Application of strain MFBS21 in improving tolerance of plants to metal ions

Abstract

The invention relates to the technical field of microorganisms, and discloses application of a strain MFBS21 in improving tolerance of plants to metal ions. The strain MFBS21 is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC AB 2020278T, the preservation time is 2020.11. 22, the preservation address is Wuhan University, Wuhan, China, and the strain MFBS21 is classified and named as Runula metallidurans sp. Nov. The strain MFBS21 is named as Runula metallidurans sp. Nov. The strain MFBS 21 can tolerate 600 [mu] M of lead, nickel or manganese ions, and the IAA yield of the strain MFBS 21 is as high as 25.57 mg / L, so that the strain MFBS 21 not only can enhance the tolerance of plants to heavy metal environmental stress, but also can promote the growth of the plants under the heavy metal pollution condition, is used for preparing a heavy metal polluted soil remediation agent, and has great application value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to the application of strain MFBS21 in improving the tolerance of plants to metal ions. Background Technology

[0002] For a long time, the mining of heavy metals in mining areas has caused serious damage to the soil and environment around the mining areas due to factors such as lack of mining technology, funding, and management, resulting in a large number of ecological and environmental problems. Mining wastelands generally contain large amounts of heavy metals, with tailings and abandoned low-grade ores having the highest heavy metal content. Heavy metals are released and migrate into the soil and rivers through surface biogeochemical processes. This water contaminated with heavy metals then enters farmland through irrigation and enters the human body through the food chain, thus posing a serious threat to the health and living environment of residents near the mining areas.

[0003] Heavy metals are a significant pollutant in agricultural environments and products. Common remediation technologies for heavy metal pollution in soil include physical remediation, chemical remediation, and bioremediation. Physical remediation mainly includes measures such as soil replacement, soil exchange, and deep tillage. By mixing the soil with polluted soil through these methods, the content of heavy metals in the soil can be reduced, thus minimizing their toxicity to the soil-plant system. Chemical remediation involves introducing soil conditioners, which reduce the bioavailability of heavy metals through adsorption, oxidation-reduction, and precipitation. Bioremediation utilizes the flocculation, absorption, accumulation, and enrichment of heavy metals in wastewater by microorganisms or plants, including biosorption, bioflocculation, and phytoremediation. Chemical and physical methods suffer from limitations such as technological constraints, high economic costs, and a high risk of secondary pollution. Bioremediation, due to its safety, harmlessness, and high efficiency, has become a current research focus.

[0004] To address the above technical problems, this invention provides a heavy metal-tolerant strain MFBS21 and its applications. Summary of the Invention

[0005] The purpose of this invention is to provide the application of strain MFBS21 in improving the tolerance of plants to metal ions. Strain MFBS21 can tolerate 600 μM of lead, nickel, or manganese ions, and its IAA yield is as high as 25.57 mg / L. Therefore, strain MFBS21 can not only enhance the tolerance of plants to heavy metal environmental stress, but also promote plant growth under heavy metal pollution conditions. It has great application value for preparing heavy metal polluted soil remediation agents.

[0006] To achieve the above objectives, this invention provides a heavy metal-tolerant strain MFBS21, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC AB 2020278.T The deposit date is October 22, 2020, the deposit location is Wuhan University, Wuhan, China, and the classification name is... Runella metallidurans sp. nov . It should be noted that strain MFBS21 has been published by the China Center for Type Culture Collection. If the public is interested, they can contact the China Center for Type Culture Collection of Wuhan University to obtain it.

[0007] Furthermore, the 16S rDNA sequence of strain MFBS21 is shown in SEQ ID NO.1.

[0008] Furthermore, the IAA yield of strain MFBS21 was 25.57 mg / L.

[0009] Furthermore, strain MFBS21 is tolerant to 600 μM lead, nickel, or manganese ions.

[0010] To achieve the above objectives, the present invention also provides the application of a heavy metal-tolerant strain MFBS21 in improving the tolerance of plants to metal ions, wherein the metal ions are lead, nickel or manganese ions.

[0011] Furthermore, the concentration of the metal ions, namely lead, nickel, or manganese ions, is 0-600 mg / kg.

[0012] Furthermore, the present invention also provides the application of a heavy metal-tolerant strain MFBS21 in the preparation of microbial preparations that enhance the tolerance of plants to metal ions, wherein the metal ions are lead, nickel or manganese ions, and the concentration of the metal ions is 0-600 mg / kg.

