Rhizobium YM-1, bacterial liquid and application of rhizobium YM-1 and bacterial liquid

Abstract

The invention discloses a strain of rhizobium sp. YM-1, a bacterial solution and application of the rhizobium sp. YM-1 and the bacterial solution. The preservation number of the strain of the rhizobium sp. YM-1 is CCTCC No: M 2025295. The strain is separated from argy wormwood root nodules planted in severely cadmium-polluted soil, the strain is used for root soaking or root irrigation treatment of hyperaccumulator sedum plumbizincicola, the cadmium transfer coefficient and the enrichment coefficient of the sedum plumbizincicola can be remarkably improved, and the biomass of the sedum plumbizincicola can be remarkably improved. The strain has a wide application prospect in the field of phytoremediation of heavy metal polluted environments.

Description

Technical Field

[0001] The present invention belongs to the field of microbial enhanced hyperaccumulator phytoremediation of heavy metal pollution, and particularly relates to a rhizobium YM-1, a bacterial liquid, and their applications in improving the cadmium accumulation ability of plants and promoting plant growth. Background Art

[0002] Hyperaccumulator extraction is a green, sustainable, highly tolerant, and soil structure-preserving in-situ remediation technology for cadmium-contaminated soils. Sedum plumbizincicola has a strong cadmium accumulation ability and is an excellent phytoremediation plant resource for cadmium-contaminated soils with application prospects. However, its small biomass and low single-season remediation efficiency limit its large-scale application and promotion. How to improve the phytoremediation efficiency has always been a difficult point in the field of phytoremediation research in recent years.

[0003] In recent years, microbial enhanced phytoremediation has been one of the research hotspots in cadmium-contaminated soil remediation technologies. In particular, the applications of plant growth-promoting rhizobacteria (PGPR) and heavy metal-tolerant bacteria have received extensive attention. Improving the cadmium transport and accumulation ability of plants and promoting plant growth can significantly improve the cadmium remediation efficiency of Sedum plumbizincicola.

[0004] The rhizobium Rhizobium sp. YM-1 of the present invention can significantly improve the ability of Sedum plumbizincicola to transport cadmium from roots to shoots, and can also produce indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, fix nitrogen, dissolve phosphorus, produce siderophores, etc., to promote plant growth, and significantly increase the cadmium accumulation in the shoots of Sedum plumbizincicola, which cannot be achieved by other microorganisms simultaneously. It has great potential in microbial enhanced hyperaccumulator phytoremediation of cadmium-contaminated soils. Summary of the Invention

[0005] The primary objective of the present invention is to provide a rhizobium YM-1 in view of the problems and deficiencies of the existing technology, so as to promote the absorption and transport of cadmium by hyperaccumulator plants and their biomass, and improve the remediation effect of hyperaccumulator plants on heavy metal pollution.

[0006] The objective of the present invention is achieved in the following manner.

[0007] A rhizobium (Rhizobium sp.) YM-1, with a deposit number of CCTCC No: M 2025295.

[0008] The rhizobium has the abilities to produce IAA, ACC deaminase, siderophores, and fix nitrogen and dissolve phosphorus.

[0009] The rhizobium also has the ability to promote plant growth, especially hyperaccumulator plants, and further promote the growth of Sedum plumbizincicola; it further includes increasing the biomass of plant roots and stems and leaves.

[0010] The rhizobia mentioned above also have the ability to promote plants, especially hyperaccumulator plants, and more specifically to promote cadmium accumulation in Sedum plumbizincicola, further including increasing the cadmium transport coefficient of plants and promoting the cadmium accumulation in the above-ground parts of plant stems and leaves.

[0011] The object of the second aspect of the present invention is to provide the application of the rhizobia or their bacterial solutions, including the application in at least one of producing IAA, ACC deaminase, siderophore, as well as nitrogen fixation and phosphorus solubilization.

[0012] The application of the rhizobia or their bacterial solutions is specifically the application of promoting plants, especially promoting the growth of hyperaccumulator plants, and more specifically promoting the growth of Sedum plumbizincicola. It includes increasing the biomass of plant roots and stems and leaves.

[0013] The object of the third aspect of the present invention is to provide the application of the rhizobia or their bacterial solutions, specifically promoting plants, especially hyperaccumulator plants, and more specifically promoting cadmium accumulation in Sedum plumbizincicola. It includes increasing the cadmium transport coefficient of plants and promoting the cadmium accumulation in the above-ground parts of plant stems and leaves.

