Pseudomonas geniculata H225 and application thereof in cadmium reduction and selenium enrichment

Abstract

The invention relates to the technical field of microorganisms, in particular to Pseudomonas geniculata H225 and application thereof in cadmium reduction and selenium enrichment. The invention provides Pseudomonas geniculata H225, and the preservation number of the Pseudomonas geniculata H225 is CGMCC (China General Microbiological Culture Collection Center) No.25640. The invention also provides a preparation method of the Pseudomonas geniculata H225. The invention also provides an application of the Pseudomonas geniculata H225 in relieving cadmium stress of crops. Pseudomonas geniculata H225 is applied to the high-cadmium selenium-rich soil, then crops are planted, cadmium accumulation of the crops can be reduced, and the selenium content of the crops is increased; namely, cadmium accumulation in crops is reduced, and the absorption amount of selenium is improved.

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 is a general term for dark gray or black carbonaceous rocks, siliceous rocks, mudstones and metamorphic rocks rich in organic carbon and sulfide minerals, formed in a special marine anoxic-poor sedimentary environment. Black rock series are rich in metal elements such as cadmium (Cd), nickel (Ni), zinc (Zn), molybdenum (Mo), as well as metal sulfides and organic matter. They are easily weathered when exposed to air and humid environments, causing heavy metals and other trace elements rich in black rock series to be leached and migrate into the surrounding soil environment with surface water, causing a series of environmental problems.

[0003] Cadmium is a heavy metal with strong biological toxicity and is a Class I carcinogen. Due to its strong migration ability, cadmium is easily enriched in agricultural products through the soil-crop system and enters the human body through dietary exposure. Excessive accumulation can induce high blood pressure, kidney damage, reproductive system dysfunction, teratogenicity and carcinogenicity, posing a major threat to food security and human health.

[0004] Selenium is an essential nutrient element for the human body confirmed by the World Health Organization and the International Nutrition Organization. It has antioxidant, anti-cancer, immunity-enhancing, and cardiovascular disease-preventing effects. Insufficient selenium intake can lead to Keshan disease, Kaschin-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; the level of selenium in the soil depends largely on the parent material, and the selenium content in the soil is between 0.01~2.0 mg / kg. Therefore, when the black rock system in the geological high background area is used as planting soil, reducing the cadmium content in the edible parts of the crop to an acceptable range and increasing the selenium content to an acceptable selenium-rich level as much as possible are the two core goals of safe and efficient use of soil in geological high background 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, it has a higher cadmium accumulation efficiency than other leafy vegetables such as lettuce and spinach. Therefore, in terms of cadmium pollution prevention and control, Shanghai green can be used as a key research and governance target. By optimizing planting methods, selecting low-accumulation varieties or taking soil remediation measures, its cadmium absorption and accumulation can be reduced, providing a demonstration for the safe production of other high-accumulation vegetables.

[0006] The bioavailability of cadmium and selenium in soil is affected by soil physical and chemical processes, including adsorption-desorption, precipitation-dissolution, and oxidation-reduction. Numerous factors such as soil water content, soil particle size, organic matter content, pH value, and mineral content also influence the occurrence forms and migration processes of cadmium and selenium in soil. In acidic soils with low pH values, cadmium ions are prone to transform from stable states to available states, accelerating the migration of cadmium ions in soil and the transfer process of cadmium in the food web, posing a more serious potential hazard to human health. An increase in protons on the cell surface (decrease in pH value) will reduce the electrostatic drive of cadmium, hindering the biosorption and bioprecipitation of cadmium on the surface of microbial cells.

[0007] The uptake of selenium by plants is affected by the physicochemical properties of the soil (oxidation-reduction state, pH, and microbial activity). The concentration of selenium in crops depends to a certain extent on the soil acidity. Since selenium exists in anionic form, electrostatic forces may dominate the adsorption interactions in the soil. Selenium can be adsorbed onto double-layer hydroxides or anionic clays through electrostatic interactions, and these interactions make selenium very sensitive to pH value. As the pH value decreases, the adsorption of selenium increases because the negatively charged clay surface and the edges of aluminum and iron oxides decrease, resulting in stronger electrostatic interactions. Therefore, as the pH value increases and the content of clay and iron oxides in the soil decreases, the mobility and bioavailability of inorganic selenium in the environment also increase. In summary, pH is an important factor regulating the migration of cadmium and selenium in soil.