[0013] Furthermore, the present invention also provides a microbial preparation for improving the tolerance of plants to metal ions, wherein the active ingredient of the microbial preparation is the above-mentioned strain MFBS21.

[0014] The advantages and positive effects of the strain MFBS21 described in this invention in improving the tolerance of plants to metal ions are as follows: 1. The IAA yield of strain MFBS21 in this invention is 25.57 mg / L, indicating that strain MFBS2 can regulate cell growth and differentiation, thereby promoting plant growth and enhancing the plant's tolerance to environmental stress.

[0015] 2. Experiments in this invention show that strain MFBS21 can tolerate 600 μM lead, nickel or manganese ions and promote plant growth under heavy metal pollution conditions, indicating that strain MFBS21 has great remediation potential for heavy metal lead and nickel contaminated soil and can be used to prepare heavy metal contaminated soil remediation agents.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 This is a plate of strain MFBS21 cultured on PYG medium for 5 days in an embodiment of the present invention; Figure 2 The above are the scanning electron microscopy results of strain MFBS21 in the embodiments of the present invention; Figure 3 The Gram staining results of strain MFBS21 in the embodiments of the present invention; Figure 4 The polar lipid profile of strain MFBS21 in this embodiment of the invention is shown, where A is the amino lipid profile, B is the phospholipid profile, and C is the total lipid profile. Figure 5 This is the whole genome phylogenetic tree of strain MFBS21 in this embodiment of the invention; Figure 6 Qualitative detection of IAA production by strain MFBS21 in this embodiment of the invention; Figure 7 This is a standard curve of IAA content in an embodiment of the present invention; Figure 8 The results show the tolerance of strain MFBS21 to heavy metal ions in the embodiments of the present invention; Figure 9 This invention provides an example of strain MFBS21's resistance to Pb in plants. 2+ The results showed that A was 200 mg / kg, B was 400 mg / kg, and C was 600 mg / kg. Figure 10 The results of plant tolerance to Ni for strain MFBS21 in the embodiments of the present invention are shown, where A is 200 mg / kg, B is 400 mg / kg, and C is 600 mg / kg.

[0018] Information on the preservation of biological materials Strain MFBS21 (MFBS21 T It is deposited at the China Center for Type Culture Collection, accession number CCTCC AB2020278 T The deposit date is October 22, 2020, the deposit location is Wuhan University, Wuhan, China, and the classification name is... Runella metallidurans sp. nov. . Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0022] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0023] Example 1: Isolation and Identification of Strain MFBS21 1. Isolation and screening of strain MFBS21: Strain MFBS21 was collected and isolated from fermented bedding material in a pig farm in Yan'an City. The specific procedure was as follows: The leachate from the fermented bedding material was collected, serially diluted with sterile water, and then plated onto PYG medium (peptone, 5 g / L; yeast extract, 0.2 g / L; glucose, 5 g / L; sodium chloride, 0.5 g / L; magnesium sulfate, 1.5 g / L; pH=7.5). It was incubated at 25°C for 5 days. Single colonies were picked and purified using the streak plating method to obtain pure strains. Plates cultured on PYG medium for 5 days are shown below. Figure 1 As shown.

[0024] 2. Multiphasic classification and identification of fungal strains: (1) Phenotypic characteristics: The growth of strain MFBS21 was observed under different temperatures, salinities, and pH values. The range of achievable growth temperature and the optimal growth temperature range were determined at 4℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, and 50℃. The salinity range for growth was 0%, 0.5%, 1%, 2%, 3%, 4%, 5%, and 10% NaCl gradients (w / v). The pH range for growth was 3-12, with 1 pH value measured for each gradient. Bacterial morphology was observed using a scanning electron microscope. Figure 2 Gram staining was performed using a Gram staining kit. Figure 3 Scanning electron microscopy (SEM) images show that the bacteria are elongated rod-shaped cells, approximately 0.5-0.8 μm × 5.7-7.9 μm in size, and that they produce a large amount of secretions. Gram staining reveals that the bacteria are pale red, indicating that they are Gram-negative.

[0025] The strain MFBS21 can grow at 20-40℃, with the optimal growth at 25-30℃; it can grow at pH 7-10, with the optimal growth at pH 8-9; and it can grow at NaCl concentrations of 0-3%, with the optimal growth at 0-0.5%.