[0014] The object of the fourth aspect of the present invention is to provide the bacterial solution. The strain YM-1 is inoculated into a YM medium without Congo red, cultured at 25 - 30 °C with a rotation speed of 170 - 180 rpm for 24 - 72 h to obtain the bacterial solution, and the pH value of the bacterial solution is 7.0 ± 0.2.

[0015] The rhizobia Rhizobium sp. YM-1 of the present invention is isolated from the root nodules of Artemisia argyi planted in severely cadmium-polluted soil. The strain YM-1 has a relatively fast growth rate, showing milky white on the plate and in the liquid medium, and can grow rapidly at an initial pH of 6.8 - 7.0 and a temperature of 25 - 35 °C.

[0016] The strain YM-1 of the present invention is inoculated into a YM medium (1.0 g / L of yeast extract, 10 g / L of mannitol, 0.5 g / L of dipotassium hydrogen phosphate, 0.2 g / L of magnesium sulfate, 0.1 g / L of sodium chloride, 0.05 g / L of calcium chloride, 0.02 g / L of boric acid, 0.02 g / L of sodium molybdate dihydrate, 1000 mL of deionized water, and its pH is 6.8 - 7.0), cultured at 25 - 35 °C with a rotation speed of 170 - 180 rpm for 24 - 72 h to obtain the bacterial solution, and the pH value of the bacterial solution is 7.0 ± 0.2.

[0017] The above-mentioned bacterial solution is a substance obtained by culturing rhizobia YM-1 in a microbial medium, containing rhizobia YM-1 and the metabolites of rhizobia YM-1.

[0018] The rhizobium Rhizobium sp. YM-1 of the present invention has a deposit number of CCTCC No: M 2025295. It was deposited at the China Center for Type Culture Collection (CCTCC) on February 25, 2025, and the deposit address is Wuhan University, Wuhan City, Hubei Province.

[0019] The rhizobium Rhizobium sp. YM-1 provided by the present invention has a short culture cycle, simple culture medium components, low cost, and the pH of the culture medium reaches 7.0 ± 0.2 during the stationary phase. The rhizobium Rhizobium sp. YM-1 can significantly improve the ability of Sedum plumbizincicola to transport cadmium from the roots to the shoots, and can also produce indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, fix nitrogen, dissolve phosphorus, produce siderophores, etc. to promote plant growth, and significantly increase the cadmium enrichment amount in the shoots of Sedum plumbizincicola, which cannot be achieved by other microorganisms at the same time, and has great potential in the microbial enhanced phytoremediation of cadmium-contaminated soil. Brief Description of the Drawings

[0020] Figure 1 It is the morphological structure of the strain Rhizobium sp. YM-1 on the plate;

[0021] Figure 2 It is the phosphorus-solubilizing ability of the strain Rhizobium sp. YM-1;

[0022] Figure 3 It is the nitrogen-fixing ability of the strain Rhizobium sp. YM-1;

[0023] Figure 4 It is the siderophore-producing ability of the strain Rhizobium sp. YM-1;

[0024] Figure 5 It is the IAA yield of the strain Rhizobium sp. YM-1;

[0025] Note: Different letters indicate significant differences (p < 0.05) of the same plant under different treatments.

[0026] Figure 6 It is the ACC deaminase-producing ability of the strain Rhizobium sp. YM-1;

[0027] Figure 7 It is the effect of inoculating the strain Rhizobium sp. YM-1 on the biomass of the stems, leaves and roots of Sedum plumbizincicola;

[0028] Note: Different letters indicate significant differences (p < 0.05) of the same plant under different treatments. Detailed Embodiments

[0029] The following are examples for further illustrating the present invention, but the protection scope of the present invention is not limited by these examples.

[0030] Example 1: Isolation and screening of the strain Rhizobium sp. YM-1

[0031] This strain was isolated and screened from the root nodules of Artemisia argyi grown in severely cadmium-polluted soil. The specific steps are as follows: Collect the surface soil (0-20 cm) of the farmland, air-dry it naturally in a cool and ventilated place, grind it and pass it through a 2-mm nylon sieve to remove impurities such as stones and plant residues. Uniformly spray the cadmium chloride solution on the soil to adjust the cadmium concentration in the soil to 100 mg / kg. After fully mixing, place it outdoors under rain-sheltered conditions for natural aging for 60 days, and turn it regularly during this period to ensure uniform distribution of pollutants. Put the aged polluted soil into flowerpots (5 kg per pot), transplant the Artemisia argyi seedlings with consistent growth, and conduct conventional water and fertilizer management. After 60 days of cultivation, take out the roots of Artemisia argyi completely and carefully peel off the root nodules growing on the roots.

