugation at 12000 rpm for 10 min and washed 2-3 times with sterile water. The washed precipitate was resuspended in sterile water and disrupted twice using a low-temperature ultra-high pressure cell disruptor, followed by filtration through a 0.22 μm filter membrane, and the filtrate was collected. Hexane was added to the filtrate for extraction, retaining the aqueous phase, and the precipitate was collected by centrifugation at 12000 rpm for 10 min. Finally, the precipitate was freeze-dried to obtain selenium nanoparticle powder, which was analyzed by scanning electron microscopy (SEM).

[0038] The scanning electron microscopy (SEM) analysis results of selenium nanoparticles synthesized by *Citrobacter freundii* TR37 are shown in Figure 5. The selenium nanoparticles synthesized by strain TR37 in LB liquid medium containing sodium selenite are uniformly distributed and exhibit a regular spherical shape. The particle size of the synthesized selenium nanoparticles ranges from 99 to 287 nm. The zeta potential is -32.4 ± 0.26 mV, indicating that this strain can synthesize stable bio-selenium nanoparticles.

[0039] Example 7: Infrared Spectroscopic Analysis of Nano-Selenate The nano-selenium prepared in Example 5 was subjected to infrared spectroscopy using the potassium bromide pellet method. Spectroscopic conditions: temperature 15–25 °C, wavelength range 400–4000 cm⁻¹. -1 Step length 4.0cm -1 Scan 3 times.

[0040] The infrared spectral analysis results of selenium nanoparticles synthesized by *Citrobacter freundii* TR37 are shown in Figure 6. The various absorption bands in the FTIR spectrum and their corresponding assignments are as follows: 3427 cm⁻¹ -1 The absorption peak at 2955 cm⁻¹ is due to the stretching vibration of the OH group. -1The absorption peak at 1644 cm⁻¹ represents the sum of the stretching vibrations of the CH moieties in the methyl and methylene groups, primarily located on the protein side chains. -1 The peak at 1546 cm⁻¹ indicates the presence of a C=O amide group (amide I); -1 The peak at 1421 cm⁻¹ corresponds to the bending of the NH₄⁺ plane of protein amide II; -1 The weak, broad peak centered on the 1246 cm⁻¹ includes stretching of CN, bending of CH, and symmetrical stretching of ionized carboxyl groups; -1 The peak at 1045 cm⁻¹ corresponds to the asymmetric stretching vibration of the amide III and OP=O regions, and is one of the characteristics of proteins; -1 The broad peak at the position is located in the typical region of the CO / CC / CN vibrational modes characteristic of polysaccharides and / or proteins. FTIR clearly shows the possible attachment of extracellular polymers such as proteins and polysaccharides on the surface of the bio-nano selenium synthesized by Citrobacter freundii TR37.

[0041] Example 8: Effect of the combined use of Citrobacter freundii TR37 and nano-selenium on the growth of coriander. Select healthy, plump coriander (coriander) seeds and place them in a sterile Erlenmeyer flask. Add a 3% hydrogen peroxide solution and soak for 10 minutes. Rinse repeatedly three times with sterile water and wipe the seeds dry with filter paper. Place 20 sterilized coriander seeds evenly in each plastic dish. Add water daily using a weighing method until constant weight and incubate at room temperature. Thin the seedlings starting three weeks after sowing, leaving 8 seedlings per pot. At weeks 5, 7, and 9, spray the coriander with a nano-selenium foliar spray (10 mL 20 mg / L nano-selenium solution) and / or drench the roots with a bacterial solution (10 mL OD200 solution). 600 After treating the coriander with a bacterial suspension of 1, the plant height was recorded as the length from the rootstock septum to the highest point of the plant when the plant was grown for ten weeks. The coriander was then cut into above-ground and underground parts, and their fresh weight was measured separately.

[0042] Among them, the CK group was the blank group (the soil tested was taken from the planting base of the College of Selenium Science and Engineering of Wuhan University of Light Industry, the soil pH was 6.7, and the soil cadmium content was 50 μg / kg as determined by ICP-MS); Cd group: Cadmium chloride solution was added to the soil of the control group, mixed thoroughly, and left to stand for 2 months to allow the soil to age and reach equilibrium. The cadmium content in the soil was determined to be 2.54 mg / kg by ICP-MS. Cd+Se group: Based on the cadmium treatment group, only the leaves were sprayed with nano-selenium; Cd+TR37 group: In addition to the cadmium treatment group, only the root drench was treated with TR37 bacterial solution. Cd+Se+TR37 group: Based on the cadmium treatment group, nano-selenium leaf spray and bacterial solution root irrigation were carried out.

