Kosakonia radicincitans and application thereof

A technology of nitrogen-fixing bacteria and nitrogen-free medium, applied in the field of microorganisms, can solve the problem of not being able to widely act on other crops, and achieve the effects of good ammonium secretion performance and nitrogen-fixing activity.

Active Publication Date: 2017-02-15
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the high host specificity of thi

Method used

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  • Kosakonia radicincitans and application thereof
  • Kosakonia radicincitans and application thereof
  • Kosakonia radicincitans and application thereof

Examples

Experimental program
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Embodiment 1

[0048] Embodiment 1: Screening and obtaining of bacterial strains

[0049] Take the root system, wash it with water and drain it. Soak it in 70% alcohol for 30 seconds, rinse it with sterile water for 3 times, and then soak it in 5%-20% NaClO solution with 1 drop of surfactant for 5-10 minutes. Then soak again in 70% alcohol for 30s, rinse with sterile water 5 times. Sterile water for the last rinse was applied to the LB solid plate. If there is no colony growth, it means that the disinfection is complete, otherwise, re-sampling and separation. Put the sterilized materials and 10 mL of phosphate buffer solution into a sterile mortar and grind them for 5 minutes, take 1 mL of the supernatant as the original solution, make a 10-fold serial dilution with sterile water, and then take 0.1 mL was spread on an Ashby nitrogen-free plate, cultured in an incubator at 28°C for 2 to 3 days, and single colonies with different colony shapes were selected, and the plate was streaked repea...

Embodiment 2

[0050] Embodiment 2: microscopic examination and electron microscope observation of bacterial strain

[0051] Pick Kosakonia radicincitans GXGL-4A from the inoculation loop under sterile conditions, smear it on a glass slide, make a slice and perform Gram staining, and observe the morphology of the bacteria under a microscope.

[0052] Cultivate the strain overnight with LB liquid medium, draw 1mL of the bacterial solution into a sterilized 1.5mL centrifuge tube, centrifuge at room temperature at 4000r / min for 2min, suck out the supernatant, add sterile water to resuspend the precipitate, and immediately absorb a small amount of bacterial solution Put it on a copper plate, place it in the air to dry, and use a scanning electron microscope (Tecnai G2spiritBiotwin, 120kV Bio-TEM) to observe the morphology of nitrogen-fixing bacteria after drying and fixing the sample.

[0053] Electron micrograph of the nitrogen-fixing bacterium Kosakonia radicincitans GXGL-4A figure 1 As shown...

Embodiment 3

[0054] Example 3: Identification of ammonium secretion characteristics of bacterial strains

[0055] Cultivate the GXGL-4A strain in LB medium at 37°C and 180r / min overnight, and the culture medium is 10 -5 After one-fold dilution, spread on Ashby nitrogen-free solid medium and grow for 4 days. Under nitrogen-free conditions, nitrogen-fixing bacteria GXGL-4A will use nitrogen in the air for nitrogen fixation, and finally secrete part of the fixed nitrogen out of the cell in the form of ammonia nitrogen. Therefore, use a pipette gun (under sterile conditions, cut off the Tip head with a monolithic knife) to absorb slightly viscous secretions, add 10 times the volume of sterile water to dilute, and pass the diluted solution through a 0.45 μm sterile filter. Membrane sterilized, collected into a sterile Eppendorf tube is the sample to be tested. The concentration of ammoniacal nitrogen in the samples was determined by indophenol blue-spectrophotometry.

[0056] (1) Preparation...

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Abstract

The invention relates to kosakonia radicincitans and an application thereof. The kosakonia radicincitans GXGL-4A is preserved in China General Microbiological Culture Collection Center on June 2, 2016 with registered preservation number of CGMCC No.12588, and the sequence of a 16S rRNA gene part is shown as SEQ ID NO.1. The nitrogen-fixing bacterium GXGL-4A provided by the invention has an excellent microbial nitrogen-fixing capacity; and in a non-nitrogen Ashby medium, a nitrogen-fixing enzyme activity is 232.94+/-8.82nmol of C2H4/(mL.h). In addition, the K.radicincitans GXGL-4A provided by the invention has a good ammonium-secreting capacity; and by cultivating the GXGL-4A on the non-nitrogen Ashby solid medium, the content of the detected extracellular ammonium nitrogen is 2.51 [mu]g/mL. The strain (the kosakonia radicincitans), which has a capsule, can resist bad external environmental conditions such as ultraviolet ray, drought and the like, and the strain has a significant effect of promoting the growth of such crops as corn, rice and the like. The strain can be used for preparing bacterial manure and can reduce the application amount of a nitrogen fertilizer, so as to achieve effects of increasing production, preventing eutrophication of a water body, protecting environment and the like.

Description

technical field [0001] The invention relates to the technical field of microbes, in particular to a nitrogen-fixing bacterium Kosakonia radicincitans GXGL-4A capable of secreting ammonia nitrogen and promoting the growth of crops such as corn and rice and its application. Background technique [0002] Nitrogen is the basic constituent element of various key organic molecules such as protein, chlorophyll and nucleic acid in organisms, and it is also an essential nutrient element in the process of plant growth and development. In agricultural production, nitrogen is an important symbol to measure the status of soil nutrients and soil fertility. Because organisms take away a large amount of inorganic nitrogen from the soil every year, and water will also wash away a large part of soluble nitrogen, in order to supplement the loss of nitrogen in the soil, the most convenient and effective way is to use industrial chemical nitrogen fertilizer. According to the statistics of the U...

Claims

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Application Information

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IPC IPC(8): C12N1/20A01P21/00C05F11/08C12R1/01
CPCC05F11/08C12N1/205C12R2001/01
Inventor 陈云鹏李琼洁程杰杰孙帅欣
Owner SHANGHAI JIAO TONG UNIV
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