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Saline-alkaline tolerant ammonium secreting type authigenic nitrogen-fixing bacterial strain and application thereof

A technology of salt-alkali tolerance and nitrogen-fixing bacteria, which is applied in the direction of application, bacteria, and biocides, can solve the problems of large impact and low nitrogen fixation efficiency, and achieve the effects of increasing biomass, promoting growth, and widening the application range

Active Publication Date: 2021-03-12
山东土地集团威海有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to solve the technical problems of the existing non-symbiotic nitrogen-fixing bacteria rhizosphere biological stress and abiotic stress and other environmental factors that have a great influence and low nitrogen fixation efficiency, and provide a saline-alkali-resistant secreting agent with strong nitrogen-fixing and ammonium secreting capabilities. Ammonium-type autogenous nitrogen-fixing bacteria strain and its application

Method used

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  • Saline-alkaline tolerant ammonium secreting type authigenic nitrogen-fixing bacterial strain and application thereof
  • Saline-alkaline tolerant ammonium secreting type authigenic nitrogen-fixing bacterial strain and application thereof
  • Saline-alkaline tolerant ammonium secreting type authigenic nitrogen-fixing bacterial strain and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: the isolation of saline-alkali-resistant nitrogen-fixing bacteria

[0028] 1. Ashby nitrogen-free medium: mannitol 10g, KH 2 PO 4 0.2g, MgSO 4 ·7H 2 O 0.2g, NaCl 0.2g, CaSO 4 2H 2 O 0.2g, CaCO 3 5g, pH 7.2.

[0029] Salt-tolerant Ashby nitrogen-free medium: mannitol 10g, KH 2 PO 4 0.2g, MgSO 4 ·7H 2 O 0.2g, NaCl 50g, CaSO 4 2H 2 O 0.2g, CaCO 3 5g, pH 7.2.

[0030] 2. Initial screening of nitrogen-fixing bacteria:

[0031] Weigh 10g of the soil sample from the coastal saline-alkali land in Weifang, Shandong, put it into a 250mL Erlenmeyer flask containing an appropriate amount of glass beads and 100mL of normal saline, vibrate at 180r / min for 1h, let it stand for 20min, and serially dilute and spread it on the Ashby nitrogen-free medium for solid culture base. The plate coated with the bacterial solution was incubated upside down in a constant temperature incubator at 37°C for 3 days. The nitrogen-fixing bacteria were isolated and purif...

Embodiment 2

[0034] Embodiment 2: the identification of nitrogen-fixing bacteria MX-3

[0035]The genomic DNA of strain MX-3 was extracted, and the full-length sequence of the 16S rRNA gene of the genomic DNA of the strain was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). PCR products were sent to Huada Gene Company for sequencing. Two-way sequencing was used for splicing, and a 16S rDNA gene sequence close to 1.5Kb in length was obtained. The 16S rDNA gene sequence results were compared with the GenBank database using EzBioCloud Identify service (https: / / www.ezbiocloud.net / identify) and NCBI Nucleotide BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) For comparison, select effectively named type strains as a reference.

[0036] The culture morphological characteristics of the strain are: Gram-negative; cells (18h) are oval-shaped, with pointed ends (2×3-4 μm), single cells, paired cells or 6-8 cell chains appear in liqui...

Embodiment 3

[0037] Embodiment 3: the culture condition optimization of saline-alkali-resistant soil nitrogen-fixing bacteria

[0038] Inoculate the saline-alkali-tolerant nitrogen-fixing bacteria MX-3 into Ashby nitrogen-free medium, and cultivate it for 3 days at 37°C with shaking at 180r / min.

[0039] 1. Effect of temperature on the growth of strain MX-3

[0040] The strains were inoculated into Ashby nitrogen-free liquid medium, placed at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, and shaken at 180r / min for 3 days, and then the bacterial suspension was measured by an ultraviolet spectrophotometer OD 600 absorbance value.

[0041] 2. The effect of pH on the growth of strains

[0042] The strains were inoculated into Ashby nitrogen-free liquid medium with pH 5, 6, 7, 8, 9, 10, cultured with shaking at 200 r / min at 37°C for 3 days, and then the OD of the bacterial suspension was measured with a visible light spectrophotometer 600 absorbance value.

[0043] 3. The effect of salt concentratio...

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Abstract

The invention relates to a saline-alkaline tolerant ammonium secreting type authigenic nitrogen-fixing bacterial strain and application thereof to solve technical problems that existing non-symbioticnitrogen-fixing bacterial rhizosphere biological stress, non-biological stress and other environmental factors are very large in influence and low in nitrogen-fixing efficiency; and the invention provides a saline-alkaline tolerant ammonium secreting type authigenic nitrogen-fixing bacterial strain named as Azotobacter salinetris MX-3. The preservation institution is China General MicrobiologicalCulture Collection Center, and the preservation number is CGMCC No. 19426. The invention also provides application of the saline-alkaline tolerant ammonium secreting type authigenic nitrogen-fixing bacterial strain. The saline-alkaline tolerant ammonium secreting type authigenic nitrogen-fixing bacterial strain can be applied to the field of saline-alkaline tolerant strains.

Description

technical field [0001] The invention relates to a nitrogen-fixing bacterial strain and its application, in particular to a saline-alkali-resistant ammonium-secreting self-generated nitrogen-fixing bacterial strain and its application. Background technique [0002] There are some microorganisms in nature that can convert N in the atmosphere 2 reduced to NH 3 , this phenomenon is called biological nitrogen fixation. The study of nitrogen-fixing bacteria and its nitrogenase has become a hot spot in agricultural production application and scientific research. According to the relationship between nitrogen-fixing bacteria and higher plants, biological nitrogen fixation can be divided into three types: symbiotic nitrogen fixation, self nitrogen fixation, and combined nitrogen fixation. Symbiotic nitrogen-fixing bacteria have high nitrogen fixation efficiency, usually 75-300 kg of pure nitrogen / (ha·year), which can provide 100% nitrogen source for legumes. However, symbiotic ni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A01N63/20A01P21/00C12R1/065C12R1/11C12R1/41
CPCC12N1/20A01N63/20
Inventor 王志春席悦仲乃琴赵盼傅深展
Owner 山东土地集团威海有限公司
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