[0008] Traditional physical and chemical remediation technologies (such as chemical precipitation, adsorption, ion exchange, etc.) can effectively remove cadmium, but they often have problems such as high cost, by-product pollution, and poor adaptability. In recent years, using microorganisms to remediate soil pollution has become a cost-effective and environmentally friendly method.

[0009] Microbial remediation, including key mechanisms such as biosorption, bioaccumulation, and biotransformation, has received extensive attention as a low-cost and efficient remediation method. Such as Stenotrophomonas, Bacillus, Pseudomonas, Azotobacter, Escherichia coli, Mycobacterium and Serratia and other bacteria, due to their rapid reproduction, high tolerance, and remarkable flexibility, are widely used for cadmium (Cd) remediation. Alkalizing bacteria are considered a class of environmental microorganisms that can effectively increase the pH value. Alkalizing bacteria Bacillus altitudinis XT-4 strain has been used for the bioremediation of cadmium, which can increase the pH value and reduce the solubility of cadmium in the culture medium. Similarly, alkalizing bacteria Cupriavidus sp. Cd02 strain can effectively increase the soil pH value and reduce the content of exchangeable cadmium. Ralstonia sp. YDR strain has good alkalizing ability, through the adsorption and absorption of cadmium, bacteria-assisted antioxidant action, and the regulation of cadmium ions (Cd 2+ ), and hydrogen ions (H+ The regulation of flow promotes 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 the rice system, and the alkalizing mechanism of alkalizing strains has not been revealed. Developing novel alkalizing bacteria to regulate soil pH, reduce the risk of cadmium enrichment in crops, and improve the bioavailability of selenium is of great significance for the safe utilization of agricultural land and the development of selenium resources in geological high-background areas.

[0010] Disclosed in CN119331776 A Stenotrophomonas sp. 215 can reduce cadmium accumulation in wheat, but it lacks the ability to alkalize and cannot specifically produce hydrogen sulfide, resulting in limited remediation efficiency.

[0011] The invention of CN118773054A, "Cadmium-tolerant plant growth-promoting rhizobacteria and their bacterial agents and applications", discloses a cadmium-tolerant PGPR strain, classified and named Pseudomonas chlororaphis Pseudomonas chlororaphis , which does not produce hydrogen sulfide and can effectively relieve cadmium stress in tomatoes.

[0012] The invention of CN110076193B, "Pseudomonas libanensis strain MY and its application in the remediation of heavy metal-contaminated saline soil", provides a strain of Pseudomonas libanensis ( Pseudomonas libanensis ) MY. This rhizosphere-promoting bacterium has strong heavy metal (cadmium, chromium, copper, nickel, lead, and zinc) resistance and salt tolerance, can significantly promote the growth of plants on heavy metal-contaminated saline soil, reduce the toxicity of heavy metals and salts to plants, improve the absorption capacity of plants for heavy metals and sodium ions in the soil, and promote the plant remediation efficiency. This strain produces hydrogen sulfide.

[0013] The invention of CN101531970B, "A strain of Pseudomonas and its use in biological reduction and biosorption", provides Pseudomonas ( Pseudomonas alcaliphila sp.MBR). Under aerobic conditions, this bacterium can not only reduce nitrate to produce ammonia, adsorb metal ions, but also reduce metal ions and non-metal ions in the solution to elemental substances. This strain 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, relieve cadmium toxicity, and increase the selenium content of crops, so as to improve the safety of agricultural production and the economic value of crops in geological high-background areas.

[0015] To solve the above technical problems, the present invention provides a Pseudomonas geniculata ( Pseudomonas geniculata‌ ) H225, with the deposit number CGMCC No.25640.

[0016] The strain H225 of the present invention has a deposit name of: Pseudomonas geniculata Pseudomonas geniculata‌ H225, depositary institution: China General Microbiological Culture Collection Center, deposit 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 application of the above-mentioned Pseudomonas geniculata H225 in alleviating cadmium stress in crops, that is, alleviating the toxic effect of cadmium on plant growth.

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

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

[0020] The high-cadmium and selenium-rich soil mentioned above refers to: When pH ≤ 5.5, cadmium content > 0.3 mg / kg, and selenium content > 0.4 mg / kg; When 5.5 < pH ≤ 6.5, cadmium content > 0.4 mg / kg, and selenium content > 0.4 mg / kg; When pH > 6.5, cadmium content > 0.6 mg / kg, and selenium content > 0.4 mg / kg.

[0021] The high-cadmium and selenium-rich soil especially refers to the high-cadmium and selenium-rich soil with a high geological background.

