Pseudomonas geniculate H225 and its application in cadmium reduction and selenium enrichment

Abstract

The present invention relates to the field of microbial technology, and in particular to Pseudomonas genu H225 and its application in cadmium reduction and selenium enrichment. Pseudomonas geniculata‌ ) H225, deposited with CGMCC No. 25640. The present invention also provides the use of the aforementioned Pseudomonas genuformis H225 for alleviating cadmium stress in crops. Applying Pseudomonas genuformis H225 to high-cadmium, selenium-rich soils, followed by crop planting, can reduce cadmium accumulation and increase selenium content in the crops; that is, it reduces cadmium accumulation in the crops and increases selenium absorption.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Pseudomonas geniculate H225 and application thereof in cadmium reduction and selenium enrichment. Background Art

[0002] Black rock series are a general term for dark gray or black carbonaceous rocks, siliceous rocks, argillaceous rocks, and metamorphic rocks rich in organic carbon and sulfide minerals. They formed in unique marine anoxic and oxygen-poor sedimentary environments. Black rock series are rich in metallic elements such as cadmium (Cd), nickel (Ni), zinc (Zn), and molybdenum (Mo), as well as metal sulfides and organic matter. They are susceptible to weathering when exposed to air and humid environments. This leaches heavy metals and other trace elements from these rock series, which migrate into the surrounding soil with surface water, causing a series of environmental problems.

[0003] Cadmium is a highly biotoxic heavy metal and a Class I carcinogen. Due to its strong migration capacity, cadmium easily accumulates in agricultural products through the soil-crop system and enters the human body through dietary exposure. Excessive accumulation can induce hypertension, kidney damage, reproductive system dysfunction, teratogenicity, and carcinogenicity, posing a significant threat to food security and human health.

[0004] Selenium is recognized by the World Health Organization and the International Nutrition Organization as an essential nutrient for humans. It has antioxidant, cancer prevention, immunity-boosting, and cardiovascular disease-preventing effects. Insufficient selenium intake can lead to Keshan disease, Kashin-Beck disease, and white muscle disease. The average abundance of selenium in the Earth's crust is extremely low, only 0.05-0.09 mg / kg. Selenium content in soils largely depends on the parent material, ranging from 0.01 to 2.0 mg / kg. Therefore, when using black rock systems as crop soil in areas with high geological backgrounds, reducing cadmium content in edible parts of crops to an acceptable range and maximizing selenium content to an acceptable level of selenium enrichment are two key objectives for the safe and efficient use of soil in these areas.

[0005] Vegetables are an indispensable part of the global diet, and their safety directly affects human health. However, some leafy vegetables are prone to accumulate harmful elements in heavy metal pollution environments, becoming an important source of cadmium exposure in the diet. Brassica rapa subsp. chinensis Due to its strong root absorption capacity and internal migration characteristics, bok choy has a higher cadmium accumulation efficiency than other leafy vegetables such as lettuce and spinach. Therefore, bok choy can be a key research and treatment target for cadmium pollution prevention and control. By optimizing planting methods, selecting low-accumulation varieties, or implementing soil remediation measures, its cadmium absorption and accumulation can be reduced, providing a model for the safe production of other high-accumulation vegetables.

[0006] The bioavailability of cadmium and selenium in soil is influenced by physical and chemical processes, including adsorption-desorption, precipitation-dissolution, and oxidation-reduction. Numerous factors, including soil moisture content, particle size, organic matter content, pH, and mineral content, also influence the occurrence and migration of cadmium and selenium in soil. In acidic soils with low pH, cadmium ions readily transform from a stable state to an available state, accelerating their migration within the soil and the transfer of cadmium within the food web, posing potentially more serious risks to human health. An increase in protons on the cell surface (lower pH) reduces the electrostatic drive for cadmium, hindering its biosorption and bioprecipitation on microbial cell surfaces.

[0007] Plant selenium uptake is influenced by soil physicochemical properties (redox state, pH, and microbial activity). Selenium concentrations in crops depend, to some extent, on soil pH. Because selenium exists in anionic form, electrostatic forces likely dominate adsorption interactions in soil. Selenium can adsorb onto double hydroxides or anionic clays through electrostatic interactions, and these interactions make selenium very sensitive to pH. Selenium adsorption increases with decreasing pH because negatively charged clay surfaces and aluminum and iron oxide edges are reduced, leading to stronger electrostatic interactions. Consequently, the mobility and bioavailability of inorganic selenium in the environment increase with increasing pH and decreasing clay and iron oxide content in soil. In summary, pH is an important factor regulating the mobility of cadmium and selenium in soil.