[0043] Depend on Figure 7 As shown in Figure a, both the application of nano-selenium and the addition of TR37 bacterial solution alone can increase the height of coriander plants. When the two are applied together, the height of coriander plants is 54.15% higher than that of the cadmium-treated group. Figure 7 Figure b shows that, compared with the cadmium-treated group, the aboveground biomass of coriander increased by 43.09%, 43.50%, and 73.09% in the nano-selenium-treated group, the TR37 bacterial solution-treated group, and the combined nano-selenium and TR37 bacterial solution-treated group, respectively, while the underground biomass increased by 28.06%, 27.86%, and 73.79%, respectively.

[0044] Example 9: Effect of combined use of Citrobacter freundii TR37 and nano-selenium on coriander carotenoids Weigh 0.1 g of coriander leaves from each treatment group in Example 7, rinse the surface with deionized water, and dry with filter paper. Add 10 mL of 95% ethanol to a mortar and grind thoroughly until the tissue turns white. Then centrifuge at 4000 rpm for 10 min, and measure the absorbance of the supernatant at 470 nm, 649 nm, and 665 nm. The calculation formula is as follows: Chlorophyll a content: Ca (mg / L) = 13.95 × A 665 -6.88×A 649 Chlorophyll b content: Cb (mg / L) = 24.96 × A 649 -7.32×A 665 Carotenoid content: Car (mg / L) = (1000 × A) 470 -2.05×Ca-114.8×Cb) / 245 Carotenoids can maintain the functional homeostasis of photosynthetic organs and can also act as precursors of signaling molecules such as abscisic acid, mediating the response of coriander to cadmium stress. As shown in Figure 8, compared with the cadmium-treated group, the carotenoid content in the group treated with nano-selenium combined with strain TR37 increased by 16.10%.

[0045] Example 10: Effect of the combined use of Citrobacter freundii TR37 and nano-selenium on the soluble sugar and soluble protein content of coriander. 1. Weigh 0.2 g of coriander leaves from each treatment group in Example 7, wash and dry them, chop them, mix them well, add 5 mL of distilled water, extract in a boiling water bath for 30 minutes, cool, centrifuge at 3000 rpm for 10 minutes, take the supernatant, and dilute to 25 mL as the sample solution to be tested. Take 0.5 mL of the sample solution to be tested in a test tube, add 1.5 mL of distilled water, add 1 mL of 9% phenol solution, mix well, and allow it to react completely. Then quickly add 5 mL of concentrated sulfuric acid to the test tube, shake well, and let it stand at room temperature for 30 minutes until the color development is complete. Measure the absorbance at 485 nm. Calculation formula: Soluble sugar (mg / g) = [(sugar content calculated from the standard curve / volume of the test solution used for testing) × total volume of extract × dilution factor] / sample mass Depend on Figure 9 As shown in Figure a, the soluble sugar content in the nano-selenium combined with strain TR37 increased by 22.94% compared to the cadmium-treated group. Soluble sugars are osmotic regulators in coriander, which can enhance cell water-holding capacity, alleviate cadmium ion stress in the soil, and serve as a storage form of photosynthetic products, providing energy support for coriander growth.

[0046] 2. Weigh 0.5 g of coriander leaves, grind them into a homogenate with 5 mL of buffer, centrifuge at 10000 rpm for 10 min, take 1 mL of supernatant into a test tube, then add 5 mL of Coomassie Brilliant Blue G-250 solution, mix thoroughly, let stand at room temperature for 5 min, and measure the absorbance at 595 nm. Calculation formula: Soluble protein content (mg / g) = (Measured protein concentration × Total volume of extract) / Sample mass.

[0047] Coomassie Brilliant Blue G-250 dye formulation: Weigh 100 mg of Coomassie Brilliant Blue G-250, dissolve it in 50 mL of 95% ethanol, add 100 mL of 85% phosphoric acid solution, and dilute to 1 L with distilled water.

[0048] Under the stimulation of adverse factors, plants typically initiate stress response mechanisms. Results of the soluble protein content determination experiment ( Figure 9 (Figure b) In the cadmium-treated group and the nano-selenium sprayed group, the soluble protein content of coriander increased by 5.14% and 5.81% respectively compared with the control group. However, in the TR37-treated group and the TR37-plus-nano-selenium-treated group, the soluble protein content of coriander decreased significantly by 4.94% and 6.36% respectively, possibly because the addition of TR37 alleviated the synthesis of stress proteins.

[0049] Example 11 Effect of combined use of Citrobacter freundii TR37 and nano-selenium on cadmium content in the aboveground and underground parts of coriander The coriander from each treatment group in Example 7 was rinsed with distilled water and baked in an oven at 70 °C until constant weight. 0.1 g each of the ground coriander aerial and underground powders were weighed, added to concentrated nitric acid, and microwave digested. After digestion, the volume was adjusted to 10 mL with 2% nitric acid, and the Cd content was determined by ICP-MS.