[0022] As a further improvement of the application of the present invention: promoting crop growth.

[0023] As a further improvement of the application of the present invention: improving the pH of high-cadmium and selenium-rich soil, thereby realizing the ecological restoration of high-cadmium and selenium-rich soil. Specifically: increasing the pH of high-cadmium and selenium-rich soil, thereby reducing the cadmium accumulation in crops and increasing the selenium level in crops, thereby realizing the safe utilization of high-cadmium and selenium-rich soil.

[0024] As a further improvement of the application of the present invention: the crops are, for example, Shanghaiqing ( Brassica rapa subsp. chinensis ).

[0025] The present invention also simultaneously provides a microbial preparation for alleviating cadmium toxicity in Shanghaiqing and increasing selenium accumulation in crops, and the active ingredient of the microbial preparation contains Pseudomonas geniculata H225.

[0026] The present invention also provides a method for alleviating cadmium toxicity and increasing selenium accumulation in Shanghaiqing: applying Pseudomonas geniculata H225 to the soil and then planting Shanghaiqing.

[0027] A new strain of Pseudomonas geniculata was isolated from the black rock series soil in the geological high-background area in the present invention. Its morphological characteristics are as follows: forming round, semi-transparent, smooth and light yellow colonies on LB medium; through molecular phylogenetic analysis, this strain is classified into the genus Pseudomonas ( Pseudomonas ), and it is named Pseudomonas geniculata ( Pseudomonas geniculata‌ ) H225, and the preservation number is CGMCC No. 25640.

[0028] The culture medium of strain H225 is LB medium, and the suitable culture conditions for this strain are a temperature of 20-30 °C. After culturing at room temperature with shaking until OD 600 is about 1.0 (the strain concentration is 2×10 9 CFU / mL), then the bacterial liquid is applied to the crop growth substrate (soil). In pot experiments, the concentration of the strain inoculated per gram of soil is 2×10 8 CFU / g to 2×10 9 CFU / g. The amount of the strain required for soil cultivation can be converted accordingly according to the amount of the strain required for the above pot experiments.

[0029] In summary, the present invention provides Pseudomonas geniculata H225 capable of producing hydrogen sulfide and the alkaloid piperidine and its application in reducing cadmium accumulation and increasing selenium accumulation (reducing cadmium and enriching selenium) in the geological high-background area.

[0030] The present invention has the following technical advantages: 1. Pseudomonas geniculata H225 has a unique metabolic mechanism, which can: 1) produce the alkaloid piperidine through metabolism, increase the soil pH, and reduce the bioavailability of cadmium in the soil; 2) produce hydrogen sulfide through metabolism, combine with cadmium ions in the soil to form a precipitate, and further reduce the cadmium absorption of crops from the soil; 3) promote the absorption of selenium by crops and improve the nutritional value of crops.

[0031] That is, Pseudomonas geniculata H225 of the present invention can simultaneously reduce cadmium accumulation, alleviate cadmium toxicity and promote selenium enrichment.

[0032] 2. When Pseudomonas geniculata H225 is applied to cadmium-polluted soil, it can significantly alleviate the toxic effect of cadmium stress on crop growth. Using this strain can reduce the cadmium content in vegetables, increase the selenium content, and improve the economic value of crops. It has the characteristics of simple operation, low cost and environmental friendliness, and is an effective measure suitable for cadmium pollution prevention and control in the geological high-background area.

[0033] 3. Research of the present invention shows that the application of Pseudomonas geniculata H225 provided by the present invention can alleviate the toxic effect of soil cadmium on the growth of Brassica chinensis, and the specific manifestations include: reducing the cadmium accumulation in Brassica chinensis, increasing the selenium content, and optimizing the growth indexes.

[0034] The results of pot experiments show that after applying Pseudomonas geniculata H225 to the high-cadmium and selenium-rich soil with high geological background, the cadmium content of vegetables (represented by Brassica chinensis) significantly decreases (decreases by 26.7% - 44.7%), while the selenium content significantly increases (increases by 60.5% - 245%), and it effectively promotes the growth of plants and increases the yield.

[0035] Compared with the traditional chemical remediation method, the present invention provides a green bioremediation strategy with low cost and no secondary pollution. It can not only alleviate cadmium stress, but also improve the nutritional value of agricultural products, providing a sustainable microbial solution for safe agricultural production in areas with high geological background.