[0008] While traditional physical and chemical remediation techniques (such as chemical precipitation, adsorption, and ion exchange) can effectively remove cadmium, they often suffer from high costs, byproduct contamination, and poor adaptability. In recent years, the use of microorganisms to remediate soil contamination has emerged as a cost-effective and environmentally friendly method.

[0009] Microbial remediation, including key mechanisms such as biosorption, bioaccumulation, and biotransformation, has attracted widespread attention as a low-cost and efficient remediation method. Stenotrophomonas, Bacillus, Pseudomonas, Azotobacter, Escherichia coli, Mycobacterium and Serratia Bacteria such as alkalizing bacteria are widely used in cadmium (Cd) remediation due to their rapid reproduction, high tolerance and remarkable flexibility. Alkalinizing bacteria are considered to be a type of environmental microorganisms that can effectively increase pH. Bacillus altitudinis The XT-4 strain has been used for cadmium bioremediation, increasing pH and reducing the solubility of cadmium in the culture medium. Cupriavidus sp. Cd02 strain can effectively increase soil pH and reduce the content of exchangeable cadmium. Ralstonia sp. YDR strain has good alkalization ability, through the adsorption and absorption of cadmium, bacterial-assisted antioxidant effect and cadmium ion (Cd 2+ ) and hydrogen ions (H+ ) flow, promoting lower cadmium accumulation in rice. In theory, alkalizing bacteria have the potential to reduce the bioavailability of cadmium, especially in the rhizosphere of plants. However, research on the efficacy, practicality and mechanism of alkalizing bacteria in cadmium remediation has mainly focused on rice systems, and the alkalization mechanism of alkalizing strains has not yet been revealed. The development of new alkalizing bacteria to regulate soil pH, reduce the risk of cadmium enrichment in crops and improve selenium bioavailability is of great significance for the safe use of agricultural land and selenium resource development in areas with high geological background.

[0010] CN119331776 A disclosed Stenotrophomonas sp. 215 can reduce cadmium accumulation in wheat, but its lack of alkalization ability and inability to specifically produce hydrogen sulfide limit its remediation efficiency.

[0011] The invention of CN118773054A, "Cadmium-resistant rhizosphere growth-promoting bacteria, their agents and applications", discloses a cadmium-resistant PGPR strain, which is classified and named Pseudomonas chlororaphis Pseudomonas chlororaphis , does not produce hydrogen sulfide. It can effectively alleviate cadmium stress in tomatoes.

[0012] The invention of CN110076193B "Lebanese Pseudomonas strain MY and its application in the remediation of heavy metal contaminated saline soil" provides a Lebanese Pseudomonas strain ( Pseudomonas libanensis MY, a rhizospheric growth-promoting bacterium with strong resistance to heavy metals (cadmium, chromium, copper, nickel, lead, and zinc) and salt, can significantly promote plant growth in heavy metal-contaminated saline soils, reduce the toxicity of heavy metals and salt to plants, enhance plants' ability to absorb heavy metals and sodium ions in the soil, and promote phytoremediation efficiency. This strain produces hydrogen sulfide.

[0013] The invention of CN101531970B "A strain of Pseudomonas and its use in bioreduction and biosorption" provides Pseudomonas ( Pseudomonas alcaliphila sp. MBR). Under aerobic conditions, this bacterium can reduce nitrates to produce ammonia, adsorb metal ions, and reduce metal and non-metal ions in solution to elemental substances. This strain also produces hydrogen sulfide. Summary of the Invention

[0014] The technical problem to be solved by the present invention is to provide a microbial strain that can reduce cadmium accumulation in crops, alleviate cadmium toxicity and increase the selenium content in crops, so as to improve the agricultural production safety and crop economic value in areas with high geological background.

[0015] In order to solve the above technical problems, the present invention provides a Pseudomonas genu Pseudomonas geniculata‌ )H225, its deposit number is CGMCC No.25640.