[0050] Depend on Figure 10 It was found that the cadmium content in both the aboveground and underground parts of coriander in all treatment groups was lower than that in the Cd group. The cadmium content in the aboveground parts of coriander in the nano-selenium spray treatment group, the TR37 bacterial solution treatment group, and the nano-selenium-TR37 treatment group was significantly reduced by 23.93%, 19.51%, and 31.08%, respectively. Figure 10 (Figure a) shows that the cadmium content in the underground parts decreased significantly by 27.84%, 28.81%, and 37.36%, respectively. Figure 10 (See Figure b in the text). Both nano-selenium and TR37 bacterial solution can effectively reduce the cadmium content in both the aboveground and underground parts of coriander, with the best cadmium reduction effect when the two are treated together. This may be because strain TR37 reduces the bioavailability of cadmium in the soil and downregulates the expression of cadmium transport proteins after nano-selenium is absorbed and utilized, thereby reducing the accumulation of cadmium in the plant.

[0051] Example 12: Effects of the combined use of Citrobacter freundii TR37 and nano-selenium on the antioxidant enzyme activity and membrane lipid peroxidation of coriander. To investigate the alleviating effect of Citrobacter freundii TR37 and nano-selenium on coriander under cadmium stress, the levels of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), total antioxidant activity, and malondialdehyde (MDA) were quantitatively analyzed. All assays were performed according to the kit instructions.

[0052] Under cadmium stress, accumulated cadmium ions in plants interfere with the electron transport chain and induce a surge in reactive oxygen species (ROS), leading to membrane peroxidation and oxidative damage. Peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), as enzymatic antioxidants in plants, play crucial roles in ROS scavenging. SOD can catalyze the conversion of superoxide anion radicals induced by cadmium stress into hydrogen peroxide and oxygen, thereby enhancing the defense function of coriander. Figure 11 As shown in Figure a, compared to the cadmium-treated group, the combined use of *Citrobacter freundii* TR37 and nano-selenium significantly increased SOD enzyme activity by 83.99%. CAT can efficiently catalyze the decomposition of high-concentration hydrogen peroxide produced by SOD into water and oxygen, preventing its conversion into highly destructive hydroxyl radicals via the Fenton reaction. Figure 11As shown in Figure c, compared with the control group, the CAT activity of coriander in cadmium-contaminated soil decreased by 63.10%. The CAT activity of coriander in the nano-selenium spray group, the TR37 bacterial solution treatment group, and the combined treatment group significantly increased by 32.26%, 53.23%, and 61.29%, respectively. POD has a high affinity for the substrate, can continuously scavenge low concentrations of H2O2 to reduce oxidative damage, and simultaneously catalyzes the polymerization of lignin precursors to strengthen the cell wall and enhance the physical barrier against cadmium ions. Figure 11 As shown in Figure b, compared with the cadmium-treated group, the POD activity of coriander increased by 7.89%, 15.79%, and 33.78% in the group sprayed with nano-selenium, the TR37 bacterial solution treatment group, and the combined treatment group, respectively. Total antioxidant capacity (T-AOC) refers to the combined ability of all antioxidant substances (including enzymes and non-enzymes) in a plant, reflecting the plant's overall ability to scavenge reactive oxygen species. When Citrobacter freundii TR37 was used in combination with nano-selenium, the total antioxidant capacity was 6.32 ± 0.07 μmol / g, significantly increased by 40.55% compared to the cadmium-treated group. Figure 12 Sustained exposure to cadmium stress disrupts the endogenous redox homeostasis of plants. Malondialdehyde (MDA) is a marker of membrane lipid peroxidation, and its increase is usually associated with oxidative stress. Cadmium stress increases the MDA content in coriander, while the combined use of Citrobacter freundii TR37 and nano-selenium significantly reduces the MDA content in coriander under cadmium stress by 31.11%. Figure 11 (Figure d in the figure) alleviated the oxidative stress damage of cadmium to coriander.

[0053] For any points not covered above, existing technologies shall apply.

[0054] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Citrobacter freundii, characterized in that, The aforementioned Citrobacter freundii is Citrobacter freundii ( Citrobacter freundii TR37, accession number CCTCC NO: M2026452.

2. A microbial inoculum containing Citrobacter freundii TR37 as described in claim 1.

3. The application of Citrobacter freundii TR37 as described in claim 1 in the reduction of Se(IV).

4. The application of Citrobacter freundii TR37 as described in claim 1 in the preparation of bio-nano selenium.

5. The application of Citrobacter freundii TR37 as described in claim 1 in promoting plant growth.

6. An application of the combined use of *Citrobacter freundii* TR37 and nano-selenium as described in claim 1 for cadmium reduction, characterized in that... The term "cadmium reduction" refers to reducing the stress of cadmium ions on plant growth or reducing the cadmium content in plants.

7. An application of the combined use of Citrobacter freundii TR37 as described in claim 1 and nano-selenium to promote coriander growth under cadmium stress.

8. The application as described in claim 7, characterized in that, Coriander was treated with nano-selenium foliar spray and bacterial solution root irrigation.

Images