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A high-efficiency nitrogen-fixing Rhizobium meliloti strain and its molecular marker screening method

An alfalfa and rhizobia technology, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of inability to achieve high yield, low nitrogen fixation efficiency, mixed rhizobia strain types, etc., and achieve good affinity , planting yield increase, good adaptability effect

Active Publication Date: 2017-12-08
HEBEI UNIVERSITY
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  • Description
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Problems solved by technology

[0003] The purpose of the present invention is to provide a high-efficiency nitrogen-fixing alfalfa rhizobium strain and its molecular marker screening method to solve the problems of mixed types of rhizobia strains obtained by existing screening methods, low nitrogen fixation efficiency, and failure to achieve high yield when applied to alfalfa production. question

Method used

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  • A high-efficiency nitrogen-fixing Rhizobium meliloti strain and its molecular marker screening method
  • A high-efficiency nitrogen-fixing Rhizobium meliloti strain and its molecular marker screening method
  • A high-efficiency nitrogen-fixing Rhizobium meliloti strain and its molecular marker screening method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] (1) Sampling, separation and purification: For the alfalfa planted in the surrounding areas of Huanghua, Cangzhou, Hebei, we selected the plants with good growth and robustness, and the root nodules were sampled at the early flowering stage of alfalfa. At each sampling location, a 5-point sampling method was used. Sampling, the sampling area of ​​each point is 1m 2 , select plump pink nodules for each plant, 3 large nodules, and two sites, each with 45 nodules, and a total of 90 nodules were obtained from the two sites;

[0026] To isolate and purify the selected nodules, the specific steps are as follows:

[0027] (a) Select the intact and unbroken nodules and put them into sterile water to fully soak them;

[0028] (b) Disinfection of the nodule surface: 95% ethanol for 3 minutes—0.5% hypochlorous acid for 3 minutes—sterile water for 6-8 times;

[0029] (c) Plate streaking separation: crush the sterilized nodules with a toothpick, pick the bacterial liquid in the no...

Embodiment 2

[0062] Example 2 Identification of strains

[0063] The preservation name of the strain provided by the present invention is Sinorhizobium meliloti 729-2-4, the preservation unit is CGMCC, the preservation number is CGMCC No. 9755, and the preservation date is October 10, 2014. The address of the preservation unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0064] The high-yielding alfalfa rhizobia strain provided by the present invention has an optimum growth temperature of 28° C., aerobic culture, and colonies on YMA medium are translucent, milky white, round, with thin edges, thick and thin middles, and grow. The time is 48-72 hours, and the colony size is 3-5mm; the bacterial cell is rod-shaped, with hydroxy fatty acid particles with strong refraction, accounting for 1 / 3 of the cell volume, slightly rounded at both ends, and 2-3μm × 3- 5 μm, Gram-negative bacteria, Gram stained red.

Embodiment 3

[0065] Example 3 Application of new strains

[0066]Experimental method: the strain obtained in Example 1 of the present invention is inoculated on alfalfa planted in the field (around Huanghua), and its inoculation method, inoculation area and sampling detection method are as shown in step 8 of Example 1; The experimental conditions were inoculated with the American strain (AM185H2) and the Australian strain (AU128) as a comparative experiment.

[0067] Experimental results:

[0068] Table 4 Application results of bacterial species

[0069]

[0070] Note: Tukey test is used, a b c are different subsets, the above data are sampled at five points in the experiment, and the average value of the indicators measured in three parallel experiments.

[0071] It can be seen from the table that the strains from Australia and the United States are not as good as the strains screened by the present invention in improving the yield of alfalfa. It can be seen that the screening metho...

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Abstract

The invention discloses a high-efficiency nitrogen-fixing Rhizobium meliloti strain. The preservation name of the strain is Sinorhizobium meliloti 729‑2‑4, the preservation unit is CGMCC, the preservation number is CGMCC No.9755, and the preservation date is 2014. October 10; the strain is a rhizobia strain that can establish a good symbiotic relationship with alfalfa and has strong nitrogen-fixing ability, which can effectively increase the planting yield of alfalfa; at the same time, the invention also provides the molecular marker of the rhizobia strain The screening method is scientific and reasonable, simple and easy to operate, and can accurately screen high-efficiency nitrogen-fixing rhizobia strains that are suitable for the local climate, soil, and a good symbiotic relationship with alfalfa.

Description

technical field [0001] The invention relates to a rhizobium strain and a screening method thereof, in particular to a high-efficiency nitrogen-fixing alfalfa rhizobia strain and a molecular marker screening method thereof. Background technique [0002] Alfalfa, original name: Medicago sativa, also known as: Alfalfa, Latin name: Medicago sativa, Leguminosae is a perennial grass plant with strong roots, deep into the soil layer, and developed root necks. It is a forage with good palatability, high nutritional value and high development value. The growth of alfalfa mainly depends on the symbiotic relationship between Rhizobium and its symbiotic nitrogen fixation. Studies have shown that the establishment of the symbiotic relationship between rhizobia and alfalfa is the result of the tripartite interaction between bacteria, plants and the environment, so no matter in the origin or the introduction site of alfalfa, only the rhizobia adapted to the local environment and the alfal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/6869C12N1/205C12R2001/01
Inventor 刘晓云刘朋飞刘桂霞
Owner HEBEI UNIVERSITY
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