[0016] The strain H225 of the present invention is deposited under the name of: Pseudomonas geniculate Pseudomonas geniculata‌ H225, deposited at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC NO. 25640, deposit date: September 2, 2022.

[0017] The present invention also provides the use of the Pseudomonas genu H225 in alleviating cadmium stress in crops, that is, alleviating the toxic effects of cadmium on plant growth.

[0018] As an improvement of the application of the present invention: by applying Pseudomonas geniculate H225 in the soil, the accumulation of cadmium in crops can be reduced and the absorption of selenium can be increased, that is, cadmium reduction and selenium enrichment can be achieved.

[0019] As a further improvement of the application of the present invention, applying Pseudomonas genu H225 to high-cadmium and selenium-rich soil and then planting crops can reduce cadmium accumulation in crops and increase the selenium content of crops; that is, reduce cadmium accumulation in crops and increase selenium absorption.

[0020] The high-cadmium and selenium-rich soil refers to:

[0021] When pH≤5.5, cadmium content>0.3 mg / kg and selenium content>0.4 mg / kg;

[0022] 5.5<pH ≤6.5, cadmium content>0.4 mg / kg, and selenium content>0.4 mg / kg;

[0023] When pH>6.5, cadmium content>0.6 mg / kg and selenium content>0.4 mg / kg.

[0024] High-cadmium-selenium-rich soil, especially refers to high-cadmium-selenium-rich soil with high geological background.

[0025] As a further improvement of the application of the present invention: promoting crop growth.

[0026] As a further improvement to the application of the present invention, the pH of high-cadmium-selenium-rich soil is improved to achieve ecological restoration of high-cadmium-selenium-rich soil. Specifically, increasing the pH of high-cadmium-selenium-rich soil reduces cadmium accumulation in crops and increases selenium levels in crops, thereby achieving safe utilization of high-cadmium-selenium-rich soil.

[0027] As a further improvement of the application of the present invention: the crop is, for example, a vegetable, Brassica rapa subsp. chinensis ).

[0028] The present invention also provides a microbial preparation for alleviating cadmium toxicity in Shanghai blueberries and increasing selenium accumulation in crops. The active ingredient of the microbial preparation contains Pseudomonas genu H225.

[0029] The present invention also provides a method for alleviating cadmium toxicity of Shanghai bluebell and improving its selenium accumulation: applying Pseudomonas geniculate H225 in the soil and then planting Shanghai bluebell.

[0030] The present invention isolated a new strain of Pseudomonas geniculate from black rock soil in a geologically high background area. Its morphological characteristics are: forming round, translucent, smooth, light yellow colonies on LB culture medium; molecular phylogenetic analysis showed that the strain was classified as Pseudomonas ( Pseudomonas ), named Pseudomonas geniculate ( Pseudomonas geniculata‌ )H225, the deposit number is CGMCC No.25640.

[0031] The culture medium of H225 strain is LB medium, and the suitable culture condition of this strain is 20~30℃. 600 is about 1.0 (the strain concentration is 2×10 9 CFU / mL), and then apply the bacterial solution to the crop growth medium (soil). In potted plants, the concentration of the inoculated strain per gram of soil is 2×10 8 CFU / g~2×10 9 CFU / g. The amount of strain required for soil cultivation can be converted accordingly based on the amount of strain required for potted cultivation mentioned above.

[0032] In summary, the present invention provides Pseudomonas genu H225 capable of producing hydrogen sulfide and the alkaloid piperidine, and its application in reducing cadmium accumulation and increasing selenium accumulation (cadmium reduction and selenium enrichment) in crops in areas with high geological background.

[0033] The present invention has the following technical advantages:

[0034] 1. Pseudomonas genuformis H225 has a unique metabolic mechanism that can: 1) produce the alkaloid piperidine through metabolism, thereby increasing soil pH and reducing the bioavailability of cadmium in the soil; 2) produce hydrogen sulfide through metabolism, which combines with cadmium ions in the soil to form a precipitate, further reducing the absorption of cadmium in the soil by crops; and 3) promote the absorption of selenium by crops, thereby increasing the nutritional value of crops.

[0035] That is, the Pseudomonas geniculate H225 of the present invention can simultaneously reduce cadmium accumulation, alleviate cadmium toxicity and promote selenium enrichment.