[0036] In summary, although existing research has 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, and it is difficult to take into account both crop safety and nutritional value. The Pseudomonas geniculata H225 of the present invention effectively reduces cadmium accumulation in crops with its unique metabolic mechanism and dual regulatory effects (i.e., increasing pH through alkaloids and generating hydrogen sulfide to precipitate cadmium), while improving the bioavailability of selenium, thus alleviating cadmium toxicity and improving the quality of agricultural products. So far, there has been no report on the microbial remediation technology that can significantly reduce cadmium content and increase selenium accumulation while increasing the yield for Brassica chinensis, a vegetable with high cadmium enrichment. The discovery of H225 provides an innovative and efficient biological solution for farmland pollution remediation and the development of functional agricultural products in areas with high geological background. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The following further elaborates on the specific implementation manners of the present invention with reference to the accompanying drawings.

[0038] Figure 1 It is the colony morphology diagram of strain H225.

[0039] Figure 2 It is the phylogenetic tree based on the 16S rRNA sequence of strain H225.

[0040] Figure 3 It is the pH change after culturing with cadmium ions added to LB medium and under cadmium stress of strain H225; Figure 3 In it: (A) is the pH change after culturing in LB medium with 0 - 200 mg / L cadmium ion stress at 180 r / min for 48 h; (B) pH change after adding strain H225 to LB medium under 0 - 200 mg / L cadmium ion stress and culturing for 48 h at 180 r / min; (C) Linear relationship between OD 600 and pH of the strain after adding 0 - 200 mg / L cadmium ion stress.

[0041] Figure 4 It is the GC - MS spectrum of the metabolites of strain H225, and the substance pointed by the arrow represents the characteristic peak of alkaloid Piperidines.

[0042] Figure 5 To confirm that strain H225 produces hydrogen sulfide gas during the culturing process.

[0043] Figure 6 It is the figure showing the effect of applying strain H225 on the growth of Shanghaiqing plants in the pot experiment; Figure 6 Among them: (A) Photo of the plant growth trend; (B) Plant height; (C) Fresh weight of leaves.

[0044] Figure 7 It is Figure 6 The statistical chart of the pH of the soil for Shanghaiqing growth and the cadmium and selenium contents in the plant leaves in; Figure 7 Among them: (A) pH of the soil for Shanghaiqing growth; (B) Cadmium concentration; (C) Selenium concentration. Specific implementation mode

[0045] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0046] The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0047] Example 1: Isolation and identification of strain H225 LB agar medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar, dissolved in 1000 mL of ultrapure water. Carry out routine sterilization treatment before use (sterilize at 1.1 atmospheres and 121 °C for 20 min), and the pH is neutral.

[0048] Weigh 10 g of the soil developed from the high-cadmium and selenium-rich black rock series in Xiaguang Town, Kaihua County, Quzhou City, Zhejiang Province, and put it into a triangular flask containing 100 mL of sterilized normal saline. Shake it at 180 r / min on a constant temperature shaker at 28 °C for 30 minutes, let it stand for 5 minutes, suck out the supernatant and dilute it step by step (10 - 1000 times). According to the conventional dilution plate method, separate it on LB agar medium to obtain several test strains. Among them, the colony characteristics of strain H225 are shown in Figure 1 As shown, the colonies appear in small dots, being round, semi-transparent, smooth, and light yellow colonies.

[0049] The determination of the 16S rRNA gene sequence of strain H225 was completed by Hangzhou Branch of Beijing Tsingke Biotechnology Co., Ltd. After submitting this sequence to the nucleotide database of the National Center for Biotechnology Information (NCBI) in the United States for comparative analysis, a phylogenetic tree of strain Pseudomonas geniculate H225 was drawn by Mega software, and the result is shown in Figure 2 As shown. The Bootstrap support rate of this phylogenetic tree is relatively high (96% - 98%), indicating that these evolutionary relationships are relatively stable and highly credible. The sequence similarity of the 16S rRNA gene of strain H225 and Pseudomonas geniculata strain XJUHX 18 reaches 99.72%, so it is classified into the species of Pseudomonas geniculata ([[]] Pseudomonas geniculata ).

[0050] Based on the above colony characteristics and molecular biology identification, strain H225 was identified as Bacteria, Pseudomonadota, Gammaproteobacteria, Pseudomonadales, Pseudomonadaceae, genus Pseudomonas ), species of Pseudomonas geniculata ( Pseudomonas geniculata ). Therefore, it was named Pseudomonas geniculata Pseudomonas geniculata H225.