[0026] (2) Chemical characteristics: Polar lipids were extracted and separated after freeze-drying of bacterial cells and analyzed using two-dimensional thin-layer chromatography. The results are as follows: Figure 4 As shown. From Figure 4 The results show that the polar lipids of this bacterium are mainly phosphatidylethanolamine, four unknown aminophospholipids (AL1-AL4), four unknown polar lipids (L1-L4), and one unknown phospholipid (PL1). The fatty acid composition of the whole-cell fatty acid extract was analyzed by gas chromatography using the MIDI microbial identification system, and the results are shown in Table 1.

[0027] Table 1. Differences in fatty acids between strain MFBS21 and the reference strain.

[0028] The values ​​in Table 1 represent the percentage of each fatty acid in the total fatty acids, and only fatty acids present in more than 1% of at least one strain are shown. “–” indicates not detected; “TR” indicates trace (<1%).

[0029] (3) Genomic characteristics: The 16S rRNA sequence of strain MFBS21 was amplified by PCR using standard primer pairs 27F and 1492R. Sequencing was performed using the Sanger method, and sequence alignment was conducted. Based on the 16S rRNA sequence alignment results, the complete genome sequences of similar strains were downloaded, and a phylogenetic tree was constructed using the NJ method based on the complete genome sequences. The results are as follows: Figure 5 As shown. From Figure 5 As can be seen, strain MFBS21 forms a stable branch with high support with Runella zeae DSM19591, indicating that MFBS21 is the closest in evolutionary distance to Runella zeae DSM19591, and it can be determined that MFBS21 belongs to the genus Runella.

[0030] The 16S rDNA sequence of strain MFBS21 is as follows (SEQ ID NO.1):

[0031] The comparison results in the EzBioCloud 16S database show that it is most similar to the species Runella zeae NS12. T The similarity was 98.58%, and the most similar microorganism found in the NCBI comparison was Runella zeae NS12. T The similarity was 98.28%. Both were below the threshold of 98.7% for distinguishing new species of microorganisms, therefore strain MFBS21 was considered a new species of the genus Runella. The dDDH and ANI values ​​of strain MFBS21 and similar strains were calculated using the Genome-to-Genome Digital Calculator (GGDC), Orthologous, and Average Nucleotide Identity Tool (OAT), respectively. The results are shown in Table 2: the maximum dDDH and ANI of this strain compared to the reference strain were 47% and 92.4386%, respectively, which are far lower than the generally accepted species classification thresholds of 70% and 95-96%, further supporting the view that strain MFBS21 is a new species of the genus Runella.

[0032] Table 2 Comparison of dDDH and ANI values ​​between strain MFBS21 and similar strains

[0033] The phenotypic differences between strain MFBS21 and the reference strain are compared below, and the results are shown in Table 3.

[0034] Table 3 Phenotypic differences between strain MFBS21 and the reference strain

[0035] In Table 3, 1 is strain MFBS21, 2 is Runella zeae DSM 19591, 3 is Runella aurantiacaYX9, 4 is Runella soli 15J11-1, and 5 is Runella palustris HMF3829. "+" indicates positive, "-" indicates negative, and "w" indicates weak positive.

[0036] Gene annotation and database comparison were performed on the sequencing results of strain MFBS21 to obtain potential heavy metal resistance genes, and the results are shown in Table 4: Table 4 Heavy metal resistance genes

[0037] In summary, strain MFBS21 differs from similar strains in some phenotypic, chemical, and genomic characteristics, confirming that strain MFBS21 is a new species of the genus Runella.

[0038] Example 2: Study on the production of indoleacetic acid by strain MFBS21 Indole-3-acetic acid (IAA) is an endogenous auxin widely found in plants, belonging to the indole class of compounds. IAA plays a crucial role in plant growth and development, regulating cell growth and differentiation. Therefore, microorganisms that produce IAA can promote plant growth and enhance their tolerance to environmental stresses.

[0039] Prepare 100 ml of sterilized LB medium (peptone, 10 g / L; yeast extract, 5 g / L; sodium chloride, 10 g / L). Prepare a 1 g / L tryptophan solution using sterile water, and filter it through a 0.22 μm sterile membrane for sterilization. Add 10 ml of the tryptophan solution to 100 ml of LB medium to prepare LB medium with a final tryptophan concentration of 100 mg / L. Inoculate OD. 600 One ml of MFBS21 bacterial suspension with a concentration of 0.5 was cultured at 25°C and 180 rpm for 3 days with shaking for qualitative and quantitative detection of indole-3-acetic acid (IAA).