[0036] 2. Pseudomonas genuformis H225, when applied to cadmium-contaminated soil, can significantly alleviate the toxic effects of cadmium stress on crop growth. This strain can reduce cadmium levels in vegetables, increase selenium levels, and enhance the economic value of crops. Its ease of use, low cost, and environmental friendliness make it an effective cadmium pollution prevention and control measure in areas with high geological backgrounds.

[0037] 3. The research of the present invention shows that the use of Pseudomonas geniculate H225 provided by the present invention can alleviate the toxic effect of soil cadmium on the growth of Shanghai bluebells, which is specifically manifested in: reducing cadmium accumulation in Shanghai bluebells, increasing selenium content, and optimizing growth indicators.

[0038] The results of potted experiments showed that after applying Pseudomonas genu H225 to high-cadmium and selenium-rich soil with a high geological background, the cadmium content of vegetables (represented by bok choy) was significantly reduced (by 26.7%~44.7%), while the selenium content was greatly increased (increased by 60.5%~245%), effectively promoting plant growth and increasing yield.

[0039] Compared with traditional chemical remediation methods, the present invention provides a low-cost, secondary pollution-free green bioremediation strategy that can not only alleviate cadmium stress, but also enhance the nutritional value of agricultural products, providing a sustainable microbial solution for safe agricultural production in areas with high geological backgrounds.

[0040] In summary, although existing studies have explored the application of some microorganisms in cadmium pollution remediation, most strains have limitations in increasing soil pH, reducing cadmium bioavailability, and promoting selenium enrichment, making it difficult to simultaneously take into account crop safety and nutritional value. The present invention's Pseudomonas genu H225, with its unique metabolic mechanism and dual regulatory effects (i.e., increasing pH through alkaloids and generating hydrogen sulfide to precipitate cadmium), effectively reduces crop cadmium accumulation while increasing selenium bioavailability, thereby alleviating cadmium toxicity while improving agricultural product quality. To date, there have been no reports of microbial remediation technologies for Shanghai green, a high-cadmium-accumulating vegetable, that can significantly reduce cadmium content and enhance selenium accumulation while increasing yield. The discovery of H225 provides an innovative and efficient biological solution for farmland pollution remediation and the development of functional agricultural products in high-geological background areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0042] Figure 1 This is the colony morphology of strain H225.

[0043] Figure 2 This is the phylogenetic tree based on the 16S rRNA sequence of strain H225.

[0044] Figure 3 The pH changes after adding cadmium ions to LB medium and culturing strain H225 under cadmium stress;

[0045] Figure 3 middle:

[0046] (A) pH changes after LB medium was added with 0-200 mg / L cadmium ion stress and cultured at 180 rpm for 48 h.

[0047] (B) pH changes after adding 1% H225 strain to LB medium stressed with 0-200 mg / L cadmium ions and culturing at 180 rpm for 48 h.

[0048] (C) is the OD of the strain after adding 0~200 mg / L cadmium ion stress 600 The linear relationship between pH.

[0049] Figure 4 This is the GC-MS graph of the metabolites of strain H225, where the substances indicated by arrows represent the characteristic peaks of alkaloids piperidines.

[0050] Figure 5 To confirm that the H225 strain produced hydrogen sulfide gas during the culture process.

[0051] Figure 6 This is a diagram showing the effect of applying H225 strain on the growth of Shanghai green plants in a potted experiment;

[0052] Figure 6 middle:

[0053] (A) is a photo of plant growth trend;

[0054] (B) is plant height;

[0055] (C) is the fresh weight of leaves.

[0056] Figure 7 for Figure 6 Statistical chart of pH of soil where Chinese bok choy grows and cadmium and selenium content in leaves of the plants;

[0057] Figure 7 middle:

[0058] (A) is the pH of the soil where the Shanghai Green grows;

[0059] (B) is the cadmium concentration;

[0060] (C) is the selenium concentration. DETAILED DESCRIPTION

[0061] The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0063] Example 1: Isolation and identification of strain H225

[0064] LB agar medium: Dissolve 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar in 1000 mL of ultrapure water. Sterilize the medium at 1.1 atmospheres, 121°C for 20 min, to a neutral pH before use.