[0032] After rinsing the complete root nodules with clean water, immediately perform surface disinfection on the root nodules. The surface disinfection steps are as follows: Disinfect the roots in 4% NaClO solution for 5 minutes and then rinse with sterile water, then soak them in 75% alcohol for 2 minutes and then rinse with sterile water. This process is repeated 5 times. After surface disinfection, blot the surface moisture with sterile filter paper, then cut off the epidermis with a sterile scalpel and put it into a sterile mortar for grinding. After grinding, transfer the ground sample into a 50-mL centrifuge tube, add 10 mL of sterile water for dilution to obtain a tuber root inoculation suspension. Dilute the prepared tuber root inoculation suspension at gradients of 10 －2 、10 －3 、10 －4 、10 －5 、10 －6 and then coat them on the rhizobium medium YM respectively, and place them in a constant temperature incubator at 30 °C for cultivation. Observe once every 24 hours, and select single colonies for multiple streak isolation and purification. The strain YM-1 has a relatively fast growth rate and appears milky white ( Figure 1 ) on the plate. Use the 16S universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGCTACCTTGTTACGACTT-3’) for PCR amplification, and sequence it by Sangon Biotech (Shanghai) Co., Ltd. Perform BLAST sequence alignment through the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). The results show that the strain YM-1 belongs to the genus Rhizobium.

[0033] Example 2: Plant growth-promoting characteristics of the strain Rhizobium sp. YM-1

[0034] I. Indoleacetic acid IAA

[0035] Inoculate 1% of the strain into the corresponding medium containing 100 mg / L of L-tryptophan, and shake-culture it in a shaker at 30 °C and 170 r / min for 5 days. Take 10000 rpm centrifugation of the bacterial suspension for 10 min, take 1 mL of the supernatant and mix it with 2 mL of Salkowski color reagent, place it in the dark at room temperature for 30 min. If the color turns pink, it indicates that the strain has the ability to produce indole acetic acid, and read the absorbance at 530 nm with a UV spectrophotometer. Use the IAA standard solution as the positive control and the sterile medium as the negative control. At the same time, configure the IAA standard curve with concentrations of 0, 10, 20, 30, 40, 50, 60, 70 mg / L, and calculate the content of IAA produced by the strain according to the standard curve. Measure it once every 24 h for 5 days.

[0036] II. Nitrogen fixation ability

[0037] Prepare a nitrogen-free medium: 10 g of glucose, 5.0 g of calcium carbonate, 0.2 g of sodium chloride, 0.2 g of magnesium sulfate heptahydrate, 0.2 g of potassium dihydrogen phosphate, 0.1 g of calcium sulfate, 20 g of agar powder, 1.0 L of distilled water, pH = 7.2 ± 0.2. Sterilize it at 115 °C for 30 min, pour the plate when it cools to 50 - 60 °C, inoculate and streak, and incubate it in an incubator at 30 °C for 6 days to measure the colony size.

[0038] III. Phosphate solubilization ability

[0039] Weigh 20.55 g / L of the phosphate solubilization medium. It is normal to have some insoluble substances. Sterilize it at 121 °C for 15 min. Inoculate the strain on the NBRIP medium plate and incubate it in an incubator at 30 °C for 6 days. Observe whether there is a clear zone around the colony.

[0040] IV. ACC deaminase

[0041] Adopt the ACC sole nitrogen source method. After inoculating the activated strain on the DF medium with (NH4)2SO4 as the nitrogen source for 3 - 4 days, transfer the strain to the ADF medium with ACC as the nitrogen source, and repeat inoculation on the ADF medium. If the strain can survive, it has the function of secreting ACC deaminase.