[0040] Qualitative detection: Take 1 ml of bacterial culture and add 1 ml of Salkowski colorimetric solution (1 ml of 0.5 mol / L FeCl3 and 50 ml of 35% HClO4 mixed thoroughly). Incubate in the dark for 30 minutes. A red color indicates a positive result, signifying the production of IAA (inorganic acid). Figure 6 ).

[0041] Quantitative test: Centrifuge 5 ml of bacterial culture at 8000 r / min for 10 min, take 3 ml of supernatant into a clean test tube, add 3 ml of colorimetric solution, place in the dark for 30 min, and then measure the OD under a spectrophotometer. 530 Calculate IAA production based on the standard curve.

[0042] The standard curve is plotted as follows: Weigh an appropriate amount of IAA (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., 98%, biotechnology grade), dissolve it in a small amount of ethanol, and then prepare a 100 mg / L stock solution using sterile distilled water. Then, perform a gradient dilution to prepare working solutions with concentrations of 0, 1, 2.5, 5.0, 10.0, 12.5, 25.0, 50.0, and 100.0 mg / L. Take eight dry test tubes (sizes 0-7), add 3 mL of the IAA concentration working solution to each tube, and then add 3 mL of Salkowski's colorimetric solution (1 mL 0.5 mol / L FeCl3, 50 mL 35% HClO4). Mix well, incubate in the dark for 30 min, and then measure the absorbance (OD) at 530 nm. 530 A standard curve was plotted with OD values ​​on the x-axis and IAA concentration (mg / L) on the y-axis. Figure 7 ).

[0043] Quantitative experiments were conducted to determine the OD levels of strain MFBS21. 530 The absorbance was 0.1487. Substituting this into the standard curve formula Y = 170.88X + 0.1598 (where Y is the IAA yield in mg / L, and X is the OD), we get the absorbance. 530 (Based on absorbance values), it can be calculated that strain MFBS21 produced up to 25.57 mg / L of IAA under the experimental conditions.

[0044] Quantitative analysis showed that strain MFBS21 produced up to 25.57 mg / L of IAA under the experimental conditions.

[0045] Example 3: Heavy metal resistance of strain MFBS21 First, the following heavy metal salt reagents (analytical grade) – MnCl₂·4H₂O, CuSO₄·5H₂O, ZnSO₄·7H₂O, PbC₄H₆O₄·3H₂O, CdCl₂·5 / 2H₂O, NiCl₂·6H₂O, and K₂CrO₄ – were dissolved in an appropriate amount of sterile water to prepare a solution with a heavy metal ion concentration of 100 mM. This solution was then filtered through a 0.22 μm filter for sterilization. In a sterile 48-well plate, 1.0 ml of sterile PYG liquid medium was added to each well. The first row and last column served as blank controls. An appropriate amount of heavy metal solution was added to each of the first seven columns to achieve the heavy metal ion concentrations shown in the figure. OD₂ was added to each well. 600 15 μL of MFBS21 bacterial suspension (0.5 g / L) was incubated at 25°C with shaking at 180 rpm for 3 days. The results are as follows: Figure 8 As shown. From Figure 8 It can be seen that strain MFBS21 can tolerate 600 μM lead, nickel and manganese ions.

[0046] Example 4: Effects of strain MFBS21 on plant tolerance to heavy metals To investigate the effect of strain MFBS21 on plant tolerance to heavy metals, alfalfa was selected as the target plant, and its growth in vermiculite containing 600 mg / kg of PbC4H6O4·3H2O and NiCl2·6H2O was examined.

[0047] Vermiculite was first sterilized, then dispensed into small flowerpots, weighed, and supplemented with appropriate amounts of heavy metal salt solution to achieve concentrations of 200 mg / kg, 400 mg / kg, and 600 mg / kg. Alfalfa seeds were disinfected with a 5% sodium hypochlorite solution, washed twice with sterile water, and germinated on water agar. Seeds that germinated and showed uniform growth were selected for planting, with 6 plants per pot. Strains MFBS21 were cultured in PGY medium for 3 days, and the bacterial cells were collected by centrifugation, resuspended in sterile water, and their OD was adjusted. 600 =1.5, add 60 ml of bacterial suspension to each pot. The plant growth period is 45 days, with 12 hours of light and 12 hours of darkness per day. The experiment is set up with 3 groups, each with 3 replicates. The control group CK is supplemented with water as needed. The heavy metal groups are supplemented with heavy metal salt concentrations of 200 mg / kg, 400 mg / kg, and 600 mg / kg. The treatment groups are inoculated with MFBS21 bacterial suspension at the same time as the heavy metal salt is added.