[0065] Weigh 10 g of soil from the high-cadmium and selenium-rich black rock system in Xiaguang Town, Kaihua County, Quzhou City, Zhejiang Province, and place it in a conical flask filled with 100 mL of sterile saline. Shake it at 180 rpm for 30 minutes on a 28°C constant temperature shaker. Let it stand for 5 minutes, then aspirate the supernatant and dilute it serially (10-1000 times). According to the conventional dilution plate method, isolate it on LB agar medium to obtain several test strains. The colony characteristics of strain H225 are shown in Figure 1 As shown, the colonies appeared as small dots, which were round, translucent, smooth, light yellow colonies.

[0066] The 16S rRNA gene sequence of strain H225 was determined by the Hangzhou branch of Beijing Qingke Biotechnology Co., Ltd. The sequence was submitted to the nucleotide database of the National Center for Biotechnology Information (NCBI) for comparison and analysis, and the strain was mapped using Mega software. Pseudomonas geniculate The phylogenetic tree of H225 is shown in the following figure. Figure 2 The Bootstrap support rate of this phylogenetic tree is high (96%~98%), indicating that these evolutionary relationships are relatively stable and have high credibility. Pseudomonas geniculata The sequence similarity of strain XJUHX 18 reached 99.72%, so it was classified as Pseudomonas geniculates ( Pseudomonas geniculata ).

[0067] Based on the above colony characteristics and molecular biological identification, strain H225 was identified as Bacteria, Pseudomonadota, Gammaproteobacteria, Pseudomonadales, Pseudomonadaceae, and Pseudomonas. Pseudomonas geniculate Pseudomonas species ( Pseudomonas geniculata ). Therefore, it was named Pseudomonas geniculate Pseudomonas geniculata H225.

[0068] The strain H225 of the present invention is deposited under the name of: Pseudomonas geniculate Pseudomonas geniculata‌ H225, deposited at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC NO. 25640, deposit date: September 2, 2022.

[0069] Example 2: Analysis of physiological and biochemical characteristics of strain H225

[0070] At 28°C and 180 rpm, LB liquid culture medium without H225 strain was cultured at different cadmium concentration gradients (0-200 mg / L) for 48 hours. The pH of the culture medium before and after culture was measured. The results are as follows: Figure 3 (A), the left and right columns in the figure represent the pH values before and after cultivation, respectively, and the pH value (y) and cadmium concentration (x) are fitted. The front (B) curve represents the fitting result before cultivation, and the back (A) curve represents the fitting result after cultivation, where R 2 This reflects the correlation. 1% H225 strain was added to LB liquid culture medium with the same gradient cadmium concentration and cultured with shaking for 48 hours (culture conditions are the same as above). The pH of the culture medium was measured and the results were as follows: Figure 3 (B). Then, Figure 3 (B) pH value (Y) and OD of each treatment group 600 (X) is fitted and R is calculated 2 To evaluate the correlation, the results are as follows Figure 3 (C).

[0071] The results show that if Figure 3 As shown in the figure: after adding cadmium ions, the pH of the culture medium showed a significant downward trend, and the degree of pH decrease was significantly negatively linearly correlated with the cadmium ion concentration, that is, the addition of cadmium ions will lead to a decrease in the pH of the culture medium. In addition, the initial pH of the LB liquid culture medium was maintained at around 7.0, and when the H225 strain was inoculated, the environmental pH increased significantly, and the lowest pH also reached above 8.0, indicating that H225 can still increase the environmental pH under the stress of different cadmium ion concentrations. Further analysis found that the OD value of the H225 strain under different cadmium concentration conditions was significantly higher than that of the H225 strain. 600 There was a significant positive linear correlation between bacterial growth and pH (R 2= 0.9302), indicating that the increase in pH may be related to the metabolic activity of strain H225, and the magnitude of the effect is proportional to the growth rate of the strain. This characteristic indicates that strain H225 can still grow under cadmium stress conditions and significantly increase the environmental pH, which may be related to the release of its metabolites, thus having the potential to regulate pH in heavy metal-contaminated environments.

[0072] To explore the possible mechanism of pH increase, strain H225 was cultured at 28°C and 180 rpm for 48 h, and the cultured bacterial solution was analyzed by gas chromatography-mass spectrometry (GC-MS). The results showed that the metabolites of strain H225 contained characteristic peaks of piperidines (such as Figure 4 ), indicating that strain H225 may release alkaloids (such as piperidine) through metabolism, leading to alkalinization of the culture medium.

