Stenotrophomonas 215 and application thereof in relieving cadmium stress of crops

Abstract

The invention discloses stenotrophomonas 215 and application thereof in relieving cadmium stress of crops, and belongs to the technical field of microorganisms. The stenotrophomonas sp. Is separated from paddy field soil, and is named as stenotrophomonas sp. 215, and the preservation number of the stenotrophomonas sp. 215 is GDMCC (China General Microbiological Culture Collection Center) No.65475. The stenotrophomonas 215 provided by the invention is applied to cadmium-contaminated soil, can relieve the toxic action of cadmium stress on crop growth, especially for wheat production, can remarkably reduce the content of cadmium in wheat growing in the cadmium-contaminated soil by utilizing the strain, can remarkably improve the wheat yield, has obvious effects of relieving cadmium stress and promoting growth, and has good application prospects. The method is an environment-friendly alleviation measure.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to an oligotrophic bacterium 215 and an application thereof in alleviating cadmium stress of crops. Background Art

[0002] With the rapid development of industrialization and urbanization, soil environmental safety issues are becoming increasingly serious. Cadmium (Cd) pollution is the most prominent among soil heavy metal pollution. Because cadmium has strong resistance to chemical and microbial degradation in soil, it exists in the soil for a long time and is transferred to the human body through the food chain, which seriously threatens human health and may induce lung cancer, renal dysfunction and bone diseases.

[0003] Wheat (Triticum aestivum L.) is the staple food for more than half of the world's population, and its safety has attracted much attention. However, wheat and its derivatives are the main sources of dietary cadmium exposure. Compared with other cereals such as rice and corn, wheat is more efficient in absorbing and accumulating cadmium, which is mainly attributed to its stronger root-to-stem cadmium transport capacity. Therefore, taking effective measures to minimize its absorption and accumulation of cadmium is key to ensuring wheat productivity and food safety.

[0004] At present, the remediation technology of soil heavy metal pollution mainly includes physical remediation, chemical remediation and biological remediation methods, among which microbial remediation has attracted much attention because of its green and environmental protection. Microbial remediation mainly relies on its biological transformation, adsorption and fixation of heavy metals, thereby alleviating the toxicity of heavy metals to crops. For example, Pseudomonas sp. K32 promotes plant growth by producing auxins, nitrogen fixation and phosphate dissolution. When inoculated in cadmium-contaminated soil where rice is grown, it has the effect of promoting the growth of rice seedlings and reducing cadmium absorption (Pramanik et al., Chemosphere, 2021, 129819). In addition, Trichoderma asperellum F1 has the ability to solidify copper in the soil, thereby reducing its absorption by corn and its transport to the aboveground part of the plant, alleviating the toxic effects of copper on corn (Amanullah et al., Plants, 2023, 958).

[0005] Regarding the remediation of cadmium pollution in wheat-growing soil, existing technologies such as patent document CN 116555079A disclose a non-decarboxylating Leclercia (Leclercia adecarboxylata) FCH-CR2 and its application in treating heavy metal pollution. The microorganism applied to the plant growth matrix can reduce the accumulation of cadmium in wheat leaves and roots and improve wheat's tolerance to heavy metals, but its effect on wheat yield and grain cadmium content has not been reported.

[0006] So far, there has been no report on microorganisms that can increase wheat yield while reducing the cadmium content in grains, and there is still a shortage of effective microorganisms against cadmium toxicity in wheat. Summary of the invention

[0007] The purpose of the present invention is to provide a microorganism that can effectively alleviate the cadmium poisoning of crops, apply the microorganism to the remediation of cadmium pollution in wheat-growing soil, and reduce the cadmium content of grains while increasing wheat yield.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention isolates a new Stenotrophomonas from paddy field soil, and its morphological characteristics are: forming round, translucent, smooth white colonies on R2A culture medium; its 16S rRNA sequence is shown in SEQ ID NO.1, and the strain is identified as belonging to the genus Stenotrophomonas in molecular phylogenetic taxonomy, and is named Stenotrophomonas sp. 215, which was preserved in Guangdong Provincial Microbiological Culture Collection Center on November 12, 2024, with a preservation address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a preservation number of GDMCC No. 65475.

[0010] Furthermore, the culture medium of the oligotrophomonas is R2A culture medium, and round, translucent, smooth white colonies appear on the R2A culture medium.

[0011] Furthermore, the culture conditions of the oligotrophic monocytogenes are 20-30°C.

[0012] The present invention has found that inoculating Stenotrophomonas sp. 215 into cadmium-contaminated soil can alleviate the toxic effect of cadmium on plant growth.

[0013] Therefore, the present invention provides the use of the oligotrophic mononas in alleviating cadmium stress of crops.

[0014] Furthermore, the crop is wheat.

[0015] Furthermore, the phenotypes of wheat under cadmium stress conditions include: plant growth inhibition, reduced grain yield, and increased grain cadmium content.