[0051] The strain H225 of the present invention has the deposit name: Pseudomonas geniculata Pseudomonas geniculata‌ H225, depositary institution: China General Microbiological Culture Collection Center, deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC NO. 25640, deposit date: September 2, 2022.

[0052] Example 2: Analysis of the physiological and biochemical characteristics of strain H225 At 28 °C and 180 r / min, the LB liquid medium without the addition of strain H225 was cultured for 48 hours under different cadmium concentration gradients (0 - 200 mg / L), and the pH of the medium before and after culture was measured. The results are as Figure 3 shown in (A). The left and right columns in the figure represent the pH values before and after culture respectively. The pH value (y) and cadmium concentration (x) were fitted. The curve in front (B) represents the fitting result before culture, and the curve behind (A) represents the fitting result after culture, where R 2 reflects the correlation. 1% of strain H225 was added to the LB liquid medium with the same gradient of cadmium concentration. After shaking culture for 48 hours (under the same culture conditions), the pH of the medium was measured. The results are as Figure 3 shown in (B). Subsequently, for Figure 3 each treatment group in (B), the pH value (Y) and OD 600 (X) were fitted, and R 2 was calculated to evaluate the correlation. The results are as Figure 3 shown in (C).

[0053] The results show that, as Figure 3 shown: after adding cadmium ions to the medium, the pH showed an obvious 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 would lead to a decrease in the pH of the medium. In addition, the initial pH of the LB liquid medium was maintained at about 7.0, while when strain H225 was inoculated, the environmental pH increased significantly, and the lowest pH also reached above 8.0, indicating that H225 could still increase the environmental pH under the stress of different cadmium ion concentrations. Further analysis found that under different cadmium concentration conditions, there was a significant positive linear correlation between the OD 600 (bacterial growth) of strain H225 and the pH (R 2 = 0.9302), indicating that the increase in pH might be related to the metabolic activities of strain H225, and the magnitude of its effect was proportional to the growth degree 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 - polluted environments.

[0054] To explore the possible mechanism of the increase in pH, strain H225 was cultured at 28 °C and 180 r / min for 48 h. The cultured bacterial liquid was analyzed by gas chromatography - mass spectrometry (GC - MS). The results showed that there were characteristic peaks of Piperidines in the metabolites of strain H225 (as Figure 4 shown), indicating that strain H225 might cause the alkalization of the medium by metabolically releasing alkaloid substances such as piperidine.

[0055] In a sealed flask containing 20 mL of LB liquid medium, cadmium (Cd), selenium (Se), and 1% strain H225 were added according to different treatment groups. After inoculating 1% strain H225, the culture was incubated at 28 °C and 180 r / min for 48 h. After the incubation, a filter paper soaked in an aqueous solution of lead nitrate (Pb(NO3)2) with a mass concentration of 0.1% was fixed at the rubber stopper of the flask, and the flask was left standing for 10 min to observe the change in the color of the filter paper (as Figure 5 shown). The experimental groups were as follows: CK (control group): Only inoculated with strain H225, without Cd or Se.

[0056] Cd: Added 3 mg / L cadmium, without inoculating strain H225.

[0057] Cd + H225: Added 3 mg / L cadmium and inoculated with 1% strain H225.

[0058] Se: Added 1 mg / L selenium, without inoculating strain H225.

[0059] Se + H225: Added 1 mg / L selenium and inoculated with 1% strain H225.

[0060] Cd + Se: Added 3 mg / L cadmium and 1 mg / L selenium, without inoculating strain H225.

[0061] Cd + Se + H225: Added 3 mg / L cadmium and 1 mg / L selenium, and inoculated with 1% strain H225.

[0062] Result analysis: The experimental results showed that black precipitates were formed on the lead nitrate test paper in the experimental groups containing H225, indicating that strain H225 could stably produce hydrogen sulfide (H2S) regardless of the presence of cadmium and selenium. The lead ions (Pb 2+ ) in the filter paper combined with sulfide ions (S 2- ) to form lead sulfide (PbS) precipitates, presenting a black color, further verifying the H2S production ability of strain H225.

[0063] The above results indicated that strain H225 had the ability to regulate the pH of the medium ( Figure 3 ), could increase the environmental pH by producing alkaloids such as piperidine ( Figure 4 ), and at the same time had the metabolic characteristic of producing hydrogen sulfide ( Figure 5 ), which was beneficial for its cadmium reduction and selenium accumulation bioremediation in high-cadmium and selenium-rich soils.