[0073] Cadmium (Cd), selenium (Se), and 1% H225 strain were added to a sealed bottle containing 20 mL of LB liquid medium according to the different treatment groups. After inoculation with 1% H225 strain, the culture was incubated at 28°C and 180 rpm for 48 hours. After the incubation period, a filter paper soaked in a 0.1% lead nitrate (Pb(NO3)2) aqueous solution was fixed to the rubber stopper of the sealed bottle. The filter paper was left to stand for 10 minutes and the color change of the filter paper was observed (e.g., Figure 5 The experimental groups are as follows:

[0074] CK (control group): inoculated with only H225 strain, without Cd or Se.

[0075] Cd: 3 mg / L cadmium was added, and the H225 strain was not inoculated.

[0076] Cd + H225: Add 3 mg / L cadmium and inoculate 1% H225 strain.

[0077] Se: 1 mg / L selenium was added, and the H225 strain was not inoculated.

[0078] Se + H225: Add 1 mg / L selenium and inoculate with 1% H225 strain.

[0079] Cd + Se: 3 mg / L cadmium and 1 mg / L selenium were added, and the H225 strain was not inoculated.

[0080] Cd + Se + H225: Add 3 mg / L cadmium and 1 mg / L selenium, and inoculate 1% H225 strain.

[0081] Result analysis:

[0082] The experimental results showed that the experimental group containing H225 formed a black precipitate on the lead nitrate test paper, indicating that the H225 strain could stably produce hydrogen sulfide (H2S) regardless of whether cadmium and selenium were present. 2+ ) and sulfide ions (S 2- ) combined with the lead sulfide (PbS) to form a black precipitate, which further verified the H2S-generating ability of the H225 strain.

[0083] The above results show that strain H225 has the ability to regulate the pH of the culture medium ( Figure 3 ), which can increase the environmental pH by producing alkaloids such as piperidine ( Figure 4 ), and also has the metabolic characteristics of producing hydrogen sulfide ( Figure 5 ), which can be beneficial for its cadmium-reducing and selenium-enriched bioremediation in high-cadmium and selenium-enriched soils.

[0084] Example 3: Potted experiment on alleviating cadmium stress and promoting selenium accumulation in soil H225 with high geological background

[0085] The soil used in the potted plant experiments was a high-cadmium, selenium-rich soil derived from the black rock system in Xiaguang Town, Kaihua County, Quzhou City, Zhejiang Province. After collection, the soil was air-dried to a moisture content of ≤10% and filtered through a 2 mm sieve for later use. The high-cadmium, selenium-rich soil contained 4.447 mg / kg cadmium and 0.406 mg / kg selenium, with a pH of 5.

[0086] The experiment was conducted in a greenhouse in Hangzhou, Zhejiang Province from October to November 2024, with three treatment groups:

[0087] 1) Control CK group: high-cadmium and selenium-rich soil developed from black rock series;

[0088] 2) 10 8 CFU / g soil treatment group: The concentration of H225 strain per gram of soil was 10 8 CFU / g;

[0089] 3) 10 9 CFU / g soil treatment group: The concentration of H225 strain per gram of soil was 10 9 CFU / g.

[0090] Take an inoculation loop of strain H225 and culture it in 100 mL (placed in a 250 mL conical flask) LB liquid medium at 28°C and 180 r / min for 48 h as the inoculum; inoculate the inoculum into LB liquid medium at a volume ratio of 1% and culture it at 28°C and 180 r / min for 10 h to obtain a concentration of 2×10 9 CFU / mL of bacterial solution.

[0091] 25 mL and 250 mL of H225 bacterial suspension were centrifuged at 4500 rpm for 10 min to obtain bacterial cells. The bacterial pellet was dispersed in 50 mL of purified water, stirred evenly, and immediately dispersed in 500 g of high-cadmium and selenium-rich soil with a high geological background.

[0092] Each treatment was replicated three times. Each replicate consisted of three healthy, plump seeds of the Shanghai Qing (Brochureum guianensis) planted in a plastic pot containing 500 g of soil. Plants were grown under natural light and temperature, with regular watering to ensure adequate moisture for normal growth. Plant height and aboveground fresh weight of the Shanghai Qing (Brochureum guianensis) were measured 45 days after planting. The pH of the harvested soil was also measured, and the cadmium and selenium contents of the edible leaves of the Shanghai Qing (Brochureum guianensis) were determined using microwave digestion.