[0016] The present invention shows that the use of the Stenotrophomonas sp. 215 provided by the present invention can alleviate the toxic effect of soil cadmium on wheat growth, and has the characteristics of promoting wheat growth, increasing yield and reducing the heavy metal cadmium content of grains. Specifically, the manifestations of alleviating wheat cadmium stress include: promoting wheat plant growth in cadmium-contaminated soil, increasing wheat yield and reducing the cadmium content of grains.

[0017] Furthermore, the application includes: applying the Stenotrophomonas sp. 215 to a crop growth matrix.

[0018] Furthermore, the Stenotrophomonas sp. 215 was inoculated into R2A liquid culture medium and cultured at room temperature for 5 days by shaking, and then the bacterial solution was applied to the crop growth matrix.

[0019] Furthermore, 2 to 5 × 10 strains were inoculated per gram of growth medium. 6 indivual.

[0020] The present invention also provides a microbial preparation for alleviating cadmium toxicity in wheat, wherein the active ingredients of the microbial preparation include the oligotrophic mononas.

[0021] The present invention has the following beneficial effects:

[0022] The present invention provides a new Stenotrophomonas sp. 215, which is applied to cadmium-contaminated soil and can significantly alleviate the toxic effects of cadmium stress on crop growth. In particular, for wheat production, the strain can significantly reduce the cadmium content in wheat grown in contaminated soil, significantly increase wheat yield, and significantly alleviate cadmium stress and promote growth. The method for alleviating heavy metal poisoning of wheat using Stenotrophomonas sp. 215 provided by the present invention has the characteristics of easy operation, low cost and significant effect, and is an environmentally friendly mitigation measure. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the colony morphology of strain 215.

[0024] Figure 2 Phylogenetic tree based on the 16S rRNA sequence of strain 215.

[0025] Figure 3 These are photos of the effects of 215 on wheat plant growth in a pot experiment, where (A) shows the grains and (B) shows the phenotype of the wheat plant.

[0026] Figure 4 for Figure 3Statistical chart of wheat plant phenotype, yield and grain cadmium content, where (A) is plant height, (B) is ear length, (C) is single plant yield, and (D) is grain cadmium content.

[0027] Figure 5 Photos of the effects of 215 on wheat grain growth in a field trial.

[0028] Figure 6 for Figure 5 Statistical chart of wheat yield and grain cadmium content, where (A) is the number of grains per ear, (B) is the grain weight per ear, (C) is the yield per plant, (D) is the number of ears per plot, (E) is the grain yield per plot, and (F) is the Cd content of the grain. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0031] Example 1: Isolation and identification of strain 215

[0032] R2A agar medium: 0.5 g yeast extract powder, 0.5 g peptone, 0.5 g casein hydrolyzate, 0.5 g glucose, 0.5 g soluble starch, 0.3 g potassium dihydrogen phosphate, 0.024 g anhydrous magnesium sulfate, 0.3 g sodium pyruvate, 15 g agar, add 1000 mL water to dissolve. Perform routine sterilization before use.

[0033] Weigh 10g of paddy field soil from Fuyang, Hangzhou, Zhejiang Province into a conical flask filled with 100mL sterile saline, shake it at 150r / min on a 28℃ constant temperature shaker for 30 minutes, let it stand for 5 minutes, draw the supernatant and dilute it step by step (10 to 1000 times), and separate it on R2A agar medium according to the conventional dilution plate method to obtain several test strains. The colony characteristics of strain 215 are shown in Figure 1 As shown, the colonies appear as small dots, round, smooth white or milky white colonies, and appear translucent on the culture medium.

[0034] The 16S rRNA gene sequence of strain 215 was determined by Zhejiang Shangya Biotechnology Co., Ltd. The sequencing result is shown in SEQ ID NO. 1. The sequence was compared with the database of the National Center for Biotechnology Information of the United States. The result is shown in Figure 2As shown, the 16S rRNA gene sequence of strain 215 was 99.72% identical to that of Stenotrophomonas pavanii strain LMG25348 and was classified into the genus Stenotrophomonas.

[0035] Based on the above colony characteristics and molecular biological identification, strain 215 was identified as Bacteria, Pseudomonadota, Gammaproteobacteria, Lysobacterales, Lysobacteraceae, Stenotrophomonas, and therefore named Stenotrophomonas sp. 215. It was deposited in Guangdong Microbiological Culture Collection Center on November 12, 2024, with the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No. 65475.

[0036] Example 2: Potted experiment of applying strain 215 to alleviate cadmium stress in wheat

[0037] The soil for the potted experiment was collected from the experimental field of the Zijingang Campus of Zhejiang University in Hangzhou, Zhejiang Province. After the soil was collected, it was naturally air-dried and filtered through a 2 mm sieve for later use. The experiment was carried out in the greenhouse of the Zijingang Campus of Zhejiang University (Hangzhou, Zhejiang, China) from February to August 2024. There were three treatment groups: 1) CK: natural experimental field soil; 2) Cd: cadmium was added to the experimental field soil until the cadmium concentration in the soil was 20 mg kg -1 ; 3) Cd+215, cadmium contaminated soil (20 mg kg -1 ) was added with 200 mL of oligotrophomonas 215 bacterial solution. The 215 bacterial solution in the experiment was obtained by shaking culture in R2A liquid medium at room temperature for 5 days, and the inoculation concentration was 2.2×10 6 / g soil. Each treatment was repeated three times, and each replicate contained a plastic pot filled with 1kg soil, sown with 5 full Yangmai 20 seeds, and grown under natural light and temperature. When the wheat matured, the plant height, ear length, ear weight and single plant yield of wheat were measured. The harvested wheat grains were digested with concentrated nitric acid, and the cadmium content was determined by atomic absorption spectrophotometry.