[0064] Example 3: Pot experiment on applying H225 to high-cadmium and selenium-rich soils with high geological background to alleviate cadmium stress and promote selenium accumulation The soil for the pot experiment was taken from the high-cadmium and selenium-rich soil developed from the black rock series in Xiaguang Town, Kaihua County, Quzhou City, Zhejiang Province. After the soil was collected, it was air-dried naturally (to a moisture content ≤ 10%) and filtered through a 2-mm sieve for standby. This high-cadmium and selenium-rich soil had a cadmium content of 4.447 mg / kg, a selenium content of 0.406 mg / kg, and a pH of 5.

[0065] The experiment was carried out in a greenhouse in Hangzhou, Zhejiang Province from October to November 2024. A total of 3 treatment groups were set up: 1) Control CK group: High-cadmium and selenium-rich soil developed from the black rock series; 2) 10 8 CFU / g soil treatment group: The concentration of strain H225 in each gram of soil was 10 8 CFU / g; 3) 10 9 CFU / g soil treatment group: The concentration of strain H225 in each gram of soil was 10 9 CFU / g.

[0066] One inoculation loop of strain H225 was cultured in 100 mL (placed in a 250-mL conical flask) of LB liquid medium at 28 °C and 180 r / min for 48 h to obtain an inoculation solution; the inoculation solution was inoculated into the LB liquid medium at a volume ratio of 1% and cultured at 28 °C and 180 r / min for 10 h to obtain a bacterial solution with a concentration of 2×10 9 CFU / mL.

[0067] 25 mL and 250 mL of the H225 bacterial solution were respectively centrifuged at 4500 r / min for 10 min to obtain bacterial cells. The bacterial cell precipitate was dispersed in 50 mL of pure water, stirred evenly, and immediately dispersed in the high-cadmium and selenium-rich soil (500 g) with a high geological background.

[0068] Each treatment was repeated three times. Each repetition included a plastic pot containing 500 g of soil. Three healthy and plump Shanghaiqing seeds were sown in each pot and placed under natural light and temperature to grow. Water was applied regularly to ensure the water required for the normal growth of the plants. After 45 days of planting, the plant height and fresh weight of the above-ground part of Shanghaiqing were measured. At the same time, the pH of the harvested soil was measured, and the cadmium and selenium contents of the edible leaf parts of Shanghaiqing were measured using the microwave digestion method.

[0069] In this example, the effects of applying strain H225 on the growth, yield, and soil element content of Shanghaiqing are as shown in Figure 6 and Figure 7 shown. The results showed that in the untreated CK group, the growth of Shanghaiqing plants was slow, and yellowing occurred in some leaves. After applying strain H225, the growth of Shanghaiqing was significantly improved. Compared with the CK group, after applying 108 After the treatment with CFU / g strains, 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 leaf fresh weight 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%. 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 of the leaves was 10 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.

[0070] 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.

[0071] The present invention was found in the process of invention: cadmium-resistant PGPR strains ( Pseudomonas chlororaphis ), Pseudomonas libanensis MY, and Pseudomonas alcaliphila sp. MBR do not involve alkaloid piperidine in their fermentation products; potted plants were grown with reference to Example 3. As far as the soil is concerned, these strains did not significantly improve the pH. As far as the leaf selenium content is concerned, the strain-added group only slightly increased compared with the CK group, with an increase of less than 20%. As far as the leaf fresh weight is concerned, the strain-added group only slightly increased compared with the CK group, with an increase of less than 15%.

[0072] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Pseudomonas geniculate Pseudomonas geniculata )H225, characterized by: Deposit number: CGMCC No.25640.

2. Use of Pseudomonas geniculate H225 as described in claim 1 in alleviating cadmium stress in crops.

3. The use according to claim 2, characterized in that: By applying Pseudomonas geniculate H225 to the soil, cadmium accumulation in crops can be reduced and selenium absorption can be increased.

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

5. The use according to any one of claims 2 to 4, characterized in that: Promote crop growth.

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

7. The use according to claim 6, 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.

8. The use according to claim 3, characterized in that: The crop is the vegetable Bok Choy.

9. 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 geniculate H225 as claimed in claim 1.

10. A method for alleviating cadmium toxicity and increasing selenium accumulation in Shanghai blue, characterized in that: Pseudomonas geniculate H225 was applied to the soil, and then Bohemia sinensis was planted.

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