[0093] In this example, the effects of applying the H225 strain on the growth, yield and soil element content of the bok choy are as follows: Figure 6 and Figure 7 The results showed that in the untreated CK group, the plants of Shanghai Qing grew slowly and some leaves showed yellowing. However, after the application of H225 strain, the growth of Shanghai Qing was significantly improved. 8 After the treatment with CFU / g strain, the plant height of Shanghai Qing increased by 11.9%, and the fresh weight of leaves increased by 83.0%. 9 The plant height of the CFU / g treatment group increased by 11.3%, and the fresh weight of leaves increased by 102.4%, indicating that the strain has a significant promoting effect on the growth of Shanghai green ( Figure 6 ). At the same time, 45 days after planting, if Figure 7 As shown in the figure, compared with the control group (CK) without strain application, the soil pH value increased after the application of H225 strain. 8 The pH of the CFU / g group increased by 0.21, 10 9 The CFU / g group increased by 0.44, indicating that the strain may regulate soil pH through metabolites and improve the growth environment of Shanghai Qing. In addition, the cadmium and selenium contents in the leaves of Shanghai Qing also changed significantly. Compared with the CK group, 10 8 After treatment with CFU / g, the Cd content in leaves decreased by 44.7%, and the 9 The CFU / g group decreased by 26.7%, indicating that the H225 strain can effectively reduce the absorption of cadmium by Shanghai Qing. At the same time, the selenium content in the leaves was 8 CFU / g increased by 60.5% after treatment, 10 9 The CFU / g increased by 245% after treatment, indicating that the strain promoted the enrichment of selenium in Shanghai green.

[0094] In summary, the application of H225 strain can effectively promote the growth of Shanghai green, reduce cadmium accumulation, increase selenium content, and improve soil pH, providing an efficient microbial regulation strategy for the ecological restoration of high-cadmium and selenium-rich soils.

[0095] The present invention was found in the process of invention: cadmium-resistant PGPR strains ( Pseudomonas chlororaphis None of the fermentation products of Pseudomonas libanensis MY, Pseudomonas alcaliphila sp. MBR contain the alkaloid piperidine; potted plants were grown in accordance with Example 3. These strains did not significantly increase soil pH. Leaf selenium content in the strain-added group was only slightly higher than in the CK group, with an increase of less than 20%. Leaf fresh weight was also only slightly higher in the strain-added group, with an increase of less than 15%.

[0096] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. Pseudomonas geniculates ( Pseudomonas geniculata ) H225, characterized by: preservation number CGMCC No.25640.

2. The use of Pseudomonas geniculate H225 according to claim 1 in alleviating cadmium stress in crops, characterized in that: Applying Pseudomonas genu H225 to the soil can reduce cadmium accumulation and increase selenium absorption in crops; The strain H225 can increase pH and generate hydrogen sulfide to precipitate cadmium through alkaloids; the alkaloid is piperidine.

3. The use according to claim 2, characterized in that: Applying Pseudomonas genuformis H225 to high-cadmium and selenium-rich soil and then planting crops can reduce cadmium accumulation in crops and increase the selenium content of crops.

4. The use according to claim 2 or 3, characterized in that: Promote crop growth.

5. The use according to claim 4, characterized in that: Improve the pH of high-cadmium-selenium-rich soil, thereby achieving ecological restoration of high-cadmium-selenium-rich soil.

6. The use according to claim 5, characterized in that: Increase the pH of high-cadmium-selenium-rich soil, thereby reducing crop cadmium accumulation, improving crop selenium levels, and achieving safe utilization of high-cadmium-selenium-rich soil.

7. The use according to claim 2, characterized in that: The crop is the vegetable Bok Choy.

8. A microbial preparation for alleviating cadmium toxicity and increasing selenium accumulation in Shanghai blue, characterized in that: The active ingredient of the microbial preparation comprises the Pseudomonas genu H225 as described in claim 1.

9. A method for alleviating cadmium poisoning and increasing selenium accumulation in bok choy, characterized in that: Pseudomonas geniculate H225 was added to the high cadmium and selenium-rich soil at pH=5, and then Shanghai green was planted.

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