[0042] V. Siderophore production ability

[0043] Siderophore medium preparation:

[0044] A: Weigh 0.079 g of CAS and dissolve it in 50 mL of deionized water. Pipette 1 mL of concentrated hydrochloric acid and dissolve it in 1000 mL of deionized water, then add 0.2703 g of FeCl3·6H2O to prepare a solution containing 1 mmol / L FeCl3 and 12 mmol / L HCl. Pipette 10 mL of the 1 mmol / L FeCl3 solution and add it to the CAS solution to obtain Solution A.

[0045] B: Weigh 0.069 g of cetyltrimethylammonium bromide and dissolve it in 40 mL of deionized water to obtain Solution B.

[0046] Add Solution A to Solution B and stir well to obtain the CAS detection solution.

[0047] Prepare 0.1 mol / L phosphate buffer: Pipette 2 mL of 0.2 M phosphate buffer with pH = 6.8 into a 100 mL conical flask, add 38 mL of deionized water, and stir evenly.

[0048] Prepare CAS medium: Add 1.5 mL of 20% sucrose solution, 4.5 mL of 10% acid-hydrolyzed casein, 150 μL of 1 mmol / L CaCl2 solution, 3 mL of 1 mmol / L MgSO4, and 2.7 g of agar to 150 mL of CAS medium. Add deionized water to make up to 150 mL. Sterilize at 121 °C for 30 min and pour plates. Inoculate the strain on the CAS medium and incubate at 30 °C for 6 d. Observe the growth of the strain. If an orange siderophore secretion circle appears, it indicates that the strain has the ability to produce siderophores.

[0049] The results are as follows: NBRIP detection shows that the strain YM-1 has the ability to dissolve phosphate ( Figure 2 ), and also has the ability to fix nitrogen ( Figure 3 ); CAS detection shows that the strain YM-1 has the ability to produce siderophores ( Figure 4 ); The Salkowski colorimetric method shows that the strain YM-1 has the ability to produce auxin IAA, and its yield reaches the highest at 48 h, about 35 mg / L ( Figure 5 ); At the same time, it also has the activity of producing ACC deaminase ( Figure 6 ). The above results indicate that Rhizobium sp. YM-1 has multiple plant growth-promoting characteristics.

[0050] Table 1. Plant growth-promoting characteristics of strain YM-1

[0051]

[0052] Note: "+" indicates a positive reaction; "-" indicates a negative reaction

[0053] Example 3: Hydroponic experiment on promoting cadmium enrichment and growth of Sedum plumbizincicola by Rhizobium sp. YM-1

[0054] Select the lateral branches of Sedum plumbizincicola at the mature stage, cut off the 5-cm-long leafy branches with only one growing point and the leaves 3 cm below the lower end of the branches with sterilized scissors, soak them in water with sterilizing agent for 2 h, and finally rinse them three times with pure water. Put the treated plant branches into a hydroponic container, insert 6 Sedum plumbizincicola branches in one box, immerse the water level 1 cm below the lower end of the branches, and culture the Sedum seedlings in 1 / 4 and 1 / 2 Hoagland nutrient solutions for 5 days in sequence after 1 week of pure water culture. Set the temperature to 26 °C for 16 h during the day and 20 °C for 8 h at night.

[0055] At the same time, culture Rhizobium YM-1 in a Rhizobium liquid medium for 48 h to obtain a bacterial solution, immerse the roots of the above Sedum seedlings in the YM-1 bacterial solution for 2-3 h, and immerse the roots of the control group seedlings in sterile water for 2-3 h. Prepare 1 / 2 Hoagland nutrient solutions with two CdCl2 concentrations of 0 and 25 μM, and put the Sedum seedlings inoculated with the strain back into the nutrient solution for continued culture. Replace the nutrient solution once every 7 days. When replacing the nutrient solution each time, the plant roots need to be immersed in the bacterial solution again. After 21 days of culture, harvest after culturing with deionized water for 7 days.

[0056] Measurement of plant height and root length: Divide the plants into roots and shoots, and measure the plant height and root length.

[0057] Measurement of wet weight and dry weight: Select 3 seedlings at intervals, gently take them out, wash the roots and shoots 3 times with pure water, separate the shoots and roots with scissors, dry the surface moisture with paper towels, and weigh the total wet weight of the shoots and the total wet weight of the roots of the 3 seedlings respectively. Then, blanch the shoots and roots at 105 °C for 2 h and dry them to a constant weight at 80 °C, and weigh the dry weight. Determine the cadmium content in the shoots and roots by ICP-AES.