[0038] The photos and element contents of the effects of strain 215 on wheat growth and yield in this example are as follows Figure 3 and Figure 4As shown. The results showed that soil cadmium pollution significantly inhibited the growth and yield of wheat, as shown by a significant decrease in plant height by 22.8%, a significant decrease in ear length by 37.3%, a significant decrease in ear grain weight by 91.5%, and a significant decrease in single plant yield by 91.5%. After applying the 215 bacterial solution, the cadmium toxicity of wheat was significantly alleviated. Compared with the cadmium treatment, the plant height of the Cd+215 group increased by 29.1%, the ear length increased by 25.7%, and the ear grain weight increased by 39.6%, with a significant alleviation effect. In addition, the cadmium content in wheat grains under cadmium pollution was 3.4 mg kg -1 , applying 215 to reduce the cadmium content in wheat grain to 2.7 mg kg -1 , a decrease of 20.6%.

[0039] The above experiments prove that the application of 215 can significantly promote wheat yield and reduce the cadmium content in wheat grains.

[0040] Example 3: Field experiment of applying 215 to alleviate cadmium stress in wheat

[0041] The experimental site was selected in a farmland contaminated by heavy metals in Huanshan Village, Fuyang, Hangzhou City, Zhejiang Province. The cadmium content of farmland soil was determined according to the HJ 804-2016 "Determination of 8 Available Elements in Soil" standard and the HJ 832-2017 "Degradation of Total Metal Elements in Soil and Sediment" standard. The results showed that the total cadmium content in the soil was 1.79 mg kg -1 The effective cadmium content is 0.84 mg kg -1 , which is a severely cadmium-contaminated soil. Based on this, a field experiment was conducted, which included two treatments: 1) Mock, naturally contaminated soil; 2) 215, where a culture of Oligotrophomonas 215 was applied to the soil. The 215 culture was obtained by shaking culture in R2A liquid medium, and the inoculation concentration of 215 was 3.6×10 6 / g soil, 2m per test plot 2 Each treatment was designed with 3 replicates and arranged in random blocks. Wheat was sown in each plot, and the plants were harvested after maturity and statistical determination of relevant indicators was performed (same as in Example 2).

[0042] In this example, after various treatments were used, the growth, yield and cadmium content of wheat in the heavy metal contaminated farmland soil were shown in Figure 2. Figure 5 and Figure 6 The wheat grain yield of the untreated control plot was 119.8 gm -2 The cadmium content in the grain is 2.2 mg kg -1 After applying 215 bacterial solution, the number of grains per ear and the weight of grains per ear increased, and the yield per plant of wheat increased, and finally the yield of wheat increased to 167.9gm -2, an increase of 40.2%. In addition, the cadmium content in wheat grains was reduced to 1.9 mg kg -1 , a decrease of 13.4%. This shows that strain 215 is effective in alleviating cadmium toxicity in wheat.

[0043] Finally, it should be noted that the above examples are only specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples, 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. A oligotrophic monocytogenes, characterized in that The Stenotrophomonas sp. was isolated from paddy field soil and named Stenotrophomonas sp.

215. It was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 12, 2024, with the deposit number GDMCC No. 65475.

2. The oligotrophic mononas according to claim 1, characterized in that The culture medium of the oligotrophic mononas is R2A culture medium, and round, translucent, smooth white colonies appear on the R2A culture medium.

3. The oligotrophic mononas according to claim 1 or 2, characterized in that The culture condition of the oligotrophic mononas is 20-30°C.

4. Use of the oligotrophic monocytogenes as described in any one of claims 1 to 3 in alleviating cadmium stress in crops.

5. The use according to claim 4, characterized in that The crop is wheat.

6. The use according to claim 5, characterized in that The phenotypes of wheat under cadmium stress conditions include: plant growth inhibition, reduced grain yield, and increased cadmium content in grains.

7. The use according to claim 4, characterized in that The application comprises: applying the Stenotrophomonas sp. 215 to a crop growth matrix.

8. The use according to claim 7, characterized in that The Stenotrophomonas sp. 215 was inoculated into R2A liquid culture medium and cultured at room temperature for 5 days through shaking, and then the bacterial solution was applied to the crop growth matrix.

9. The use according to claim 7 or 8, characterized in that 2-5×10 strains were inoculated per gram of growth medium. 6 indivual.

10. A microbial preparation for alleviating cadmium toxicity in wheat, characterized in that: The active ingredient of the microbial preparation comprises the Stenotrophomonas described in any one of claims 1-3.

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