[0048] The results are as follows Figure 9 and Figure 10 As shown in the figure, compared with the control (CK), the addition of heavy metals inhibited the growth of alfalfa. However, the addition of strain MFBS21 significantly promoted plant growth, resulting in better growth not only than the group with added heavy metals but also than the CK group. This indicates that strain MFBS21 can not only promote alfalfa growth but also enhance its tolerance to heavy metals lead and nickel. It has significant remediation potential for soils contaminated with heavy metals lead and nickel.

[0049] Therefore, this invention utilizes the aforementioned strain MFBS21 to enhance plant tolerance to metal ions. Strain MFBS21 can tolerate 600 μM of lead, nickel, or manganese ions, and its IAA yield is as high as 25.57 mg / L. Thus, strain MFBS21 not only enhances plant tolerance to heavy metal environmental stress but also promotes plant growth under heavy metal pollution conditions, making it highly valuable for preparing heavy metal contaminated soil remediation agents.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of the strain MFBS21 for increasing the tolerance of a plant to metal ions, characterized in that: The metal ion is lead, nickel or manganese ion; the strain MFBS21 is preserved in China Center for Type Culture Collection, with the preservation number CCTCC AB2020278 T , the preservation time is October 22, 2020, the preservation address is Wuhan University in Wuhan, China, and the classification name is Runella metallidurans sp. nov. .

2. Use according to claim 1, characterized in that: The concentration of the metal ion is 0-600 mg / kg.

3. Use according to claim 1, characterized in that: The IAA production of strain MFBS21 was 25.57 mg / L.

4. Use of the strain MFBS21 for increasing the tolerance of a plant to lead ions, characterized in that: The lead ion concentration is 0-600 mg / kg; the strain MFBS21 is preserved in China Center for Type Culture Collection, the preservation number is CCTCC AB2020278 T , the preservation time is October 22, 2020, the preservation address is Wuhan University, Wuhan, China, and the classification name is Runella metallidurans sp. nov. .

5. Use of the strain MFBS21 for increasing the tolerance of plants to nickel ions, characterized in that: The nickel ion concentration is 0-600 mg / kg; the strain MFBS21 is preserved in China Center for Type Culture Collection, the preservation number is CCTCC AB2020278 T , the preservation time is 2020.10.22, the preservation address is Wuhan University in Wuhan, China, and the classification name is Runella metallidurans sp. nov. .

6. Use of the strain MFBS21 for increasing the tolerance of a plant to manganese ions, characterized in that: The manganese ion concentration is 0-600 mg / kg; the strain MFBS21 is preserved in China Center for Type Culture Collection, the preservation number is CCTCC AB2020278 T , the preservation time is 2020.10.22, the preservation address is Wuhan University in Wuhan, China, and the classification name is Runella metallidurans sp. nov. .

7. Use of the strain MFBS21 for the preparation of a microbial preparation for increasing the tolerance of plants to metal ions, characterized in that: The metal ion is lead, nickel or manganese ion; the strain MFBS21 is preserved in China Center for Type Culture Collection, with the preservation number CCTCC AB 2020278 T , the preservation time is October 22, 2020, the preservation address is Wuhan University in Wuhan, China, and the classification name is Runella metallidurans sp. nov. .

8. Use according to claim 7, characterized in that: The concentration of the metal ion is 0-600 mg / kg.

9. A microbial preparation for increasing the tolerance of plants to metal ions, characterized in that: The metal ion is lead, nickel or manganese ion; the effective component of the microbial preparation is strain MFBS21, which is preserved in China Center for Type Culture Collection with the preservation number CCTCC AB 2020278 T , the preservation time is October 22, 2020, the preservation address is Wuhan University in Wuhan, China, and the classification name is Runella metallidurans sp. nov. .

10. The microbial preparation according to claim 9, characterized in that: The concentration of the metal ion is 0-600 mg / kg.

Images