[0058] Analysis of test results:

[0059] Without cadmium: Compared with the control group, the shoot length of the Sedum plants with roots immersed in the YM-1 bacterial solution increased by 20.30%, and the root length increased by 11.57%; the shoot dry weight increased by 55.93%, and the root dry weight increased by 211.82% ( Figure 7 upper left), the shoot wet weight increased by 91.25%, and the root wet weight increased by 434.71% ( Figure 7 upper right).

[0060] 25 μM cadmium: Compared with the control group, the shoot length of the Sedum plants with roots immersed in the YM-1 bacterial solution increased by 23.77%, and the root length increased by 15.96%. The shoot dry weight increased by 9.19%, and the root dry weight increased by 124.60% ( Figure 7Lower left), the wet weight of the stems and leaves increased by 40.03%, and the wet weight of the roots increased by 143.30%( Figure 7 Lower right).

[0061] Under the treatment of 25 μM cadmium, compared with the control group, the cadmium content in the stems and leaves of Sedum plumbizincicola plants with YM-1 root immersion increased significantly by 60.00%, while the cadmium content in the roots decreased significantly by 41.63%; the cadmium accumulation in the above-ground part of a single Sedum plumbizincicola plant increased significantly by 76.51%; YM-1 root immersion increased the cadmium enrichment coefficient in the stems and leaves of Sedum plumbizincicola by 60.05% and decreased the cadmium enrichment coefficient in the roots by 41.63%; the cadmium translocation coefficient of Sedum plumbizincicola increased significantly by 184.62%. The above results indicate that YM-1 treatment significantly increased the cadmium enrichment of Sedum plumbizincicola and significantly improved the ability of cadmium to be transported from the underground part to the above-ground part, indicating that YM-1 has great potential in strengthening the phytoremediation of cadmium-contaminated soil by hyperaccumulators.

[0062] Table 2. Cadmium content in the above-ground part and roots of Sedum plumbizincicola after YM-1 root immersion

[0063]

[0064] Note: Different letters indicate significant differences at p < 0.05 for the same plant under different treatments.

Claims

1. A strain of Rhizobium sp. YM-1, with the preservation number of CCTCC No: M 2025295.

2. The rhizobia according to claim 1, characterized in that, It has the abilities of producing IAA, ACC deaminase, siderophore, as well as nitrogen fixation and phosphorus solubilization.

3. The rhizobia according to claim 1, characterized in that, It has the ability to promote the growth of plants, especially hyperaccumulator plants, and more specifically, to promote the growth of Sedum plumbizincicola; it further includes increasing the biomass of plant roots, stems and leaves.

4. The rhizobium according to claim 1, characterized in that, It has the ability to promote cadmium accumulation in plants, especially hyperaccumulator plants, and more specifically, to promote cadmium accumulation in Sedum plumbizincicola; it further includes increasing the cadmium transport coefficient of plants and promoting the cadmium accumulation amount in the above-ground parts of plant stems and leaves.

5. Use of the rhizobia or its bacterial liquid according to any one of claims 1 to 4, characterized in that It includes the application in at least one of producing IAA, ACC deaminase, siderophore, as well as nitrogen fixation and phosphorus solubilization.

6. Use of the rhizobium or its bacterial liquid according to any one of claims 1 to 4, characterized in that, Specifically, it is the application of promoting the growth of plants, especially hyperaccumulator plants, and more specifically, to promote the growth of Sedum plumbizincicola.

7. The application according to claim 6, wherein It includes increasing the biomass of plant roots, stems and leaves.

8. Use of the rhizobium or its bacterial liquid according to any one of claims 1 to 4, characterized in that, Specifically, it is to promote cadmium accumulation in plants, especially hyperaccumulator plants, and more specifically, to promote cadmium accumulation in Sedum plumbizincicola.

9. The application according to claim 8, characterized in that, It includes increasing the cadmium transport coefficient of plants and promoting the cadmium accumulation amount in the above-ground parts of plant stems and leaves.

10. The bacterial liquid according to any one of claims 5-9, characterized in that, Inoculate the strain YM-1 into a YM medium without Congo red, culture at 25 - 30 °C, with a rotation speed of 170 - 180 rpm, and a culture time of 24 - 72 h to obtain a bacterial solution, and the pH value of the bacterial solution is 7.0 ± 0.2.

Images